Citation
Reformation of the nuclear envelope following mitosis in chinese hamster ovary cells

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Title:
Reformation of the nuclear envelope following mitosis in chinese hamster ovary cells
Creator:
Conner, Gregory E., 1950-
Publication Date:
Language:
English
Physical Description:
ix, 88 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Cell cycle ( jstor )
Cell membranes ( jstor )
Cultured cells ( jstor )
DNA ( jstor )
Electrophoresis ( jstor )
Gels ( jstor )
Nuclear membrane ( jstor )
Phosphates ( jstor )
Phospholipids ( jstor )
Surface areas ( jstor )
Cricetulus ( mesh )
Mitosis ( mesh )
Nuclear Envelope ( mesh )
Ovum ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1978.
Bibliography:
Includes bibliographical references (leaves 83-87).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Gregory E. Conner.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
25174135 ( OCLC )
ocm25174135
0029767090 ( ALEPH )

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Full Text














REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS











By

GREGORY E. CONNER












A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY







UNIVERSITY OF FLORIDA

1978














ACKNOWLEDGEMENTS

I would like to thank Dr. Kenneth D. Noonan, my graduate

supervisor, for his endless support and encouragement during the

course of my graduate studies.

I would also like to thank the members of my supervisory

committee, Drs. C. M. Allen, C. M. Feldherr, E. M. Hoffman, and

T. W. O'Brien for their help and advice throughout this research

project. I am particularly grateful to Dr. Feldherr for his assis-

tance in obtaining the various micrographs. I would also like to
express my thanks to Dr. Allen for his assistance with thin layer

chromatography.














TABLE OF CONTENTS

ACKNOWLEDGEMENTS. ... .. ii
LIST OF TABLES. ................... .... v
LIST OF FIGURES . vi
ABBREVIATIONS USED. . ... .. vii
ABSTRACT. . ... viii
CHAPTER I-INTRODUCTION. ... 1
NE Structure . ... .. 1
Membranes of the NE. . 3
Pore Structures. . ... .. 3
Proteinaceous Lamina ................. 4
Association of Chromatin with NE 5
Isolation and Composition of NE. 5
Isolation Procedures . 5
Composition . 7
Morphological Changes in the NE
During Cell Cycle . 9
Objectives . .12
CHAPTER II-ISOLATION AND CHARACTERIZATION ... .13
Materials and Methods. .................... 13
Materials. . .. 13
Cell Culture . 13
Isolation of Nuclei and NE ... .14
Isolation of Plasma Membrane .... 15
Chemical Assays ................... ...16
Enzyme Assays................... .. .16
Electron Microscopy. . .. 17
SDS Polyacrylamide Disc Gel
Electrophoresis (PADGE). 17
Results . .18
Isolation of Nuclei. .................. 18
Morphology of the Purified Nuclear
Fraction . 22
Isolation of NE .. ... ... 26
Morphology of the Purified NE Fraction 26
Recovery of Isolated Fractions 28
Characterization and Purity of Nuclear
and NE Fractions . 28
Enzyme Activity. ... .... .......... ..30
Chemical Composition . 32
Peptide and Glycopeptide Composition .. 34
CHAPTER III-REFORMATION OF THE NUCLEAR ENVELOPE
DURING MITOSIS . ... ... .36
Materials and Methods. .................... 36
Materials. . 36
Cell Culture and Synchrony .. 36









Labeling of Cell Cultures. ... 37
Isolation of Nuclei and NE .... 38
Determination of Specific Activities .. 38
Determination of Precursor Pool
Equilibration Rates . 39
Measurement of Nuclear Surface Areas .... 40
Polyacrylamide Gel Electrophoresis .... 40
Results . .. .. 41
Cell Synchrony ... ... .. ... ... ... 41
Peptide Composition of the NE during
Different Phases of the Cell Cycle .... 43
Dilution of [3H] Leucine Labeled NE. ... 44
Incorporation of the [3H] Leucine
into NE Protein. . 53
Dilution of [3H] Choline Labeled NE ... 59
Dilution of [32p] Orthophosphate
Labeled NE Phospholipid. ... 61
Incorporation of [32p] Orthophosphate
into NE Phospholipid .. .. .. .. .. ...... .64
Rate of Precursor Pool Equilibration .. 65
Changes in Nuclear Surface Area. ... 75
CHAPTER IV-DISCUSSION . ... .. 77
APPENDIX--DERIVATION OF THE EQUATION FOR CALCULATING
THE PROPORTION OF M/G1 NE SYNTHESIZED DE NOVO
DURING THE G2-M-G1 TRANSITION ............... 82
BIBLIOGRAPHY. . .. 83
BIOGRAPHICAL SKETCH .... ..... 88
















LIST OF TABLES

I. Recovery of Protein, DNA, and
Phospholipid in Subcellular Fractions.

II. Enzyme Activities of Subcellular
Fractions ...............

III. Chemical Content of Subcellular Fractions.

IV. Dilution of [3H] Leucine Labeled NE
Protein. ............... .

V. Dilution of Labeled NE Phospholipids .


. 29


. 31

. 33


. 47

. 60














LIST OF FIGURES

1. Diagrammatic Representation of NE
Structure .


2. Flow Diagram of Nuclei and NE Isolation
Procedures. . .. 19

3. Phase Contrast Micrograph of the Purified
Nuclear Fraction. . .. 23

4. Electron Micrograph of the Purified Nuclear
Fraction. . ... .... .25

5. Electron Micrograph of the Purified NE
Fraction. . .. .... .27

6. Coomasie Blue Stained Profile of Sub-
cellular Fractions. . .35

7. Cell Cycle Parameters in Synchronized
CHO Cells . 42

8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells 45

9. Dilution of [3H] Leucine Labeled NE
Protein ............... .. ..... 50

10. SDS Gel Electrophoresis of NE Peptides
Isolated after Removal of [3H] Leucine
at the G2/M Boundary. . 52

11. Incorporation of [3H] Leucine into
NE Protein. . ... 55

12. SDS Gel Electrophoresis of [3H] Leucine
Pulse Labeled NE Peptides 58

13. Dilution of [32p] Labeled NE Phospholipid 63

14. Incorporation of [32p] Labeled NE Phos-
pholipid . 67

15. [3H] Leucine Pool Dilution. ... 70

16. [32P] Phosphatidic Acid Pool Dilution 74














ABBREVIATIONS USED

BME $-Mercaptoethanol

CHO Chinese hamster ovary

DNase I Deoxubonuclease I

EDTA ethylene diaminetetraacetic acid

EM electron microscopy

ER endoplasmic reticulum

HU hydroxyurea

LSC liquid scintillation counting

M mitosis

NE nuclear envelope

PBS phosphate buffered saline

PMSF phenylmethylsulfonyl fluoride

RER rough endoplasmic reticulum

RNase A Ribonuclease A

SDS sodium dodecyl sulfate

TM 50mM Tris, pH 7.6, 2.5mM MgC12










Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS

By

Gregory E. Conner

August 1978

Chairman: Kenneth D. Noonan
Major Department: Biochemistry

A technique for isolating nuclei and nuclear envelopes (NE) from

Chinese hamster ovary (CHO) cells has been developed. This technique

does not depend on the use of detergents to solubilize contaminating

chromatin. In this procedure NE are prepared from purified nuclei by

nuclease digestion and subsequent high salt-sucrose gradient centrifu-

gation. The nuclei and NE fractions are free of significant contamina-

tion by other subcellular organelles as judged by electron microscopy

and enzymatic analysis. Electron microscopic observation of the puri-

fied NE clearly demonstrates that the isolated material retains both a

double bilayer and the pore complexes observed in intact nuclei,

strongly suggesting that the isolation procedure described in this

dissertation permits the recovery of "intact" NE. Biochemical analysis

of the isolated nuclei and NE shows the NE fraction to be composed

primarily of protein and phospholipid, while containing only small

amounts of DNA and RNA. Examination of the peptide composition of the

NE fraction by SDS polyacrylamide gel electrophoresis reveals a very

complex coomasie blue staining profile with prominent bands in the

55,000 to 75,000 dalton molecular weight range.








Employing this isolation technique I have examined the breakdown

and reformation of the NE during a limited stage (late G2, M, and early

G1) of the replicative cycle in synchronized populations of CHO cells.

Using [ H] leucine as a precursor for protein and either [ 3H] choline

or [32P] orthophosphate as precursors for phospholipid, I have shown

that a minimum of 60% of the early G1, NE protein and a minimum of 50%

of the early G1 NE phospholipid were present during the proceeding G2

phase of the cell cycle and were reutilized in the reformation of the

NE which occurs during late M and early G1. Pulse label studies

employing [3H] leucine or [32P] orthophosphate indicate that a burst

of NE biosynthesis occurs in early G1. Autoradiographic examination

of NE peptides isolated during these pulse label and label dilution

studies shows neither preferential loss nor preferential biosynthesis

of specific NE peptides during the G2 to M or M to G1 transition. My

evidence suggests that all the peptides of the NE, which I can resolve

in one dimensional gel electrophoresis, are synthesized during this

portion of the cell cycle. Examination of NE peptides by one dimension-

al gel electrophoresis does not highlight any reproducible changes in

NE peptide composition which can be correlated with specific phase of

the cell cycle.














CHAPTER I
INTRODUCTION

The nuclear envelope (NE) is a complex subcellular organelle

which exists at the nuclear periphery and separates the genetic

machinery of the eucaryotic cell from other cellular components. During

mitosis in many plant and animal cells, the NE becomes morphologically

indistinct and reappears after daughter chromosome separation is com-

plete. The studies which will be described in this dissertation are

directed towards understanding the mechanisms) by which this large

membranous organelle disassembles and restructures during mitosis.

Before presentation of these studies, I will briefly discuss the cur-

rently available information concerning NE structure, composition and

breakdown and reformation during mitosis.

NE Structure

The structure of the NE has been probed in great detail by many

light and electron microscopic (EM) studies (for reviews see Feldherr,

1972; Kay and Johnston, 1973; Kessel, 1973; Franke, 1974; and Franke

and Scheer, 1974). The NE (shown diagramatically in Figure 1) is com-

posed primarily of two lipid bilayer membranes which contain pore

structures. Electron microscopic observation suggests that these

bilayer membranes are continuous at the pore structures leaving apparent

gaps when viewed in transverse sections. These pore complexes do con-

tain internal structure and may be involved in nuclear-cytoplasmic

exchange of molecules (Feldherr, 1972). The outer surface of the outer
















ONM-


'PL

INM-
\\ \\\ \ 1 ^ V *' 8:* r iHC










Figure 1. Diagrammatic representation of NE structure.
ONM outer nuclear membrane; INM inner
nuclear membrane; PS pore structure; PL -
proteinaceous lamina; HC heterochromatin.







nuclear membrane is often studded with ribosomes in a manner similar to

rough endoplasmic reticulum (RER) and occasionally has been observed to

be continuous with RER (Watson, 1955). The inner aspect of the inner

nuclear membrane appears to be in close association with a proteinaceous

lamina (Fawcett, 1966) and to be intimately associated with hetero-

chromatin (i.e. dense inactive chromatin).

Membranes of the NE

The membranes of the NE, when visualized by EM, are most fre-

quently found to be 60-80 A in thickness. The two membranes are normally

separated by 100-700 A except at the pore structures where they appear

to be joined, thus forming a perinuclear cisternum. The frequent local-

ization of ribosomes and the occasional continuity of the outer membrane

with RER indicates the strong morphological similarity between membranes

of the NE and RER. As will be discussed later, the enzymatic and bio-

chemical composition of the NE serves to strengthen the resemblance of

these two membranous organelles. Currently no data are available which

conclusively demonstrate that components of the NE arise from RER or

that components of the RER arise from those of the NE.

Recent studies by Feldherr et al. (1977) and Virtanen (1977,1978)

and Virtanen and Wartiovaara.(1976) suggest that the nuclear membranes

possess a property of sidedness similar to that of other cellular mem-

branes. Specifically these authors have reported that the carbohydrate

containing portions of NE glycopeptides are located on only one side,
the cisternal side, of each nuclear membrane.

Pore Structures

When pore structures of the NE are visualized by EM in transverse

sections, they appear as gaps (150 A 700 A wide) at which the nuclear








membranes seem to be fused (Figure 1). When visualized tangentially,

the pore structures appear as complexes of 8 or 9 granules arranged as

an annulus. The central zone of the pore structure is less electron

dense than the surrounding annular material and may coincide with the

gaps seen in transverse sections. The annular pore complexes have

an overall diameter of 1000 2000 A suggesting that the pore structure

extends beyond the gaps visualized in transverse sections possibly

overlapping part of the nuclear membranes. Electron dense granules

can occasionally be seen in the interior of the pores. Some data have

been obtained which suggest that these granules are material in transit

between nucleus and cytoplasm. Many authors have suggested that the

pore complexes function in the control of nucleo-cytoplasmic communi-

cation (for review see Feldherr, 1972).

Proteinaceous Lamina

In some cell types the inner aspect of the inner nuclear membrane

appears to be associated with an electron dense material. This layer,

variously called the dense lamella (Kalifat et al., 1967), zonula

nucleum limitans (Patrizi and Poger, 1967), and the fibrous lamina

(Fawcett, 1966), ranges in thickness from 2800 A in amoeba to 150 200 A

in vertebrate cell types. This layer, apparently proteinaceous in com-

position (Stelly et al., 1970), appears to be tightly associated with

the interphase NE, in that it may copurify with NE during subcellular

isolation procedures. The exact function of this proteinaceous lamina

has not been determined, however Stelly et al. (1970) have suggested

that it is responsible for the shape and rigidity of the nucleus.








Association of Chromatin with NE

Examination of the NE by EM has demonstrated a close association

of heterochromatin with the inner side of the inner nuclear membrane.

Based on technical difficulties associated with removing trace amounts

of DNA from purified NE, several authors have suggested that the asso-

ciation of DNA with the NE may play a functional role in DNA replica-

tion (e.g. O'Brien et al., 1972). Currently no solid evidence is

available which can be used to support this contention in vertebrate

cells (Franke, 1974).

Isolation and Composition of NE
Subcellular fractionation has proved to be one of the most pro-

ductive techniques employed by cell biologists.to probe the composition,

function, and biosynthesis of various components of the cell. Many

procedures for isolation of nuclei and NE from whole tissues have been

reported (for reviews see Franke, 1974; Kasper, 1974). To our know-

ledge, however, no large scale procedure for isolation of biochemically

characterized NE from cultured cells has been described. Procedures

which purport to isolate structural components from nuclei of cultured

cells (the so-called nuclear matrix [Hodge et al., 1977] or nuclear

ghost [Riley et al., 1975]) have been reported. These isolation

methods, however, are relatively harsh removing most of the lipid

bilayers of the nuclear membranes. These procedures appear to isolate

a nonmembranous portion of the NE which, it has been suggested, is

comprised of the proteinaceous lamina and pore complexes (Dwyer and

Blobel, 1976).

Isolation Procedures

Isolation of NE is attained by disruption of purified nuclei and

subsequent centrifugal separation of membranous components from the








remaining elements of the nucleus (e.g. nucleoli, chromatin, and soluble

proteins). Because separation of NE from other nuclear components is

usually dependent on the membranous characteristic of this organelle,

the purified nuclei from which NE are to be isolated, must be prepared

in a fashion which retains both nuclear membranes and yet removes other

contaminating organelles of the cell. Such nuclear isolation procedures

have been described and are usually modifications of the technique of

Chauveau et al. (1956). The lack of a well characterized preparation

of NE from cultured cells is most probably due to difficulties in iso-

lation of large quantities of "clean" nuclei which contain both nuclear

membranes. The use of detergents to remove cytoplasmic contamination

from nuclei most likely strips the nuclei of the lipid bilayers of the

nuclear membranes.

Disruption of nuclei for isolation of NE is usually accomplished

by mechanical and/or chemical treatments. The most frequently used

mechanical techniques are sonication (e.g. Franke, 1967; Kashnig and

Kasper, 1969) and hypotonic shock (e.g. Zbarsky et al., 1969; Kartenbeck

et al., 1971). High concentrations of MgCl2 (Berezney et al., 1970;

Monneron et al., 1972), sodium citrate (Bornens, 1968; Kashnig and

Kasper, 1969), NaCl and KC1 (Franke et al., 1970; Matsuura and Ueda,

1972), and heparin (Bornens, 1973,1978) have been shown to disrupt

nuclei and aid in solubilization of chromatin during NE isolation.

Deoxyribonuclease I (DNase I) has often been utilized to reduce the

viscosity of ruptured nuclei and to reduce the level of DNA contamina-

tion in isolated NE fractions (e.g. Berezney et al., 1970; Kay et al.,

1972).








After disruption of nuclei and dispersion of chromatin, differ-

ential or isopynic centrifugation is then employed to separate NE from

the remaining nuclear components (Kasper, 1974).

Composition

Franke (1974) and Kasper (1974) have extensively reviewed the

chemical and enzymatic composition of NE fractions isolated from

tissue. The NE is comprised primarily of protein and phospholipid and

has generally been described as a protein rich organelle. Reported

protein values generally vary between 60% and 75% of the total membrane

mass while phospholipid values range from 20% to 30% of the total

membrane mass (see Kasper, 1974). Phosphatidyl choline is the pre-

dominant lipid species of NE followed by phosphatidyl ethanolmine,

phosphatidyl serine and phosphatidyl inositol (Keenan et al., 1970,

1972; Khandwala and Kasper, 1971). Some neutral lipids such as

cholesterol and free fatty acids have also been reported in NE

fractions (Kleinig, 1970; Keenan et al., 1970, 1972).

Almost all isolated NE fractions contain small quantities of

DNA and RNA which vary in amount depending on the preparative pro-

cedure. Those procedures which do not strip the outer membrane of

ribosomes normally result in RNA values which are higher than those

obtained after NE isolation procedures which employ citrate or

MgC12. Small residual amounts of RNA associated with isolated NE

may reflect a functional component of the pores (Monneron and Bernhard,

1969; Dhainaut, 1970). The extent of DNase I treatment and the con-

centration of salt used in NE isolation procedures appear to affect

the quantity of DNA associated with the NE fraction.








Carbohydrates have been detected in isolated NE fractions by

chemical analysis. Kashnig and Kasper (1969) have reported that rat

liver NE contains 3%-4% carbohydrate which is made up of mannose,

glucose, and galactose. These results have been confirmed by

Franke (1977).

The enzyme composition of isolated NE has been reported to be

different by different workers although in most instances it appears

to closely resemble that of the microsomal membranes. Cytochemically

glucose-6-phosphatase (Orrenius and Ericsson, 1966; Rosen, 1969, 1970;

Leskes et al., 1971) and Mg2+ dependent ATPase (Klein and Afzelius,

1966; Yasuzumi and Tsubo, 1966) have been localized to the NE of a

variety of mammalian cell types. Typically these enzymes are present

and occasionally purified in isolated NE fractions (Franke, 1974).

The enzymatic resemblance of the NE and microsomes is strengthened by

the presence of NADH-cytochrome c reductase in NE. This activity is

not, however, increased by application of drugs (e.g. phenobarbital)

as is the enzymatic activity in microsomes (Kasper, 1971). NADPH-

cytochrome c reductase (another constituent of microsomal membranes)

has also been reported by many authors to be present in isolated NE of

liver and in NE of avian erythrocytes (Zentgraff et al., 1971), a cell

which is practically devoid of endoplasmic reticulum. The lack of

drug induction of NE NADH-cytochrome c reductase and the presence

of NE NADPH-cytochrome c reductase in ER depleted cells (avian erythro-

cytes) strongly suggests that these enzymes also do not represent ER

contamination of the isolated NE.








The cytochrome oxidase content of NE remains a controversial issue.

Several authors have reported the presence of cytochrome oxidase or

cytochrome aa3 spectrum in isolated NE (Zbarsky et al., 1969; Berezney

et al., 1972; Matsuura and Ueda, 1972; Jarasch and Franke, 1974).

Jarasch and Franke (1974) interpret their results as mitochondrial con-

tamination while Berezney et al. (1972) suggest that cytochrome oxidase

is a NE enzyme. Extensive discussions of this issue can be found in

Jarasch and Franke (1974) and Berezney et al. (1972).

At the present time, enzymatic analysis can not accurately deter-

mine NE enrichment or contamination by mitochondria or microsomes. Only

plasma membrane contamination can be accurately quantitated by use of

enzymatic markers because several plasma membrane specific enzymes

(e.g. Na+, K+, stimulated ATPase and 5' nucleotidase) have not been

reported to be present in any NE fraction. When evaluating the enrich-

ment or purity of NE fractions, it is necessary to rely heavily on the

presence of NE specific morphological features (pore structures and

double membranes) and the lack of other organellar structures such as

those of mitochondria or unbroken nuclei.

Morphological Changes in the NE
During Cell Cycle

Continuously growing cells proceed through a cycle of biochemical

and morphological events which reproducibly repeat in a fixed order

after each cell division. The events of this cell cycle have been

extensively studied (for review see Mitchison, 1971). The cell cycle

can be divided into four segments -- G1 phase, S (DNA synthetic) phase,

G2 phase, and mitosis. G1 phase is defined as that time segment which








exists between completion of cell division and initiation of DNA syn-

thesis, G2 phase is defined as that time segment which exists between

completion of DNA synthesis and initiation of cell division.

A variety of techniques are available which will induce individ-

ual cells of a culture to traverse the cell cycle in unison. These

synchronization techniques have been employed by cell biologists to

study the biochemical and morphological events which characterize

particular segments of this cycle and which are "hidden" by the random

nature of an untreated cell culture.

Mitosis is perhaps the most carefully studied morphological event

of the cell cycle. The sequence of events of mitosis is traditionally

divided into several phases based on early morphological descriptions.

Briefly, mitosis consists of four phases: prophase, metaphase, anaphase,

and telophase. The onset of prophase is marked by chromosome conden-

sation at the inner nuclear membrane. The nuclear envelope becomes

very irregular, taking on a "wave-like" appearance in the region of the

centrioles. Centriole movement to opposite sides of the nucleus (i.e.

the poles) and chromosome separation occur very early in prophase.

The nuclear envelope begins to dissociate at the poles and microtubules

of the forming spindle apparatus enter into the nucleoplasm. The

nuclear envelope breakdown proceeds toward the equatorial plane and in

late prophase the condensed chromosomes begin to assemble in the com-

pleted spindle (metaphase). At this point the NE is completely dis-

rupted. After a short pause the chromosomes separate at the centromere

into daughter chromatids and the chromatids begin moving toward the

poles anaphasee). The nuclear envelope begins to reform as this







movement begins. Rows of elongated membranous cisternae appear at

the polar aspect of the spindle. As the chromatids begin to fuse into
a chromatin mass, the row of cisternag becomes juxtaposed and extends

further toward the equatorial constriction (cytokinesis and telophase).
The NE is completely reconstructed before the finish of cytokinesis
which marks the end of the mitotic cycle.
Unfortunately very little is known concerning the fate of the

NE during mitotic breakdown nor is there good evidence relating to the
origin of the components which reform the NE in late M and early G1.

Similarly the mechanisms) whereby the NE is disassembled at the mitosis
and reformed in late mitosis have not been elucidated.
It has been suggested by a number of workers (Porter and Machado,

1960; Moses, 1964; Robbins and Gonatas, 1964; Murray et al., 1965;
Brinkley et al., 1967) that following NE breakdown, fragments of NE

mingle with and become indistinguishable from the endoplasmic reticulum

(ER). Furthermore, it has been argued that components of the ER are
utilized in reformation of the G1-NE (Porter and Machado, 1960; Moses,
1964). Other authors have suggested that the NE, or components of the

NE, persist through mitosis as distinct entities, biochemically differ-
ent from the ER, and that these components are specifically reutilized
to reform NE at the completion of M (Erlandson and De Harven, 1971;
Maruta and Goldstein, 1975; Maul, 1977). Finally some workers in the
field have suggested that the NE which reappears at the end of M is a
product of de novo synthesis of all the NE components (e.g. Jones,
1960).
The multiplicity and diversity of hypotheses which have been pre-
sented to explain NE breakdown and reformation during M are probably








due to the variety of animal and plant species in which the predom-

inantly microscopic work was performed and to the fact that the evidence

available concerning NE breakdown and reformation consists almost

entirely of morphological studies which suffer from the difficulty of

positively identifying cytoplasmic components which might be NE frag-

ments released during mitosis.

Objectives

The main objective of the studies presented in this dissertation

is to investigate the mechanism whereby the NE disassembles and reforms

during mitosis. To examine biochemical changes and the biosynthesis

of NE during the cell cycle it is necessary to develop a procedure to

use with cultured cells which would permit the isolation of "clean"

NE containing both lipid bilayers of the nuclear membranes as well as

the proteinaceous lamina.

In Chapter II of this dissertation I will report a preparation

technique which permits the isolation of nuclei from Chinese hamster

ovary (CHO) cells without the use of reagents (such as detergents) which

might disrupt membranes. The isolated nuclear fraction has been used

to purify sufficient quantities of NE to allow both biochemical and

EM studies of this organelle.

In Chapter III of this dissertation I will present a biochemical

investigation of the breakdown and reformation of the NE during mitosis

in CHO cells. Using radioactive precursors of protein and phospho-

lipid I have studied the reutilization and biosynthesis of NE in the

late G2-M-early G1 portion of the cell cycle.













CHAPTER II
ISOLATION AND CHARACTERIZATION

Materials and Methods

Materials

All tissue culture materials were purchased from Grand Island

Biological Company (Grand Island, New York). Tissue culture plastic-

ware was obtained from Corning Glass Works (Corning, N.Y.). Ultra-

pure sucrose, [methyl-3H] thymidine, and [5-3H] uridine were obtained

from Schwarz-Mann Division, Becton, Dickinson and Company (Orangeburg,

New York). Deoxyribonuclease I (DNase I) and ribonuclease A (RNase A)

were purchased from Worthington Biochemical Corporation (Freehold, New

Jersey) and were shown to be protease-free via the assay of Tomarelli

et al. (1949). [Methyl-3H] choline chloride was obtained from Amer-

sham Searle Corporation (Arlington Heights, Illinois). All reagents

for electrophoresis were purchased from Bio-Rad Laboratories (Rockville

Centre, New York). All other reagents were obtained from Scientific

Products (Ocala, Florida).

Cell Culture

Chinese hamster ovary cells (originally obtained from Dr. Kenneth

Ley, Sandia Laboratories, Albuquerque, New Mexico) were maintained at

370C in suspension culture in Ham's F-10 nutrient medium, supplemented

with 10% (v/v) calf serum and 5% (v/v) fetal calf serum. Cell density

was monitored daily and maintained between 1.2 x 105 4 x 105 cells/ml.








Isolation of Nuclei and NE

A flow chart of this procedure is presented in Figure 2. All

procedures were carried out at 00C unless otherwise noted. For each

NE preparation 3- 4 x 109 cells were harvested, washed twice with phos-

phate buffered saline (PBS, pH 7.2), and resuspended at 2 4 x 107

cells/ml in a homogenization buffer containing 10mM Tris (pH 7.6),

10mM KC1, and 10mM EDTA. The cells were allowed to swell for 15 min

and then lysed (in 15 ml batches) by 5-7 strokes in a Dounce homogen-

izer fitted with a tight pestle. Homogenization was continually

monitored by phase contrast microscopy to assure maximum cell lysis

with minimum nuclear breakage.

Immediately following lysis, the homogenate obtained from each

batch of cells was diluted with 2 volumes of 65% (w/w) sucrose, 50mM

Tris (pH 7.6), 7.5mM MgCl2, and kept on ice until homogenization of

the entire cell sample was completed. The diluted homogenate was cen-

trifuged in a Sorvall HB-4 rotor for 30 min at 4,936 x gmax. The

resultant crude nuclear pellet was resuspended in 24 ml of 65% (w/w)

sucrose, 50mM Tris (pH 7.6), 2.5mM MgC12 (65% sucrose-TM). Aliquots

of the crude nuclear pellet were overlayed with 6 ml of 60% sucrose-TM,

12 ml of 55% sucrose-TM, 6 ml of 50% sucrose-TM and 7 ml of 30% sucrose-

TM to form a discontinuous sucrose gradient. The gradient(s) was

subsequently spun for 1 hr at 72,100 x g in a Beckman SW27 rotor.

Nuclei banding at the 65%-60% and 60%-55% sucrose interfaces were

removed, combined, and diluted to 50% sucrose with TM buffer. Using

an SW27 rotor (72,100 x gmax for 1 hr) these nuclei were sedimented

through another discontinuous gradient containing 12 ml of 55% sucrose-

TM, 3 ml of 60% sucrose-TM and collected onto a cushion of 3 ml 65%








sucrose-TM. Material caught on the 65% sucrose-TM cushion was removed,

diluted to 10% sucrose with distilled water, and pelleted in an HB-4

rotor at 1020 x gmax for 5 min. This pellet is designated the purified

nuclear fraction.

In order to bring about nuclear lysis the purified nuclei were

resuspended in 1 ml of 10mM Tris (pH 8.5), containing O.1mM MgCl2.

After 20 min in 10mM Tris, O.1mM MgCl2, DNase I and RNase A were each

added to a final concentration of lO00g/ml and the incubation continued

for 20 min at 22C. Following lysis (which could be monitored with

phase optics) four volumes of 60% (w/w) sucrose, 50mM Tris (pH 7.6),

500mM MgC12 were added to the lysed nuclei. The solutions were

thoroughly mixed and the suspension placed in the bottom of a Beckman

SW41 centrifuge tube. A linear 20%-45% (w/w) sucrose gradient con-

taining 50mM Tris (pH 7.6) and 500mM MgCl2 was formed on top of the

sample and the gradients spun for 2 hr at 153,244 x gmax in a Beckman

SW41 rotor. Nuclear envelopes banded at 1.19 g/cc (35% sucrose in

500mM MgCl2). The purified NE's were diluted with 50mM Tris (pH 7.6)

500mM MgCl2 and then pelleted in a Beckman fixed angle 40 rotor for

30 min at 130,766 x gmax. The NE's were subsequently resuspended in

10% sodium dodecyl sulfate (SDS), 10mM Tris (pH 8.5), 1% B-Mercapto-

ethanol if they were to be analyzed by electrophoresis, or in 10%

sucrose, 10mM Tris (pH 7.6), 0.5mM MgC12 if chemical or enzymatic

analysis was to be performed on the sample.

Isolation of Plasma Membrane

Plasma membrane was isolated by a slight modification (McClure

and Noonan, 1978) of the aqueous two phase polymer technique of Brunette

and Till (1971).








Chemical Assays

All protein determinations were performed according to the pro-

cedure of Lowry et al. (1951). Phospholipid phosphorus (Rouser et al.,

1966) was measured in chloroform-methanol extracts (Rouser and

Fleischer, 1967) of various subcellular fractions. Phospholipid

content of the various fractions was extrapolated from the phosphorus

determination by multiplying the phosphorus value by a factor of 25.

Cholesterol was determined according to Glick et al. (1964). In

order to determine the RNA and DNA content of the NE fraction, cells

were grown for five generations in the presence of O.5lCi/ml [3H]

thymidine or 0.4pCi/ml [3H] uridine prior to preparation of NE. The

specific radioactivity of DNA or RNA (cpm/pg nucleic acid) was deter-

mined from the cell homogenate by liquid scintillation counting and

spectrophotometric assay. DNA content was determined by the diphenyl-

amine assay (Burton, 1956) while RNA content was determined by the

orcinol (I-San Lin and Schjeide, 1969) assay of extracts prepared

according to Fleck and Munro (1962). The specific radioactivity

obtained for DNA and RNA was then used to determine the relative

nucleic acid content of nuclei and NE.

Enzyme Assays

Glucose-6-phosphatase activity was determined according to

Franke et al. (1970) with the inorganic phosphate release being mea-

sured according to Fiske and Subbarowe (1925). 5'-Nucleotidase was

assayed via the technique of Widnell and Unkless (1969) with the

inorganic phosphate released being measured by a modification of the

technique described by Chen et al. (1956). Succinate dehydrogenase








was assayed according to Veeger et al. (1969). Cytochrome c oxidase

was assayed according to Smith (1955).

Electron Microscopy

Nuclei and NE's were fixed (00C, 2 hr) in 1.8% glutaraldehyde,

8% sucrose, 50mM phosphate (pH 7.6), and 2.5mM MgC12. Both the nuclei

and NE fractions were postfixed in the same buffer containing 1%

osmium tetroxide (22C, 1 hr). Samples were sequentially dehydrated

in increasing concentrations of ethanol and finally embedded in Spurr's

low viscosity medium (1969). Thin sections were stained with uranyl

acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965)

and then examined in an Hitachi AS8 electron microscope.

SDS Polyacrylamide Disc Gel Electrophoresis (PADGE)

All electrophoretic analyses were performed in 1.5mm thick slab

gels according to the procedure of Laemmli (1970). The gels used were

composed of a 5.6% acrylamide stacking gel overlaying a linear 7.5%

to 12.5% acrylamide gradient separation gel. The acrylamide ratio to

bis-acrylamide was maintained at 37.5:1 in both the stacking and

running gel. Purified NE's were solubilized by boiling at 1000C for

5 min in 10% SDS, 10mM Tris (pH 8.5), 1% B-mercaptoethanol (BME).

Plasma membrane was solubilized by resuspension and boiling in 2% SDS,

62.5mM Tris (pH 6.8), 10% glycerol, and 0.1% BME ("sample buffer").

Lysed nuclei, sampled after DNase I and RNase A digestion, and whole

cell homogenates were solubilized by boiling in one volume of 2x
"sample buffer." Each of the samples were then exhaustively dialyzed

against "sample buffer" containing 0.1% 6-mercaptoethanol and O.ImM

phenylmethylsulfinyl fluoride (PMSF). Inclusion of PMSF in the sample








buffer is essential since we have found that NE's dissolved in sample

buffer lacking PMSF are subject to extensive proteolysis, possibly

as the result of the co-purification of an intrinsic protease with

the purified envelopes (Carter et al., 1976).

Following electrophoretic separation of the membrane peptides

and glycopeptides, the slabs were fixed for 30 min in 10% trichloro-

acetic acid and then allowed to equilibrate overnight in 25% ethanol,

8% acetic acid. The following day the gels were stained with Coomasie

brilliant blue according to Weber and Osborn (1969) and finally

destined in 25% ethanol, 8% acetic acid.

Results

Isolation of Nuclei

The primary goal which I set for myself prior to isolating NE's

was that the purified NE's contain both the inner and outer membrane

bilayer as well as the pore complex and be free of contaminating cyto-

plasm or nucleoplasm. In order to achieve this goal, a nuclear

fraction had to be isolated which itself contained both the inner and

outer nuclear membranes and was free of substantial cytoplasmic con-

tamination. The nuclear isolation procedure which I have employed in

my work has been described in detail in the Materials and Methods

section and is presented diagrammatically in Figure 2.

In many of the nuclear isolation procedures previously published

workers have used nonionic detergents to remove cytoplasmic components

(Muramatsu, 1970) from the nuclei. Unfortunately the use of such

detergents on isolated nuclei from rat liver has been shown to remove

almost all of the nuclear membrane phospholipid and much of the protein




















Supernatant


inder of 55%-60% and
adient 60%-65% sucrose
SCARD) interfaces


rI-


60%-65% sucrose
interface


Dounce homogenize in hypotonic buffer
at 2-4 x 10/ cells/mi. Dilute with
2 volumes 65% sucrose, 50mM Tris
(pH 7.6), 7.5 mM MgC12.


Spin 4936 x gmax' 30 min
Resuspend in 65% sucrose TM and place
at the bottom of a SW27 gradient.


Spin 72,100 x gmax, 1 hr

Remove nuclei from interfaces.
Dilute to 50% sucrose TM, and place
in a SW27 gradient.

Spin 72,100 x gmax 1 hr


Remove nuclei from 60%-65% sucrose inter-
face, dilute with distilled water to 10%
sucrose


Cells


homogenate

I-----


\u.


pellet


Rema
gra
(DI!


Remainder of
gradient
(DISCARD)











pellet


pellet -



Figure 2. Flow Diagram of Nuclei and


Supernatant
(DISCARD)


Spin 1020 x gmax' 5 min


Purified Nuclear Fraction
Lyse in 10mM Tris (pH 8.5), 0.1mM MgC12,
nuclease digest, dilute with 60% sucrose,
50mM Tris (pH 7.6), 500mM MgC12 and place
at the bottom of a SW41 gradient.


Spin 153,244 x gmax' 2 hr



Remove NE (band at 1.19 g/cc). Dilute
with 50mM Tris (pH 7.6), 500mM MgCl2.


Spin 130,766 x gmax' 30 min


Purified NE Fraction


NE Isolation Procedure


Supernatant
(DISCARD)








(Aaronson and Blobel, 1974). For this reason I avoided the use of any

detergents in my nuclear isolation procedure. Instead, I developed

homogenization conditions which minimized nuclear clumping and adhesion

of cytoplasmic material to the nuclei. Specifically I found that

immediate dilution of the cell homogenate with 65% (w/w) sucrose, 50mM

Tris (pH 7.6), 7.5mM MgCl2 (Figure 2) maintained the nuclei in a

dispersed state. Isolation of "clean" nuclei from such an homogenate

could then be readily accomplished by density gradient centrifugation

if the nuclei were maintained under the salt and pH conditions outlined

in Figure 2 and handled with care so as to avoid premature lysis.

In the procedure which I developed (Figure 2) the whole cell

homogenate is diluted with 65% sucrose in 50mM Tris (pH 7.6), 7.5mM

MgCl2, and spun for 30 min at 4,936 x gmax. This differential centri-

fugation serves to collect nuclei in a pellet while leaving most other

membranous organelles in the supernatant. This crude nuclear pellet

is subsequently subjected to two sequential centrifugations on

sucrose step gradients (Figure 2) in which purified nuclei band at

the 60-65% sucrose interface. The purified nuclei are subsequently

diluted with distilled water (to give a final sucrose concentration

of 10%) and then collected as a pellet by a 5 min, low speed, differ-

ential centrifugation step (Figure 2).

Three precautions must be taken to ensure the isolation of a

"clean" nuclear fraction from CHO cells. (a) Homogenization must

be performed at pH 7.6 in the absence of divalent cations. (b)

Homogenization, resuspension, and mixing of fractions must be done

very carefully so as to avoid premature lysis or shearing of the








nuclei. Breakage of the nuclei at early stages in the isolation pro-

cedure inevitably results in nuclear clumping with concomitant

entrapment of cytoplasmic and nucleoplasmic contaminants. (c) Homog-

enization, resuspension, and mixing must be carefully monitored to

insure oneself that the nuclei are adequately dispersed. Failure to

achieve a relatively homogeneous mixture of nuclei inevitably results

in failure of the subsequent centrifugation steps to remove cytoplasmic

contaminants.

Morphology of the Purified Nuclear Fraction

As can be seen in the phase contrast micrograph presented in

Figure 3, nuclei purified via the procedure outlined in Figure 2 are

intact, contain distinct nucleoli, and show few cytoplasmic "tags."

Electron micrographs of the purified nuclear fraction show no obvious

contamination of the nuclei with mitochondria, RER, vesicles or large

sheets of plasma membrane (Figure 4A). Although rough or smooth

microsomes are not immediately detectable in the nuclear fraction,

it must be pointed out that the outer nuclear membranes of some nuclei

are blebbed. It is possible that these blebs could represent micro-

somal contamination. However it is more likely that the blebs simply

represent swelling of the outer nuclear membrane. A higher magnifi-

cation micrograph (Figure 4B) of nuclei isolated in the "purified

nuclear pellet" (Figure 2) clearly demonstrates that both the inner

(arrow) and outer (double arrow) nuclear membranes are present on

the isolated nuclei and that very few ribosomes are attached to the

outer membrane. Furthermore, the isolated nuclei have obvious pores

(triple arrow) associated with them.























***'' *.. ^D B
7 'i-.;; l
HetA-*''^Pf


Figure 3. Phase Contrast Micrograph of the Purified
Nuclear Fraction. (x1340)


~~9;r ~~


~ii*i r*


























Figure 4. Electron Micrograph of the Purified Nuclear Fraction
Purified nuclei were fixed, embedded, and stained as
described in Materials and Methods.

A. Representative section through "purified nuclear
fraction" (x3775).

B. High magnification EM of two randomly chosen nuclei
with the attention being directed to the nuclear
membranes (x24,350) (') outer bilayer; (T) inner
bilayer; C( ) pores.





25








Isolation of NE

The NE isolation procedure described in the Materials and Methods

section is a combination and modification of the procedures published

by Kay et al. (1972) and Monneron et al. (1972) for the isolation of

NE from rat liver nuclei.

As outlined in Figure 2, the NE isolation procedure begins with

the "purified nuclear fraction." In the isolation method used the

purified nuclei are lysed in 10mM Tris (pH 8.5) containing O.ImM MgCl2.

Incubation of the nuclei in this hypotonic buffer for 20 min at 0C

produces a very viscous solution which is subsequently incubated with

DNase I and RNase A for 20 min at 22C. Addition of the respective

nucleases immediately reduces the viscosity of the lysed nuclei.

Following nuclease digestion, the material is diluted with 4 volumes

of 60% sucrose, 500mM MgC12 (to remove residual chromatin [Monneron

et al., 1972]) in 50mM Tris (pH 7.6) and the mixture vortexed.

During the ensuing centrifugation (SW41 rotor at 153,244 x gmax'

2 hr), NE float up into the gradient leaving nucleic acid, soluble

proteins of the nucleoplasm, and unlysed nuclei in the dense load

zone. Under the centrifugation conditions used, the NE fraction bands

in the gradient at a density of approximately 1.19 g/cc. The NE are

subsequently removed from the gradient and pelleted (Figure 2).

Morphology of the Purified NE Fraction

Electron micrographs of sections through the purified NE frac-

tion (Figure 5) demonstrate that most, if not all, of the isolated

nuclear membranes retain the double membrane sheets (single arrow)

characteristic of the NE. Furthermore, as would be expected if one













































Figure 5. Electron Micrograph of the Purified NE Fraction

The NE fraction was fixed, embedded, and stained
as described in the Materials and Methods
section (x31,850). Regions of double membranes
(T) can be seen as well as pores in tranverse
) and tangential section (f .








were isolating intact NE's, nuclear pores can be seen in both trans-

verse (double arrow) and tangential section (triple arrow). Clearly

the presence of pores and double membranes in this purified fraction

supports its identify as a NE fraction.

Few, if any, vesicles are apparent in this preparation (Figure 5),

suggesting little or no microsomal contamination. Similarly no mito-

chondria or unbroken nuclei are observed in this "purified NE fraction."

Recovery of Isolated Fractions

Typical recoveries of protein, phospholipid and DNA obtained

during preparation of the NE are presented in Table I. Approximately

14% and 30% of the homogenate protein and DNA, respectively, are

recovered in the nuclear fraction. Based on the recovery of DNA it

appears that 30% of the total starting nuclei are isolated in the

"purified nuclear fraction" (Figure 2). My work suggests that <70%

of the starting nuclei are lost during the repeated centrifugations

(Figure 2) which I find to be necessary for preparing clean, intact

nuclei.

As can be seen in Table I the NE fraction contains 8% of the

starting nuclear protein and 50% of the starting nuclear phospholipid

while only 0.3% of the nuclear DNA is recovered in this fraction. If

one assumes that all nuclear phospholipid resides in the nuclear

membranes (Franke, 1974), one can estimate that approximately 50% of

the starting NE are isolated in the "purified NE fraction" (Figure 2).

Characterization and Purity of Nuclear and NE Fractions

In order to further confirm the relative purity of the isolated

nuclear and NE fractions, as well as to determine the composition of











Table I

Recovery of Protein, DNA, and Phospholipid in Subcellular Fractions

% of Total % of Total % of Total % of Total % of Total
Fraction Cellular DNA Cellular Protein Nuclear DNA Nuclear Protein Nuclear Phospholipid


Homogenate 100 100 -


Nuclei 30 14 100 100 100


NE 0.1 1 0.3 8 50



Recoveries were determined by assay of individual fractions sampled during the isolation
procedure.








the nuclei and NE's, I have examined the enzymatic activities and

chemical content of both fractions.

Enzyme Activity

The activity of 5'-nucleotidase was examined in homogenate,

nuclei, and NE fractions in order to evaluate possible plasma membrane

contamination of the purified NE fraction. Table II clearly shows

that little plasma membrane (as assayed by 5'-nucleotidase) is found

associated with either the nuclei or NE. Similarly, assays of

succinate dehydrogenase activity (as a marker for mitochondrial con-

tamination) indicated that neither the nuclear nor the NE fraction

has significant quantities of this enzyme associated with them

(Table II). Cytochrome c oxidase assays of homogenate, nuclei, and

NE indicated that this enzyme was a very minor component of the NE

fraction (Table II). In comparison to values obtained for purified

mitochondria, the NE fraction contains extremely low levels of this

enzyme which could reflect either minor mitochondrial contamination

(Jarasch and Franke, 1974) or that cytochrome c oxidase is a normal

component of the NE (Berezney et al., 1972).
Glucose-6-phosphatase activity, frequently reported to be a

marker of the ER (Kasper, 1974), has been cytochemically identified

in the NE of liver and purified in some NE fractions isolated from

this tissue (see Introduction). Assays of the glucose-6-phosphatase

activities of homogenate, nuclei, and NE fractions from CHO cells

(Table II) indicate that a 2 fold purification of this activity is

obtained in nuclei and NE relative to the homogenate. The lack of

further purification of this enzyme in the NE fraction relative to









Table II

Enzyme Activities of Subcellular Fractions


Homogenate Nuclei NE Mitochondria *

5'-Nucleotidase
(nmoles P04 = hydrolyzed/ 49 (6) ND (2) ND (2)
mg protein/hr)

Succinate Dehydrogenase
(nmoles succinate oxidized/ 470 (2) ND (2) ND (2)
mg protein/hr)

Cytochrome c Oxidase
(nmoles cytochrome c oxidized/ 0.7 (2) ND (2) 1.5 (2) 18.0 (2)
mg protein/min)

Glucose-6-Phosphatase
moless PO4 = hydrolyzed/ 0.41 (2) 0.75 (2) 0.80 (2)
mg protein/hr)


Each fraction was assayed as described in Materials and Methods, immediately following
isolation of NE fractions. ND -- no detectable activity. The minimum detectable activity in succinate
dehydrogenase assays was 50 nmoles succinate oxidized/mg protein/hr and the minimum detectable activity
in 5' nucleotidase assays was 3 nmoles/mg protein/hr. The minimum detectable activity in cytochrome c
oxidase assay was 0.3 nmoles cytochrome c oxidized/mg protein/min. Numbers in parentheses are the number
of assays performed. $ Digitonin treated mitochondria, isolated from bovine liver, were obtained from
Dr. T. W. O'Brien.







the nuclear fraction may be due to loss or denaturation of the

enzyme by the high salt conditions employed during the latter steps

of our isolation procedure (Kasper, 1974).

Chemical Composition

A preliminary analysis of the relative chemical composition

of the homogenate, nuclei and NE fractions are presented in Table III.

Among the points presented in Table III which should be stressed is

the fact that the "purified NE fraction" does contain small amounts

of residual DNA and RNA. Similar findings with regard to nucleic

acid composition of isolated NE have been reported in previously

published characterizations of NE from liver (Kasper, 1974) and may,

in fact, represent physiologically significant components of the NE.

It should also be noted that, not unexpectedly, if the nuclease

digestions (Figure 2) were omitted from the isolation procedure, sig-

nificantly larger amounts of DNA and RNA were recovered in the

purified NE fraction.

To confirm the membranous nature of the isolated NE fraction,

phospholipid content was determined in the homogenate, nuclei, and

NE fraction contained 200pg phospholipid/mg protein indicating that

the fraction isolated is relatively protein rich (see pg. 31 of

Fleischer and Kervina, 1974) perhaps due to the proteinaceous lamina

associated with the inner nuclear membrane. In order to demonstrate

that this relatively low phospholipid/protein ratio was not due to

incomplete extraction of phospholipid, NE's were prepared from cells

which had been maintained for 5 generations in medium containing

O.lpCi/ml [3H] choline. Ninety-seven percent of the [3H] choline











Table III

Chemical Content of Subcellular Fractions


ug DNA/mg


pg RNA/mg


pg Phospholipid/mg


pg Cholesterol/mg


Fraction Protein Protein Protein protein


Homogenate 90 220 91 ---


Nuclei 200 90 30 ---


NE 10 20 200 20


PM --- --- --- 117




Individual fractions were sampled during the isolation procedure and assayed for protein,
DNA, RNA, phospholipid or cholesterol as described in Materials and Methods. A plasma membrane
enriched fraction was prepared from CHO cells via the two-phase aqueous polymer technique of
Brunette and Till (1971).








present in the NE fraction was extracted by chloroform methanol and

accounted for in subsequent phosphorus determinations (data not

shown).

The cholesterol content of NE and plasma membrane were compared

in order to determine if there were any clear differences in choles-

terol composition between these two fractions taken from CHO cells.

As can be seen in Table III, five-fold less cholesterol (per mg pro-

tein) was found in the NE as compared to the PM. These data are in

agreement with previously published work concerning the cholesterol

content of bovine liver NE (Keenan et al., 1970).

Peptide and Glycopeptide Composition

When the purified NE fraction was examined by SDS gel electro-

phoresis (Laemmli, 1970), a complex Coomasie blue staining profile

was obtained (Figure 6) which was distinguishable from the stained

profiles obtained from whole cell homogenate, purified nuclei and

plasma membrane derived from CHO cells (Figure 6).

The major Coomasie brilliant blue staining components of the

NE fraction ranged in molecular weight from 55,000 to 75,000 daltons.

The majority of the remaining NE peptides and glycopeptides were of

a MW higher than 75,000 daltons. It should be noted (compare nuclear

with NE fraction, Figure 6) that few, if any, peptides are found in

the NE fraction which co-migrate with the histones (arrows) found

in the nuclear fraction again arguing against significant contam-

ination of the NE with DNA.
























i I











1 2 3 4 5 6




Figure 6. Coomasie Blue Stained Profiles of Subcellular
Fractions

Fractions were isolated and electrophoresed as
described in the Materials and Methods section.
Seventy-five micrograms of protein were applied
to each slot. From left to right: lane 1,
standards (phosphoryase A, 100,000 MW; bovine
serum albumin, 69,000 MW; ovalbumin, 43,000 MW;
DNase I, 31,500 MW; soybean trypsin inhibitor,
23,000 MW; and cytochrome c, 13,500 MW); lane 2,
DNase I and RNase A; lane 3, NE; lane 4, nuclei;
lane 5, plasma membrane and lane 6, whole cell
homogenate. Arrows ( ) indicate the region in
which histones migrate in these gels.














CHAPTER III
REFORMATION OF THE NUCLEAR ENVELOPE DURING MITOSIS

Materials and Methods

Materials

All tissue culture materials were purchased from Grand Island

Biological Company (Grand Island, New York). Tissue culture plastic-

ware was purchased from Corning Glass Works (Corning, New York).

[4,5-3H] L-leucine, [methyl-3H] thymidine, and ultrapure sucrose

were purchased from Schwarz Mann, Division of Becton, Dickinson and

Company (Orangeburg, New York). [Methyl-3H] choline chloride and

[32P] orthophosphate were purchased from Amersham Searle Corporation
(Arlington Heights, Illinois). All reagents for electrophoresis

were purchased from Bio-Rad Laboratories (Rockville Centre, New

York). All other reagents were obtained from Scientific Products

(Ocala, Florida).

Cell Culture and Synchrony

Chinese hamster ovary cells were grown in suspension culture

as described in Chapter II. Cells were synchronized via a modifica-

tion of the isoleucine deprivation technique first introduced by

Tobey and Crissman (1972). In our procedure suspension cultures

(usually 6 liters) were allowed to grow to stationary phase

(6-8 x 105 cells/ml) in complete media. The cells were left for 12

to 24 hr at stationary phase and then harvested and resuspended in








medium containing 10% calf serum, 5% fetal calf serum and ImM

hydroxyurea (HU). Ten hours after the addition of the medium

supplemented with HU (when the majority of the cells were arrested

at the G1/S boundary), the cultures were re-harvested and resuspended

in fresh, complete medium. Immediately upon resuspension in fresh

medium the cells began to traverse the cell cycle. Attempts to

synchronize other CHO subclones using this technique were not

successful.

In order to make comparisons between experiments, I have plotted

synchronization curves as fraction of cells divided (N-No/No) versus

time. No is the number of cells before division and N is the number

of cells at any given time during division.

Labeling of Cell Cultures

Labeling of cell cultures in preparation for isotope dilution

experiments was performed by maintaining cells for 5 generations

prior to synchronization in a particular radioactive precursor (at

the specific activity indicated) and then synchronizing the cultures

in the presence of either O.2pCi/ml [3H] leucine, O.lpCi/ml [3H]

choline, or 0.41Ci/ml [32P] orthophosphate. In order to determine

the dilution of radiolabel which occurred during the mitotic phase

of the cell cycle, the labeled cell cultures were harvested

immediately prior to entrance into M (i.e. in very late G2) and

washed twice in PBS, pH 7.2. One aliquot of cells was used to

prepare G2 NE while the remaining cells were returned to culture in

fresh media containing no radioactive precursor. After the cells had

completed M, this aliquot was used to prepare the G1 NE's.








Pulse label experiments with [3H] leucine were performed by

growing and synchronizing cultures in the absence of radiolabel.

Following release of the cells from HU, the cultures were resuspended

in media containing 3.3pg/liter leucine (25% of the leucine normally

present in F-10 medium). One hour before each sequential NE iso-

lation, an aliquot of the culture was removed from the synchronized
stock culture and [3H] leucine was added to a final concentration

of 0.2pCi/ml [3H] leucine.

Pulse labeling of cultures with [ 32P orthophosphate was per-
formed as in [ 3H] leucine pulses except that media added after

release of the cells from the HU block was complete F-10 with no
dilution of any component. [32P] Orthophosphate was added to a final

concentration of O.8pCi/ml.

Isolation of Nuclei and NE

Preparation of nuclei and NE was described in detail in
Chapter II.

Determination of Specific Activities

The specific activity of [3H] leucine labeled cell components
was determined by liquid scintillation counting (LSC) and protein

assay according to Lowry et al. (1951) after either solubilization

of the cell component in 10% sodium dodecyl sulfate (SDS), 10mM

Tris (pH 8.5), and 1% B-mercaptoethanol (BME) and dialysis for 36 hr
against 2% SDS, 62.5mM Tris (pH 6.8), 10% glycerol, 0.1% 6ME and
0.1mM phenylmethylsulfonyl fluoride (PMSF) or following precipitation

of the isolated component with 10% trichloroacetic acid (TCA). The
specific activity of [32p] or [3H] choline labeled phospholipid was








determined from phospholipid phosphorus assays (Rouser et al., 1966)

of chloroform-methanol extracts (Rouser and Fleischer, 1967) of the

specific cell component.

Determination of Precursor Pool Equilibration Rates

Cell cultures were grown for 5 generations in either 0.2pCi/ml

[3H] leucine or 0.4pCi/ml [P32] orthophosphate. The cells were then

synchronized in the presence of 0.2pCi/ml [ H] leucine or 0.4jCi/ml

[32P] orthophosphate. Five to six hours after release from HU, an

aliquot of cells was taken from the culture and the initial precursor

pool specific activity was determined as described below. Immediately

after removal of this initial aliquot, the remaining cells were

washed twice with PBS, sampled again, and returned to unlabeled

medium. Each hour for the next 4 hr after being returned to unlabeled

medium, a sample of cells was removed, and the precursor pool specific

activity was determined.

To determine the size of the [3H]1eucine pool, cells were

collected on a glass fiber filter, washed quickly with 10 volumes of

ice cold PBS, and solubilized in 0.2N NaOH for 5 minutes at 700C.

The solubilized material was chilled, precipitated with 10% trichloro-

acetic (TCA) overnight at 4C and the supernatant collected by cen-

trifugation. [3H] Leucine in the supernatant (i.e. the acid soluble

pool) was determined by LSC and total cell protein was assayed

according to Lowry et al. (1951).

Phospholipid precursor pool size was based on labeled phospha-

tidic acid. [32P] Phosphatidic acid specific activity was determined

by harvesting cells via centrifugation and immediately extracting








the harvested cells with chloroform-methanol (Rouser and Fleischer,

1967). After the Folch back washes of the chloroform-methanol

extract, phosphatidic acid was found in the lower organic phase.

Phosphatidic acid was purified by two sequential separations on

silica gel thin layer chromatography using solvent system I developed

by Skipski and Barclay (1969) for acidic phospholipids. The purified

phosphatidic acid was assayed for phosphorous (Rouser et al., 1966)

and the [32P] content determined by LSC.

Measurement of Nuclear Surface Areas

Nuclear surface areas were measured by point and intersection

planimetry on photographs of thick sections of embedded whole cells.

Samples of cells were taken from a synchronized culture 5h and 8h

after release from HU. Samples were fixed in 2% glutaraldehyde, 2%

formaldehyde and 50mM PO4 = (pH 7.4) for 30 minutes at 220C. Post-

fixation was performed in 1% OsO4, 100mM PO4 = (pH 7.4) for 30

minutes at 220C. Samples were then dehydrated and embedded as

described in Chapter II. Thick sections were cut on a Sorvall micro-

tome and stained with 0.1% toludine blue 0 in 1% sodium borate for

15 seconds at approximately 2000C. Sections were photographed using

a Wild MII microscope with camera attachment. The resulting photo-

graphs were subsequently subjected to planimetry according to

Weibel (1969).

Polyacrylamide Gel Electrophoresis

All electrophoresis was performed as described in Chapter II.

Fluorography of [3H] labeled NE gels was performed according to

Bonner and Laskey (1974).








Results

Cell Synchrony

The relative synchrony obtained by the technique outlined in

the Materials and Methods section is presented in Figure 7. [3H]

Thymidine incorporation into DNA indicated that following release

from HU, DNA synthesis was initiated immediately and that the syn-

thetic phase (S pahse of the cell cycle) lasted approximately 5 hr.

A 5 hr S phase is in good agreement with the value previously obtained

by Tobey and Crissman (1972) using these same cells. Concurrent with

cessation of DNA synthesis (i.e. 5 hr after release from HU),

mitotic cells, defined by metaphase figures appeared (Figure 7). The

mitotic phase of the cell cycle was complete in approximately 4-5 hr

(i.e. occurred 5-10 hr after release from HU). During this time

frame the cell population increased by approximately 80% (Figure 7).

It must be pointed out that from Figure 7 it is apparent that the G2

phase of the CHO cell cycle synchronized by the technique outlined

in Materials and Methods is very short. The fact that mitotic

figures begin appearing as soon as DNA synthesis is terminated

(Figure 7) does not allow accurate measurement of the G2 phase of the

cell cycle. It should also be noted that the maximum mitotic index

at any time after release from HU is 25-30% of the total cell popula-

tion. Thus, during the 4-5 hr mitotic phase of these synchronized

cells a proportion of the cells are either in the preceding G2 or

have progressed into the G1 phase and are therefore coexisting with

cells in mitosis. Since I well recognize that I do not have 100% of

the cells in mitosis at any one time, I refer to the 4-5 hr time

period during which the cell population divides as the G2-M-G1



















S/ -5

S.


o 0


o z



TIME AFTER ADDITION
OF HU(HRS)









Figure 7. Cell Cycle Parameters in Synchronized CHO Cells

A suspension culture was synchronized as
described in Materials and Methods. DNA syn-
thesis was determined by pulsing duplicate 2 ml
aliquots of cells for 10 min at 370C with
2.6 pCi/ml [3H] thymidine. Labeled cells were
then washed three times with ice cold PBS, pre-
cipitated and washed three times with ice cold
10% TCA, and radioactivity in the precipitate
was determined by LSC. Cell number was deter-
mined from 4 replicate counts using a Levy-
Hausser counting chamber. Mitotic index is
expressed as percent of cells in mitosis
observed in phase microscopy. [3H] Thymidine
incorporation into TCA insoluble material 0 0;
cell density A-A; mitotic index ID-D.








transition. It is during this transitional period of the cell cycle

that the NE breaks down and reforms.

Peptide Composition of the NE during Different Phases of the Cell
Cycle

Maul et al. (1972) have reported that changes in the number of

nuclear pores per unit nuclear surface area occur as HeLa cells pro-

gress through the cell cycle. Riley and Keller (1978) have reported

major morphological changes and minor compositional changes in non-

membranous nuclear ghosts isolated from HeLa cells at various stages

of the cell cycle. Hodge et al. (1977) have also examined polypep-

tides of the HeLa cell nuclear matrix isolated from synchronized

populations of cells at various stages of the cell cycle. These

authors reported, in agreement with Riley and Keller, that minor

changes in the composition of nuclear matrix could be noted when

nuclear matrixes, isolated from cells in various phases of the cell

cycle, were compared by electrophoresis. Sieber-Blum and Burger

(1977), using CHO cells, have compared NE, isolated from synchronized

cell cultures, by electrophoresis and found no noticeable differences

in the stained gel profiles of cell cycle specific NE fractions.

In an effort to determine whether any changes occur in CHO NE

during the cell cycle which could be detected as changes in the

coomasie blue staining profile of NE peptides and glycopeptides

separated on SDS-PAGE, I have isolated NE from cells at various stages

of the cell cycle. Specifically NE were isolated from a) cells which

were in the logarithmic phase of growth (i.e. primarily G1 cells);

b) cells which had been grown to stationary phase, diluted, allowed

to reinitiate the cell cycle in fresh HU containing media and then








collected 10 hr after dilution (i.e. cells at G1/S boundary, Figure 7);

c) cells 5 hr after release from HU (i.e. cells at the G2/M boundary);

and d) cells 10 hr after release from HU (i.e. cells in early G1).

The NE's from these four distinct cell populations were then solu-

bilized in SDS as described in Materials and Methods and equal

amounts of protein applied to individual wells of 7.5 12.5% Laemmli

discontinuous SDS-PAGE. As can be seen in Figure 8, coomasie blue

staining of the various isolates showed some differences in the

overall coomasie blue stained profile of NE's isolated from the

different stages of the cell cycle. However the cell cycle related

differences shown in Figure 8 were not consistently seen from exper-

iment to experiment suggesting that these differences are of

questionable physiologic significance. It is my own bias, based on

observations drawn from a number of experiments identical to that

outlined in Figure 8, that there are no reproducible differences in

the coomasie blue stained profile of NE derived from the four phases

of the cell cycle discussed in this section of the dissertation.

Dilution of [3H] Leucine Labeled NE

Using synchronized and uniformly labeled CHO cells I have

examined the dilution of labeled NE in order to follow synthesis of

NE peptides during the G2-M-G1 transition. Such label dilution

experiments were directed at determining whether peptides present in

NE, isolated from early G1 cells (10-12 hr after release from HU),

were synthesized de novo during the G2-M-G1 transition or whether

the peptides present in the early G1 NE pre-existed in the cell

prior to mitosis.















IS

















-













Figure 8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells

Nuclear envelopes were isolated from cells in
logarithmic growth and from cells at various
stages of the cell cycle. Nuclear envelope
fractions were solubilized in SDS and electro-
phoresis was performed as described in
Materials and Methods. Lane 1, log phase;
Lane 2, G2/M; Lane 3, M/G1; Lane 4, Gl/S.







Specifically CHO cells were grown for 5 generations in 0.2pCi/ml

[3H] leucine and then synchronized in the presence of 0.2pCi/ml [3H]
leucine as described in Materials and Methods. In late G2 (5 hr

after HU removal, Figure 7) the cell culture was harvested, washed

twice with PBS and then the 6 liters of cells divided into two equal

aliquots. From one aliquot NE were immediately isolated (described

in Table IV as the G2/M NE population). The remaining cells were

returned to fresh medium free of [ 3H] leucine but containing 13.2 mg/

liter cold leucine. Four hours later (after 80% of the cells had

completed M, Figure 7) these cells were harvested and NE immediately

isolated (described as the M/G1 NE population in Table IV). The

specific activity of these two NE fractions was then determined and

compared. As can be seen in Table IV, the average specific activity

of the M/G1 NE is approximately 25% less than the specific activity

of the NE's isolated from the G2/M cell population. These data

suggest that although some de novo synthesis of NE protein has

occurred over the 4 hr time span between the isolation of the G2/M

and M/G1 NE, the majority of the peptides present in the M/G1 NE's

pre-existed in the G2/M cells. The data presented in Table IV do

not, of course, take the precursor pool into account. Resolution

of this problem will be discussed below.

The reduction in specific activity broadly described in

Table IV was further investigated by isolating NE at a time point

(6.5 hr after release from HU) at which less than 5% of the cells

have completed M and every hour thereafter until greater than 80%

of the cells have completed M (Figure 9). Cells were grown and











Table IV

Dilution of [3H] Leucine Labeled NE Protein

Specific Activities of NE Protein (cpm/mg protein)

G2/M M/G1 Ratio (M/G1:G2/M)

5.3 x 105 3.9 x 105 0.74

9.3 x 105 6.7 x 105 0.72

6.3 x 105 4.2 x 105 0.67

2.7 x 105 2.1 x 105 0.78

1.4 x 105 1.1 x 105 0.79

x= 0.74

SD = + 0.05


Cells were grown and synchronized in media containing 0.2pCi/ml [3H] leucine and NE
isolated from half of the cell population immediately before synchronous division (G2/M NE).
Four hours later (when '80% of the cells had completed M) NE were isolated from the other
half of the cell population which had proceeded through M in the absence of [3H] leucine (M/G1 NE).
Specific activities were determined as described in Materials and Methods.








synchronized in the presence of [ H] leucine as described for the

experiments presented in Table IV. Six and one-half hours after

release from HU (Figure 9), cells were harvested and washed twice

in PBS. One aliquot of cells was used immediately for NE isolation

(G2/M NE), while the remaining cells were returned to culture in

complete medium lacking [3H] leucine. Each hour thereafter, for the

next 6 hr, an aliquot of cells was harvested, NE isolated and the

specific activity of NE protein was determined. The specific

activity of the NE's at each time point is displayed in Figure 9.

Clearly, NE's which are in the late G2 phase of the cycle (8.5 hr

after release from HU) have lost 10% of the label associated with

the envelopes suggesting that during very late G2 labeled components

of the NE are rapidly diluted with unlabeled components. As the

NE's enter M there appears to be a sharp decrease in the rate of

dilution and then as NE's enter G1 a resumption of label dilution

occurs (Figure 9). Dilution of labeled NE peptides is greatly

reduced during that period of time in which the cell population is

dividing when compared to the dilution observed in NE's isolated from

cells in the G2 and G, phases of the cell cycle.

In order to determine whether a particular peptide or group of

NE peptides were preferentially lost during the G2-M-G1 transition,

[3H] leucine-labeled NE, isolated during the experiments described in

Table IV were solubilized in SDS, labeled peptides and glycopeptides

separated on a discontinuous SDS-PAGE, and the profile examined by

fluorography. Figure 10 contains both the coomasie blue stained

profile of the SDS-PAGE and the fluorogram obtained from the labeled






















Figure 9. Dilution of [3H] Leucine Labeled NE Protein

A suspension culture was grown to stationary
phase in the presence of O.2pCi/ml [3H] leucine
and synchronized as described in Materials and
Methods in O.2pCi/ml [3H] leucine. Six and one-
half hours after release from HU NE were prepared
from an aliquot of cells. The remaining cells
were washed twice with PBS and returned to
culture in complete medium lacking [3H] leucine.
Nuclear envelopes were isolated each hour after
removal of [3H] leucine and specific activities
of each fraction were determined as described
in Materials and Methods. Fraction of cells divided
was determined from 4 replicate counting in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0-0; fraction divided D-0.





50
















4 1.0

-.9

-.8

-.7
S3-

a_ -.5 z





o2- 2
2..3


0 3
ax -




S1 ,00


6 7 8 9 10 11 12 13
TIME AFTER RELEASE
FROM HU (HRS)






















Figure 10. SDS Gel Electrophoresis of NE Peptides Isolated
After Removal of [3H] Leucine at the G2/M
Boundary

A cell culture was grown for 5 generations in
0.2pCi/ml [3H] leucine and synchronized in
0.2pCi/ml [3H] leucine as described in Materials
and Methods. Nuclear envelopes were isolated
before label removal (G2/M interface, 7 hr after
removal of HU) and after division in the absence
of label (M/G1 interface, 11 hr after removal of
HU). The isolated NE were solubilized in SDS,
electrophoresed and fluorographed as described
in Materials and Methods. (A) Fluroograph of
NE peptides isolated at G2/M (lane 1) and M/G1
(lane 2) interfaces. Equal numbers of counts
(31,000 cpm) were applied to the gel. (B)
Coomasie blue stained profiles corresponding
to the fluorogram in A. The G2/M (lane 1)
fraction contained 75pg of protein while the
M/G1 (lane 2) fraction contained 50pg protein.



















I~ K


S..


CM


V-


S- om


1~1








components. From the fluorogram presented in Figure 10 it is apparent

that, within the detection limit of the fluorographic process and

one dimensional gel electrophoresis, no individual NE peptide or

glycopeptide is preferentially lost during the G2-M-G1 transition.

Incorporation of the [3H] Leucine into NE Protein

One likely, if not the most likely, explanation for the reduced

specific activity found in M/G1 NE's relative to G2/M NE's (Table IV)

is dilution of the pre-existing (i.e. G2/M) label with unlabeled NE

components synthesized and inserted into the NE during either G2, M,

or G1 phase of the cell cycle after removal of [3H] leucine from the

culture medium. The data presented in Figure 9 suggests that some

dilution of label occurs in G2 immediately after removal of [3H]

leucine from the culture medium as well as in G1. In order to

determine whether this dilution results from phase-specific biosyn-

thesis of NE peptides, cells were synchronized as described above and

then 5.25 hr after release from HU an aliquot of cells was taken and

pulsed for 1 hr in medium containing O.21Ci/ml [3H] leucine but only

25% of the standard F-10 leucine concentration. After 1 hr in labeled

precursor an aliquot was taken from the homogenate and the NE was

isolated. Cells were pulsed with O.2pCi/ml [3H] leucine and NE iso-

lated every hour from 5.25 11.5 hr after release of cells from an

HU blockade (Figure 11).

The specific activity of each NE and homogenate fraction was

determined and the specific activities plotted as a function of time

after release from HU block. As can be seen, the specific activity

of the NE fractions remain relatively constant (t 5%) from 6 9 hr




















Incorporation of [3H] Leucine into NE Protein


A suspension
phase and syn
and Methods.
blockade into


culture was grown to stationary
chronized as described in Materials
The culture was released from HU
Media containing 25% of the normal


F-10 leucine concentration. One hour before
synchronous division (i.e. 5.25 hr after removal
from HU), an aliguot of cells was removed, pulsed
with 0.2pCi/ml [ H] leucine for one hour and
harvested for NE isolation. Each hour thereafter,
an additional aliquot of cells was pulsed for 1 hr
with 0.2pCi/ml [3H] leucine, NE isolated, and the
specific activity determined on each NE and
homogenate fraction. Fraction of cells divided
was determined from 4 replicate counting in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0 0; homogenate specific
activity A-A ; fraction divided[-[].


Figure 11.































-.9


-.8


.7


-.6


-.5 z
0
-.4 i


-.3 L


-.2


TIME AFTER RELEASE
FROM HU (HRS.)








after release from HU (by which time approximately 40% of the cells

in the population have divided). However between 9 and 10 hr after

release from HU when the majority of the cell population is in the

early portion of the G1 phase of the cell cycle (Figure 11), incor-

poration of [ H] leucine into the NE's increased 1.25 times that

seen in M and by 11 hr after release, incorporation of [3H] leucine

into the NE is 1.56 that seen in M. Taken together, the data in

Figure 9 and Figure 11 suggest that the majority of NE synthesis (and

consequently dilution of pre-existing label) occurs in late G2 and

early to mid Gl, with NE specific synthesis in M being low relative

to either of these time periods.

Determination of acid soluble [3H] leucine counts (cpm/mg protein)

in the whole cell homogenate at each time point clearly demonstrated

that the increase in specific activity of NE and homogenate protein

seen in Gl was not due to increased transport of labeled precursor

but rather was due to enhanced de novo biosynthesis.

To determine whether specific peptides of the NE might be

preferentially synthesized during the G2-M-G1 segment of the cell cycle

NE fractions, obtained from the time points taken in Figure 11, were

solubilized in SDS and the peptide and glycopeptide composition dis-

played by coomasie blue staining and fluorography on a 7.5 12.5%

discontinuous SDS-PAGE. The fluorogram presented in Figure 12A

indicates that, at the limits of detection by one dimensional SDS gel

electrophoresis and fluorography, all NE peptides are synthesized at

some point in the G2-M-G1 transition. Comparison of the coomasie

blue stained profile (Figure 12B) with the intensities of the individ-

ual bands in the exposed fluorogram (Figure 12A) suggests that the





















Figure 12. SDS Gel Electrophoresis of [3H] Leucine Pulse Labeled
NE Peptides

Nuclear envelopes isolated during the experiment
described in Figure 11 were solubilized and subjected
to electrophoresis and fluorography as described in
Materials and Methods. (A) Fluorogram depicting
[ H] leucine labeled NE isolated at the six time
points indicated in Figure 11. Lane 1, 5.75 hr
after HU removal; Lane 2, 6.75 hr after HU removal;
Lane 3, 7.75 hr after HU removal; Lane 4, 8.75 hr
after HU removal; Lane 5, 9.75 after HU removal;
Lane 6, 11 hr after HU removal. Equal numbers of
counts (11,000 cpm) were applied to each lane.
(B) Coomasie blue stained profile corresponding
to the fluorogram in (A). Lane 1, 72pg protein;
Lane 2, 74pg protein; Lane 3, 76pg protein; Lane 4,
71ig protein; Lane 5, 56ug protein; Lane 6, 42pg
protein.










I -II- (








I




S- (0


C


c
L








NE peptides were labeled approximately in proportion to their relative

staining with coomasie blue.

Dilution of [3H] Choline Labeled NE

Using [3HI choline as a precursor of phosphatidyl choline, I

have examined the relative reutilization of NE phospholipids during

the G2-M-G1 transition. As in the work with [ H] leucine labeled NE,

I have initially followed the dilution of label at two time points

within the cell cycle, the so-called G2/M stage and the M/G1 stage of

the cell cycle (Table V).

Chinese hamster ovary cell cultures were grown for 5 generations

in medium supplemented with O.lpCi/ml [3H] choline and then syn-

chronized in complete medium containing O.liCi/ml [3H] choline. Five

hours after release from HU (i.e. at the G2/M transition) the labeled

culture was harvested, washed twice with PBS, and divided into equal

aliquots. G2/M NE were prepared from one aliquot while the other

aliquot was returned to culture in complete F-10 medium without

[3H] choline but containing 0.7 mg/liter cold choline. Following
division (i.e. 9 hr after release from HU, see Figure 7), M/G1 NE

were prepared and chloroform-methanol extracts (Rouser and Fleischer,

1967) were prepared from both preparations. The specific radio-

activity of the G2/M NE and the M/G1 NE phospholipid (cpm/pg lipid

phosphorus) was determined and the specific activities compared

(Table V).

The simplest interpretation of the data in Table V would suggest

that approximately 65% of the M/G1 NE phospholipid was present in the

cell prior to mitosis while 35% of the M/G1 phospholipid was










Table V

Dilution of Labeled NE Phospholipids


NE Phospholipid Specific Activity (cpm/pg lipid phosphorus)

Radiolabel G2/M M/G1 Ratio (M/G1:G2/M)

[3H] choline 2.0 x 104 1.4 x 104 0.70

[32P] orthophosphatet 0.8 x 104 0.5 x 104 0.63


x= 0.67


Cells were grown and synchronized in media containing O.l~Ci/ml [3H] choline or 0.4pCi/ml [32p]
orthophosphate and NE isolated from half of the cell population immediately before synchronous
division (G2/M NE). Four hours later (whenm80% of the cells had completed M) NE were isolated
from the other half of the cell population which had proceeded through M in the absence of
label (M/G1 NE). Specific activities were determined as described in Materials and Methods.
$ [32p] data were calculated from experiment shown in Figure 13.








synthesized during the G2-M-G1 transition. Reutilization of label

is not considered in these data but will be discussed below.

Dilution of [32P] Orthophosphate Labeled NE Phospholipid

Since choline labels a specific phospholipid which could con-

ceivably behave differently than the majority of the NE phospholipid,

I felt it important to determine the dilution of total NE phospho-

lipid. To do this I chose to label cells to constant specific

activity with [32P] orthophosphate. In these experiments CHO cell

cultures were maintained and synchronized in complete F-10 medium

supplemented with 0.4pCi/ml [32P] orthophosphate. As in the previous

radiolabel dilution experiments (Tables IV and V, Figure 9), the

culture, labeled to constant specific activity with [32 P, was

harvested approximately 6.5 hr after release from HU (at the G2/M

transition) and washed twice with PBS. One aliquot of the washed cells

was immediately used for preparation of NE while the remaining cells

were returned to complete medium lacking [32P] and allowed to proceed

through M. Each hour after the cells were returned to unlabeled media,

a percentage of the cells was harvested, homogenate prepared, and NE

isolated. After the last isolation was completed (11.5 hr after

release from HU, Figure 13), phospholipid was extracted into chloro-

form methanol, phosphorous anlaysis was performed on each fraction,

and the NE phospholipid specific activity (cpm/ug lipid phosphorus)

determined. Figure 13 describes the time course of the change in

phospholipid specific activity in cell homogenate and the NE fraction.

Clearly the specific activity of both the NE and homogenate remains

relatively constant while the majority of the cells are in late G2























Figure 13. Dilution of [32P] Labeled NE Phospholipid

A suspension culture was grown to stationary phase
in the presence of O.4iCi/ml [32p] orthophosphate
and synchronized as described in Materials and
Methods in 0.4uCi/ml [32p] orthophosphate. Six
and one-half hours after release from HU NE were
prepared from an aliquot of cells. The remain-
ing cells were washed twice with PBS and returned
to culture in complete medium lacking [32p].
Nuclear envelope and homogenate were isolated each
hour after removal of [32P] and phospholipid
specific activities were determined on each frac-
tion as described in Materials and Methods.
Fraction of cells divided was determined from 4
replicate counting in a Levy-Hausser counting
chamber. Nuclear envelope specific activity
0-0; homogenate specific activity A-A; fraction
divided -D .





63




















1.0

-.9




I -.7

I0 /
0

0 t
--.

.4

oA _.30
x-
.2u
0 5
N .1


6 7 8 9 10 11 12
TIME AFTER RELEASE
FROM HU(HRS.)








and M and then begins to drop as the cells enter G1. After 80% of

the cells have completed division (Figure 13, 11.5 hr after release

from HU), the specific activity of the NE is approximately 65% of

the NE phospholipid specific activity before M (Figure 13 and Table V).

The simplest explanation for these data is that 65% of the phospho-

lipid present in NE 12 hr after release from HU (i.e. early G1, see

Figure 13) was present in the cell prior to M.

Interestingly the specific activity of the NE phospholipid

begins to decrease after 20-30% of the cells have entered the G1 phase

of the cell cycle whereas the specific activity of the whole cell

phospholipid drops only after %80% of the cells have completed M

(i.e. the majority of the cells are in early to mid G1). The data

in Figure 13 clearly suggest that synthesis of phospholipid to be

incorporated into NE and other cellular phospholipid increases as the

cells enter G1.
32
Incorporation of [ 32] Orthophosphate into NE Phospholipid

In order to confirm that the dilution of NE phospholipid specific

activity, seen in Table V and Figure 13, is due to synthesis of new

phospholipid, which is, in turn incorporated into NE, cell cultures

were grown to stationary phase and then synchronized as described

previously in complete medium containing no label. Seven hours after

release from HU (i.e. at the G2/M transition, Figure 14), an aliquot

of cells was removed from the stock culture and pulsed for 1 hr with

0.8pCi/ml [32P] orthophosphate. After one hour in [32 P, the labeled

culture was harvested and both NE and homogenate prepared. Similarly

treated aliquots of cells were pulsed for 1 hr with [32P] over the








next 5 hr. The specific activity of phospholipid was then deter-

mined by chloroform-methanol extraction and phosphorus analysis on

each homogenate and NE fraction. The time course of [32P] incorpora-

tion into NE and homogenate phospholipid can be seen in Figure 14.

As would have been predicted from Figure 13, after 30-40% of the

cells have completed division, the homogenate and NE phospholipid

specific activity begins to rise. This increase in specific activity

of the two fractions remains linear over the remainder of the time

course studied. These data (Figure 14) clearly suggest that a major

fraction of the phospholipids which are destined to become part of the

NE are synthesized and incorporated into the organelle during early G1.

Rate of Precursor Pool Equilibration

To more rigorously interpret the label dilution data described

throughout the previous experiments (Tables IV and V and Figures 9

and 13), I examined the kinetics of precursor pool equilibration in

synchronized cultures which were manipulated in a fashion identical

to that already reported in Tables IV and V and Figures 9 and 13.

Such data is, of course, essential if I am to make more precise

estimates of the amount of NE peptide and phospholipid synthesis (and

subsequently dilution of pre-existing label) which occurs during the

G2-M-G1 transition in our synchronized cultures.

In the experiments presented in Figure 15, CHO cells were

grown for 5 generations in medium supplemented with 0.2pCi/ml [3H]

leucine (i.e. until the peptides in the cell had reached a constant

specific activity) and then synchronized as described in Materials

and Methods in the presence of 0.2pCi/ml [3H] leucine. Five hours























Figure 14. Incorporation of [32P] into NE Phospholipid

A suspension culture was grown to stationary
phase and synchronized as described in Materials
and Methods. Seven hours after removal of HU an
aliquot of cells was removed, pulsed with O.8pCi/ml
[32p] orthophosphate for one hour and NE isolated.
Each hour thereafter (for 6 hr) another aliquot of
cells was pulsed with 0.8uCi/ml [32p] for 1 hr and
NE isolated. Phospholipid specific activity was
determined on NE and homogenate isolated after
each pulse. Fraction of cells divided was deter-
mined from 4 replicate counting in a Levy-Hausser
counting chamber. Nuclear envelope specific
activity 0-0; homogenate specific activity A-A ;
fraction divided I-0.I


















































TIME AFTER RELEASE
FROM HU(HRS.)








after release from HU an aliquot of cells was removed from the culture,

collected on a glass fiber filter, quickly washed with 10 volumes of

ice cold PBS and solubilized in 0.2 N NaOH. The remaining cells were

harvested, washed twice with PBS (as was done in all my previously

presented dilution data). Immediately after the PBS washes (6 hr

after HU removal) another aliquot of cells was collected on a glass

fiber filter, washed and solubilized. The remaining cells were

returned to complete media without [ 3H] leucine. Each hour after

returning the cells to unlabeled media an aliquot of cells was

collected, washed, and placed in 0.2 N NaOH. After all the samples

had been collected (10 hr after removal from HU, 90% of the cells

having divided, Figure 15), the filters were heated at 700C for 5 min,

cooled on ice, and precipitated overnight in 10% TCA (40C). The

specific activity of the TCA soluble [ 3H] leucine counts was then

determined and plotted as a function of time (Figure 15). As can

be seen in Figure 15, approximately 55% of the [3H] leucine, which

was in the cytoplasmic precursor pool before the cells were harvested,

was removed during the initial two PBS washes. Subsequent culturing

of the washed cells in [3H] leucine-free media reduced the soluble

[3H] leucine pool to a specific activity with was 30% of the initial
specific activity of the initial time point (Figure 13).

By first setting the initial, pre-PBS wash, soluble [3H] leucine

pool at 100% (initial time point, Figure 15) and then averaging the

fractional value of this initial pool which was measured during the

G2-M-G1 transition in the absence of label, I have calculated a mean

soluble [3H] leucine pool specific activity during the G2-M-G This
























Figure 15. [3H] Leucine Pool Dilution

A suspension culture was grown for 5 generations
in O.2uCi/ml [3H] leucine and synchronized in
O.2pCi/ml [3H] leucine as described in Materials
and Methods. Five and one-half hours after HU
removal a sample was taken and acid soluble [3H]
leucine determined as described in Materials and
Methods. The remaining cells were washed 2 times
with PBS and another sample taken for detemina-
tion of TCA soluble [3H] leucine counts. The
washed cells were then returned to unlabeled
media and samples taken each hour for determination
of TCA soluble [3H] leucine counts. Fraction of
cells divided was determined from 4 replicate
counting in a Levy-Hausser counting chamber.
Acid soluble [3H] cpm/mg total protein 0-0;
fraction dividedD-D .





70

















r 1.0

-.9

-.8

3H-LEUCINE -.7
removed
I .6

8.-
3. .52
x7-

u z
15- 0

33- i,,


1- .
.,. ....... 0
5 6 8 10
TIME AFTER RELEASE
FROM HU (HRS)








calculation results in a mean value equal to 36% (average of two

experiments) of the [3H] leucine found in the soluble pool of cells

prior to removal of label. This mean value represents the percentage

of [3H] leucine in the soluble pool of cells dividing in the absence

of label relative to those cells (before PBS washes) whose cytoplasm

had reached equilibrium with [3H] leucine in the medium. Since the

leucine pool size has been shown to remain constant in HeLa cells

during this phase of the cell cycle (Robbins and Scharff, 1966), I

have concluded that the protein synthesized following removal of

[3H] leucine from the medium, is synthesized with a specific activity
that is, on the average, 36% of the specific activity of proteins

synthesized when [3H] leucine was maintained in the culture.

Using the formula below (which is derived in the appendix), I

have estimated the amount of early G1 NE protein synthesized during

the G2-M-G1 transition. I have based these calculations on the ratio

of specific activities of G2/M NE to M/G1 NE presented in Table I.

SAG1 SAG2
SA' SAG2

where: p = proportion of G1 NE synthesized during G2-M-G1,

SAG1 = specific activity of M/G1-NE

SAG2 = specific activity of G2/M-NE
SA' = relative specific activity of pool after label removal

Setting SAG2 equal to unity gives SAG1 a value of 0.74 (Table IV).

Substituting these values into the equation we have:


S(0.74) 1.0 0.41
p = (0.36) 1.0 41







Thus when one takes into account the fact that the soluble pool

is not completely depleted of [ H] leucine after removal of [3H]

leucine from the media, one calculates that%40% of the M/G -NE

protein was synthesized during the G2-M-G1 transition. By simple sub-

traction then 160% of the M/G1 NE protein must have pre-existed M.

I have used the same experimental design and logic to examine

the dilution of the [32P] labeled phosphatidic acid pool in order to

more precisely estimate NE phospholipid biosynthesis during the

G2-M-G1 transition. Phosphatidic acid was chosen since it is a

precursor of phospholipids and is not shunted into other pathways

(Howard and Howard, 1974; Spector, 1972).

In these experiments (Figure 16) CHO cells were grown for 5

generations in 0.4 pCi/ml [32P] and then synchronized as described

in Materials and Methods in 0.4 pCi/ml [32P] orthophosphate. Six

hours after release from HU (Figure 16) an aliquot of cells was

harvested and immediately extracted with chloroform-methanol to give

me a base value for phosphatidic acid specific activity. The

remaining cells were also harvested, washed twice in PBS and another

aliquot of cell population extracted with chloroform-methanol

(Figure 16, 6.5 hr after HU removal). The remaining cells were then

returned to media containing 60 mg/liter cold phosphate but free of

[32P]. As shown in Figure 16, aliquots were harvested from the cell
population each hour through the G2-M-G1 transition and extracted

with chloroform-methanol. After all samples had been extracted, each

was Folch backwashed (Rouser and Fleischer, 1967), filtered, and

concentrated for application to thin layer chromatography plates.

























Figure 16. [32P] Phosphatidic Acid Pool Dilution

A suspension culture was grown for 5 generations
in O.4pCi/ml [32p] and synchronized in 0.4iCi/ml
[32p] as described in Materials and Methods.
Five hours after HU removal an aliquot of cells
was harvested by centrifugation and phosphatidic
acid specific activity was determined as
described in Materials and Methods. The remain-
ing cells were washed twice with PBS, another
aliquot removed for phosphatidic acid specific
activity determination and the bulk of the
cells returned to unlabeled media. Aliquots
were removed for phosphatidic acid specific
activity determination each hour thereafter
until 90% of the cells had completed division.
Fraction of cells divided was determined from
4 replicate counting in a Levy-Hausser counting
chamber. Phosphatidic acid (PA) specific
activity 0-0; fraction divided 0-D.































In
0

15-


'0



x 5
0.
a
U
('I


TIME AFTER RELEASE
FROM HU (HRS.)








Thin layer chromatography was run in solvent system I of Skipski and

Barclay (1969) for acidic phospholipids. After development, the

phosphatidic acid spot was visualized by spraying with distilled

water, scraped from the plate, eluted from the silica gel with

chloroform-methanol, and rechromatographed in the same developing

solvents. The phosphatidic acid spot was visualized in an iodine

chamber, scraped, and eluted as before. The specific activities of

phosphatidic acid at each time were determined on the eluted material

as described in Materials and Methods.

The rate of decrease in the specific activity of [32P] labeled

phosphatidic acid is presented in Figure 16. The arithmetic mean

intracellular specific activity during the G2-M-G1 transition was

calculated to be 38% (average of two experiments) of that found in

cells prior to removal of [32P] from the media. Calculating from

the observed dilution of [32P] labeled NE phospholipid (Table V) I

find the proportion of M/G1-NE phospholipid synthesized during the

G2-M-G1 transition to be 0.53.

0.67 1.0
P = 0.38 1.0 0.53

Again by subtraction, I estimate that 50% of the M/G1-NE pre-existed

M.

Changes in Nuclear Surface Area

Since a change in total nuclear surface area might reflect

synthesis of NE and at the same time affect the interpretation of

my data, I have determined mean nuclear surface areas in a population

of cells before and after the G2-M-G1 transition (i.e. at 5 hr and








8 hr in Figure 7). Cells were synchronized as described in Materials

and Methods and 5 hr after release from HU, an aliquot of cells was

removed, and prepared for sectioning as described in Materials and

Methods. After 75% of the cells had completed division (8 hr after

HU removal) another aliquot of cells was removed and prepared for

sectioning. Thick sections were taken from both cell samples,

stained, and photographed as described in Materials and Methods.

Measurements of the surface area of individual nuclei were made by

planimetry (Weibel, 1969). The nuclei in both cell populations were

found to have a mean nuclear surface area of"lO02. If we assume

that the mean nuclear surface area after the cells have completed

mitosis represents the mean surface area of "daughter" nuclei only,

then the magnitude of the G1 (8 hr after release from HU) nuclear

surface area relative to G2 nuclear surface area will reflect what

increase, if any, has occurred during the G2-M-G1 transition. If no

increase in NE occurred during the G2-M-G1 transition, this would be

reflected by a mean nuclear surface area in G1 which is exactly one-

half of the G2 mean nuclear surface area. My measurements suggest

that the total nuclear surface area has doubled during the G2-M-G1

transition (G1 and G2 mean nuclear surface areas are approximately

equal). If such a doubling in nuclear surface area was due entirely

to NE synthesis (i.e. no stretching or expansion of available NE),

I would expect to see a 50% dilution of NE protein during the G2-M-G1

transition. My label dilution experiments have indicated that the Gl

NE peptides have a specific activity which is 60% of the G2 NE while

the G1 NE phospholipids have a specific activity which is 50% that

of the G2 NE. In my opinion both my labeling data and my surface

area data are, within the limits of resolution, complementary.













CHAPTER IV
DISCUSSION

In Chapter II of this dissertation I have described a procedure

for the isolation of highly enriched nuclear and NE fractions from

CHO cells. The nuclei and NE fractions obtained by use of this

procedure have been characterized by light and electron microscopy;

chemical and enzymatic assay; as well as SDS gel electrophoresis.

These characterizations have demonstrated that the nuclei and NE

fractions isolated are distinct from each other and that both frac-

tions are distinct from the plasma membrane and whole cell homogen-

ate.

Although electron microscopy and marker enzyme assays indicate

that contamination of the nuclei and NE fraction with other organ-

elles is very low, the true enrichment of NE in the final pellet as

compared to the homogenate is impossible to determine since no

dependable enzymatic marker has been unequivocally localized to this

organelle in CHO cells. However, it must be again stressed that

the final NE fraction obtained by the procedures outlined in this

dissertation is morphologically similar to the intact NE found in

the cell in that the isolated NE's contain a double membrane bilayer

as well as the pore-lamina complex, thus strongly suggesting that

the isolated procedure described produces a highly enriched NE

fraction.








The NE fraction isolated from CHO cells is composed primarily

of protein and phospholipid having 200pg phospholipid/mg protein.

This phospholipid/protein ratio suggests that, in comparison to other

isolated cellular membranes (Fleischer and Kervina, 1974), the isolated

NE fraction is relatively protein rich. This finding may derive

from the fact that the isolation procedure described, isolates both

the nuclear membrane and the so-called proteinaceous lamina. The

inclusion of the proteinaceous lamina in the NE fraction would be

expected to increase the protein to lipid ratio.

Examination of the peptide composition of the NE fraction by

SDS gel electrophoresis demonstrates that the major coomasie blue

staining peptides of the NE fraction chromatograph with molecular

weights between 55,000 and 75,000 daltons. These peptides are

undoubtedly related to the pore-lamina and nuclear matrix proteins

identified in rat liver (Dwyer and Blobel, 1976) and HeLa cell

(Hodge et al., 1977) NE's. It is of interest to note that the

majority of the remaining NE peptides chromatograph with a molecular

weight greater than 75,000 daltons. Relatively few coomasie blue

staining bands are found to run at molecular weights below 50,000

daltons and no peptides appear to co-migrate with the histones seen

in the nuclear fraction.

It can not be emphasized too much that although the isolation

technique described required careful handling of material during the

preparation of the nuclei and NE, it does produce material in

sufficient quantity and of sufficient purity to permit biochemical

studies directed at elucidating the molecular basis of both NE

structure and biosynthesis.








In Chapter III of this dissertation the NE isolation procedure

(described in Chapter II) has been employed to examine the question

of whether or not cellular proteins and phospholipids which pre-existed

the mitotic phase of the CHO cell cycle are used by the CHO cell in

the reformation of the NE which occurs during telophase.

Sodium dodecyl sulfate gel electrophoresis of NE isolated at

various stages of the CHO cell cycle showed no consistent differences

in peptide or glycopeptide composition which could be assigned to

any phase of the cell cycle. It is of interest to note that I did not

see any cell cycle dependent changes in the peptides migrating between

55,000 and 75,000 MW, as have been reported by Hodge et al. (1977) to

occur in the HeLa cell nuclear matrix. In this regard, the data in

this dissertation agree with those of Sieber-Blum and Burger (1977)

who also did not see cycle specific changes in CHO cell NE's.

In Chapter III of this dissertation,evidence, obtained primarily

from what I have dubbed "label dilution" studies, has been presented

which strongly suggest that at least 60% of the early G1 NE protein

and at least 50% of the early G1 NE phospholipid existed in the cell

prior to mitotic breakdown and reformation (i.e. in late G2). These

data further suggest that the remaining 40% of early G1-NE protein

and the remaining 50% of early G1-NE phospholipid come from de novo

synthesis of NE components. Pulse label experiments suggest that

relatively little NE protein or phospholipid synthesis occurs in M

but rather that the majority of this NE synthesis occurs in late G2

and early G1. Unfortunately the degree of synchrony obtainable with

such a large number of cells does not allow me to determine whether








some NE components are specifically synthesized in the G2, M, or G1

phases of the cell cycle. However, my data do indicate that at some

point in the G2-M-G1 transition all of the NE peptides separable on

a one-dimensional SDS-PAGE are labeled with [3H] leucine suggesting

that no specific components) is carried through the transition while

another components) is degraded and totally resynthesized during

the G2-M-G1 transition.

Measurement of nuclear surface area in synchronized populations

of cells in G2 and G1 indicated that there was no significant differ-

ence in the average surface area of the two nuclear populations.

These data suggest that the total nuclear surface has increased

%2 fold between G2 and early G1. If one assumes that increases in

surface area are directly correlated to NE biosynthesis, these data

predict that "label dilution" experiments would detect an%50%

dilution of NE protein during the time period examined. This is in

good agreement with our calculated results of 40% dilution and 60%

reutilization of pre-existing peptides.

The 60% of the early G1-NE protein which the data suggest

pre-existed M, when taken together with the data suggesting a burst

of early G1 NE synthesis does strongly imply that a majority of

G1 NE proteins come from pre-existing cellular components. Thus,

in my opinion, these data unequivocably rule out "complete" de novo

synthesis of NE protein as being responsible for mitotic reassembly.

Unfortunately I cannot state unequivocally that the early G1 NE

protein which pre-existed M resided solely in the G2 NE. The

possibility clearly exists that G1 NE components are made prior to








M and then stored in some cellular compartment for use in restruc-

turing the NE after division. The most likely organelle for such a

"storage function" would be the ER. Testing such a possibility must

await the availability of NE specific probes which can be applied

to the cells at various phases of the cell cycle to determine whether

"NE specific" components exist in other cellular organelles prior

to or during M.

The same problems and interpretation just discussed with regard

to NE peptides apply to those experiments which indicate that 50% of

the early G1 NE phospholipid pre-existed M. This apparently higher

rate of NE phospholipid biosynthesis relative to NE protein bio-

synthesis over the same G2-M-G1 transition may reflect a high turn-

over has been reported in other tissue culture cells (Cunningham,

1972).

I feel that my data can be interpreted to rule out complete

de novo synthesis of the NE during mitotic breakdown and reformation.

I also feel that the majority of the very early G1 NE components

pre-exist mitosis. A question which should now be asked is what is

the exact fate of NE peptides during mitosis. This can be ascer-

tained only by applying probes (e.g. antibodies) specific for NE

peptides to small populations of mitotic cells. Such probes should

allow identification of the precise location of NE peptides during

M and thus determine the "storage" and reutilization of such com-

ponents.













APPENDIX

DERIVATION OF THE EQUATION FOR CALCULATING THE PROPORTION
OF M/G1 NE SYNTHESIZED DE NOVO DURING THE G2-M-G] TRANSITION


p = proportion of M/G1 NE synthesized during the G2-M-G1
transition
SAG1 = specific activity of M/G1 NE

SAG2 = specific activity of G2/M NE
SA' = mean intracellular leucine pool specific activity during

the G2-M-G1 transition assuming an initial pool specific
activity of 1.0

The NE specific activity at M/G1 (SAG1) is equal to some pro-
portion (p) which was synthesized at a diluted pool specific activity

(SA') plus some proportion (l-p) synthesized before label removal at
the G2/M NE specific activity (SAG2). This can be expressed in the
following equation:
SAG1 = p(SA') + (l-p) SAG2 (1)
Rearranging equation 1 gives:
SAG1 = p(SA') + SAG2 p(SAG2) (2)

SAG1 SAG2 = p(SA') p(SAG2) (3)
SAG1 SAG2 = p(SA' SAG2) (4)

SAG1- SAG2
SA' SAG2 p (5)














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BIOGRAPHICAL SKETCH

Gregory E. Conner was born on December 21, 1950 in Jacksonville,

Florida, where he received his primary and secondary education in

parochial schools. In June of 1972 he received a Bachelor of Arts

degree from Vanderbilt University in Nashville, Tennessee. In

September of 1973 the author began graduate studies in the Department

of Biochemistry and Molecular Biology at the University of Florida.

Upon completion of these studies he will assume a post-doctoral

position at the Rockefeller University, New York, New York.

The author is married to the former Daphne Ann Wales of

Jacksonville, Florida.









I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Kennet D Nonan, Chairman
Assistant Professor of
Biochemistry and Molecular Biology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Charles M. Allen, Jr.
Associate Professor of
Biochemistry and Molecular Biology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Carl M. Feldherr
Associate Professor of
Anatomy









I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Edward M. Hoffman /
Associate Professor o
Microbiology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Thomas W. O'Brien
Associate Professor of
Biochemistry and Molecular Biology



This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

August 1978




Dean, College of Medicine




D anV-waduate School





\ILI
CG73r



























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3 1262 08554 6207




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5 6 7 8 9 10 11
TIME AFTER RELEASE
FROM HU (HRS.)


17
was assayed according to Veeger et al. (1969). Cytochrome c oxidase
was assayed according to Smith (1955).
Electron Microscopy
Nuclei and NE's were fixed (0C, 2 hr) in 1.8% glutaraldehyde,
8% sucrose, 50mM phosphate (pH 7.6), and 2.5mM MgCl^. Both the nuclei
and NE fractions were postfixed in the same buffer containing 1%
osmium tetroxide (22C, 1 hr). Samples were sequentially dehydrated
in increasing concentrations of ethanol and finally embedded in Spurr's
low viscosity medium (1969). Thin sections were stained with uranyl
acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965)
and then examined in an Hitachi AS8 electron microscope.
SDS Polyacrylamide Disc Gel Electrophoresis (PADGE)
All electrophoretic analyses were performed in 1.5mm thick slab
gels according to the procedure of Laemmli (1970). The gels used were
composed of a 5.6% acrylamide stacking gel overlaying a linear 7.5%
to 12.5% acrylamide gradient separation gel. The acrylamide ratio to
bis-acrylamide was maintained at 37.5:1 in both the stacking and
running gel. Purified NE's were solubilized by boiling at 100C for
5 min in 10% SDS, lOmM Tris (pH 8.5), 1% B-mercaptoethanol (gME).
Plasma membrane was solubilized by resuspension and boiling in 2% SDS,
62.5mM Tris (pH 6.8), 10% glycerol, and 0.1% |3ME ("sample buffer").
Lysed nuclei, sampled after DNase I and RNase A digestion, and whole
cell homogenates were solubilized by boiling in one volume of 2x
"sample buffer." Each of the samples were then exhaustively dialyzed
against "sample buffer" containing 0.1% 6-mercaptoethanol and O.lmM
phenylmethylsulfinyl fluoride (PMSF). Inclusion of PMSF in the sample


time after release
FROM HU (HRS.)
3H CPM X15/ mg PROTEIN
{j)-\ i i | n i | i | i | i i i
FRACTION DIVIDED
I 1 I
OD (D
cn
o


3
Figure 12. SDS Gel Electrophoresis of [ H] Leucine Pulse Labeled
NE Peptides
Nuclear envelopes isolated during the experiment
described in Figure 11 were solubilized and subjected
to electrophoresis and fluorography as described in
Materials and Methods. (A) Fluorogram depicting
[3H] leucine labeled NE isolated at the six time
points indicated in Figure 11. Lane 1, 5.75 hr
after HU removal; Lane 2, 6.75 hr after HU removal;
Lane 3, 7.75 hr after HU removal; Lane 4, 8.75 hr
after HU removal; Lane 5, 9.75 after HU removal;
Lane 6, 11 hr after HU removal. Equal numbers of
counts (11,000 cpm) were applied to each lane.
(B) Coomasie blue stained profile corresponding
to the fluorogram in (A). Lane 1, 72pg protein;
Lane 2, 74pg protein; Lane 3, 76yg protein; Lane 4,
71yg protein; Lane 5, 56yg protein; Lane 6, 42yg
protein.


3
Figure 15. [ H] Leucine Pool Dilution
A suspension culture was grown for 5 generations
in 0.2pCi/ml [3h] leucine and synchronized in
0.2yCi/ml [3h] leucine as described in Materials
and Methods. Five and one-half hours after HU
removal a sample was taken and acid soluble [^H]
leucine determined as described in Materials and
Methods. The remaining cells were washed 2 times
with PBS and another sample taken for determina
tion of TCA soluble [3h] leucine counts. The
washed cells were then returned to unlabeled
media and samples taken each hour for determination
of TCA soluble [3H] leucine counts. Fraction of
cells divided was determined from 4 replicate
countings in a Levy-Hausser counting chamber.
Acid soluble [^H] cpm/mg total protein 0-0;
fraction dividedO-O .


CTi3r
ISili
3 1262 08554 oZU/


CHAPTER III
REFORMATION OF THE NUCLEAR ENVELOPE DURING MITOSIS
Materials and Methods
Materials
All tissue culture materials were purchased from Grand Island
Biological Company (Grand Island, New York). Tissue culture plastic-
ware was purchased from Corning Glass Works (Corning, New York).
3 3
[4,5- H] L-leucine, [methyl- H] thymidine, and ultrapure sucrose
were purchased from Schwarz Mann, Division of Becton, Dickinson and
3
Company (Orangeburg, New York). [Methyl- H] choline chloride and
32
[ P] orthophosphate were purchased from Amersham Searle Corporation
(Arlington Heights, Illinois). All reagents for electrophoresis
were purchased from Bio-Rad Laboratories (Rockville Centre, New
York). All other reagents were obtained from Scientific Products
(Ocala, Florida).
Cell Culture and Synchrony
Chinese hamster ovary cells were grown in suspension culture
as described in Chapter II. Cells were synchronized via a modifica
tion of the isoleucine deprivation technique first introduced by
Tobey and Crissman (1972). In our procedure suspension cultures
(usually 6 liters) were allowed to grow to stationary phase
(6-8 x 10 cells/ml) in complete media. The cells were left for 12
to 24 hr at stationary phase and then harvested and resuspended in
36


P CPM x 1 In g LIPID PHOSPHORUS
TIME AFTER RELEASE
FROM HU (HRS.)


TIME AFTER RELEASE
FROM HU (HRS)
ACID SOLUBLE 3H CPM X104 / MG. TOTAL PROTEIN


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Thomas W. O'Brien
Associate Professor of
Biochemistry and Molecular Biology
This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
August 1978


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
tljn fr.
IKenneth D. Npom
ponan, Chairman
Assistant Professor of
Biochemistry and Molecular Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
<% i?u2gA.
Charles M. A1len, Jr.
Associate Professor of
Biochemistry and Molecular Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
c
Carl M. Feldherr
Associate Professor of
Anatomy


Cells
J
homogenate
Supernatant pellet
gradient 60%-65% sucrose
(DISCARD) interfaces
Remainder of
gradient
(DISCARD)
60%-65% sucrose
interface
Dounce homogenize in hypotonic buffer
at 2-4 x 10' cells/ml. Dilute with
2 volumes 65% sucrose, 50mM Tris
(pH 7.6), 7.5 mM MgCl2.
Spin 4936 x gm 30 min
max
Resuspend in 65% sucrose TM and place
at the bottom of a SW27 gradient.
Spin 72,100 x gmax, 1 hr
Remove nuclei from interfaces.
Dilute to 50% sucrose TM, and place
in a SW27 gradient.
Spin 72,100 x g 1 hr
3max
Remove nuclei from 60%-65% sucrose inter
face, dilute with distilled water to 10%
sucrose


Figure 16. [^P] Phosphatidic Acid Pool Dilution
A suspension culture was grown for 5 generations
in 0.4pCi/ml [32p] and synchronized in 0.4pCi/ml
[32P] as described in Materials and Methods.
Five hours after HU removal an aliquot of cells
was harvested by centrifugation and phosphatidic
acid specific activity was determined as
described in Materials and Methods. The remain
ing cells were washed twice with PBS, another
aliquot removed for phosphatidic acid specific
activity determination and the bulk of the
cells returned to unlabeled media. Aliquots
were removed for phosphatidic acid specific
activity determination each hour thereafter
until 90% of the cells had completed division.
Fraction of cells divided was determined from
4 replicate countings in a Levy-Hausser counting
chamber. Phosphatidic acid (PA) specific
activity 0-0; fraction divided .


26
Isolation of NE
The NE isolation procedure described in the Materials and Methods
section is a combination and modification of the procedures published
by Kay et al. (1972) and Monneron et al. (1972) for the isolation of
NE from rat liver nuclei.
As outlined in Figure 2, the NE isolation procedure begins with
the "purified nuclear fraction." In the isolation method used the
purified nuclei are lysed in lOmM Tris (pH 8.5) containing O.lmM MgC^.
Incubation of the nuclei in this hypotonic buffer for 20 min at 0C
produces a very viscous solution which is subsequently incubated with
DNase I and RNase A for 20 min at 22C. Addition of the respective
nucleases immediately reduces the viscosity of the lysed nuclei.
Following nuclease digestion, the material is diluted with 4 volumes
of 60% sucrose, 500mM MgCl2 (to remove residual chromatin [Monneron
et al., 1972]) in 50mM Tris (pH 7.6) and the mixture vortexed.
During the ensuing centrifugation (SW41 rotor at 153,244 x 9max>
2 hr), NE float up into the gradient leaving nucleic acid, soluble
proteins of the nucleoplasm, and unlysed nuclei in the dense load
zone. Under the centrifugation conditions used, the NE fraction bands
in the gradient at a density of approximately 1.19 g/cc. The NE are
subsequently removed from the gradient and pelleted (Figure 2).
Morphology of the Purified NE Fraction
Electron micrographs of sections through the purified NE frac
tion (Figure 5) demonstrate that most, if not all, of the isolated
nuclear membranes retain the double membrane sheets (single arrow)
characteristic of the NE. Furthermore, as would be expected if one


Table V
Dilution of Labeled NE Phospholipids
NE Phospholipid Specific Activity (cpm/yg lipid phosphorus)
Radiolabel
[%] choline
[32pj orthophosphatet
G2/M
2.0 x lCf
0.8 x 104
M/G
1.4 x 10^
0.5 x 104
Ratio (M/G-j :G2/M)
0.70
0.63
x = 0.67
Cells were grown and synchronized in media containing 0.1yCi/ml [^H] choline or 0.4pCi/ml [ P]
orthophosphate and NE isolated from half of the cell population immediately before synchronous
division (G2/M NE). Four hours later (when^805i of the cells had completed M) NE were isolated
from the other half of the cell population which had proceeded through M in the absence of
label (M/G-| NE). Specific activities were determined as described in Materials and Methods,
t [32p] data were calculated from experiment shown in Figure 13.
CD
O


Labeling of Cell Cultures 37
Isolation of Nuclei and NE 38
Determination of Specific Activities 38
Determination of Precursor Pool
Equilibration Rates 39
Measurement of Nuclear Surface Areas 40
Polyacrylamide Gel Electrophoresis 40
Results 41
Cell Synchrony 41
Peptide Composition of the NE during
Different Phases of the Cell Cycle 43
Dilution of [3H] Leucine Labeled NE 44
Incorporation of the [3H] Leucine
into NE Protein 53
Dilution of [3h] Choline Labeled NE 59
Dilution of [32p] Orthophosphate
Labeled NE Phospholipid 61
Incorporation of [32p] Orthophosphate
into NE Phospholipid 64
Rate of Precursor Pool Equilibration 65
Changes in Nuclear Surface Area 75
CHAPTER IV-DISCUSSION 77
APPENDIXDERIVATION OF THE EQUATION FOR CALCULATING
THE PROPORTION OF M/G-i NE SYNTHESIZED DE NOVO
DURING THE G2-M-G-] TRANSITION 82
BIBLIOGRAPHY 83
BIOGRAPHICAL SKETCH 88
IV


7
After disruption of nuclei and dispersion of chromatin, differ
ential or isopynic centrifugation is then employed to separate NE from
the remaining nuclear components (Kasper, 1974).
Composition
Franke (1974) and Kasper (1974) have extensively reviewed the
chemical and enzymatic composition of NE fractions isolated from
tissue. The NE is comprised primarily of protein and phospholipid and
has generally been described as a protein rich organelle. Reported
protein values generally vary between 60% and 75% of the total membrane
mass while phospholipid values range from 20% to 30% of the total
membrane mass (see Kasper, 1974). Phosphatidyl choline is the pre
dominant lipid species of NE followed by phosphatidyl ethanolmine,
phosphatidyl serine and phosphatidyl inositol (Keenan et al., 1970,
1972; Khandwala and Kasper, 1971). Some neutral lipids such as
cholesterol and free fatty acids have also been reported in NE
fractions (Kleinig, 1970; Keenan et al., 1970, 1972).
Almost all isolated NE fractions contain small quantities of
DNA and RNA which vary in amount depending on the preparative pro
cedure. Those procedures which do not strip the outer membrane of
ribosomes normally result in RNA values which are higher than those
obtained after NE isolation procedures which employ citrate or
MgClg- Small residual amounts of RNA associated with isolated NE
may reflect a functional component of the pores (Monneron and Bernhard,
1969; Dhainaut, 1970). The extent of DNase I treatment and the con
centration of salt used in NE isolation procedures appear to affect
the quantity of DNA associated with the NE fraction.


44
collected 10 hr after dilution (i.e. cells at G-j/S boundary, Figure 7);
c) cells 5 hr after release from HU (i.e. cells at the G^/M boundary);
and d) cells 10 hr after release from HU (i.e. cells in early G-j).
The NE's from these four distinct cell populations were then solu
bilized in SDS as described in Materials and Methods and equal
amounts of protein applied to individual wells of 7.5 12.5% Laemmli
discontinuous SDS-PAGE. As can be seen in Figure 8, coomasie blue
staining of the various isolates showed some differences in the
overall coomasie blue stained profile of NE's isolated from the
different stages of the cell cycle. However the cell cycle related
differences shown in Figure 8 were not consistently seen from exper
iment to experiment suggesting that these differences are of
questionable physiologic significance. It is my own bias, based on
observations drawn from a number of experiments identical to that
outlined in Figure 8, that there are no reproducible differences in
the coomasie blue stained profile of NE derived from the four phases
of the cell cycle discussed in this section of the dissertation.
3
Dilution of f Hi Leucine Labeled NE
Using synchronized and uniformly labeled CHO cells I have
examined the dilution of labeled NE in order to follow synthesis of
NE peptides during the G^-M-G^ transition. Such label dilution
experiments were directed at determining whether peptides present in
NE, isolated from early G^ cells (10-12 hr after release from HU),
were synthesized de novo during the G2-M-G1 transition or whether
the peptides present in the early G-j NE pre-existed in the cell
prior to mitosis.


42
Figure 7. Cell Cycle Parameters in Synchronized CHO Cells
A suspension culture was synchronized as
described in Materials and Methods. DNA syn
thesis was determined by pulsing duplicate 2 ml
aliquots of cells for 10 min at 37C with
2.6 yCi/ml [3I1] thymidine. Labeled cells were
then washed three times with ice cold PBS, pre
cipitated and washed three times with ice cold
10% TCA, and radioactivity in the precipitate
was determined by LSC. Cell number was deter
mined from 4 replicate counts using a Levy-
Hausser counting chamber. Mitotic index is
expressed as percent of cells in mitosis
observed in phase microscopy. [^H] Thymidine
incorporation into TCA insoluble material 0-0
cell density A-A ; mitotic index -.


21
(Aaronson and Blobel, 1974). For this reason I avoided the use of any
detergents in my nuclear isolation procedure. Instead, I developed
homogenization conditions which minimized nuclear clumping and adhesion
of cytoplasmic material to the nuclei. Specifically I found that
immediate dilution of the cell homogenate with 65% (w/w) sucrose, 50mM
Tris (pH 7.6), 7.5mM MgCl2 (Figure 2) maintained the nuclei in a
dispersed state. Isolation of "clean" nuclei from such an homogenate
could then be readily accomplished by density gradient centrifugation
if the nuclei were maintained under the salt and pH conditions outlined
in Figure 2 and handled with care so as to avoid premature lysis.
In the procedure which I developed (Figure 2) the whole cell
homogenate is diluted with 65% sucrose in 50mM Tris (pH 7.6), 7.5mM
MgCl,, and spun for 30 min at 4,936 x g This differential centri-
fugation serves to collect nuclei in a pellet while leaving most other
membranous organelles in the supernatant. This crude nuclear pellet
is subsequently subjected to two sequential centrifugations on
sucrose step gradients (Figure 2) in which purified nuclei band at
the 60-65% sucrose interface. The purified nuclei are subsequently
diluted with distilled water (to give a final sucrose concentration
of 10%) and then collected as a pellet by a 5 min, low speed, differ
ential centrifugation step (Figure 2).
Three precautions must be taken to ensure the isolation of a
"clean" nuclear fraction from CHO cells, (a) Homogenization must
be performed at pH 7.6 in the absence of divalent cations, (b)
Homogenization, resuspension, and mixing of fractions must be done
very carefully so as to avoid premature lysis or shearing of the


65
next 5 hr. The specific activity of phospholipid was then deter
mined by chloroform-methanol extraction and phosphorus analysis on
32
each homogenate and NE fraction. The time course of [ P] incorpora
tion into NE and homogenate phospholipid can be seen in Figure 14.
As would have been predicted from Figure 13, after 30-40% of the
cells have completed division, the homogenate and NE phospholipid
specific activity begins to rise. This increase in specific activity
of the two fractions remains linear over the remainder of the time
course studied. These data (Figure 14) clearly suggest that a major
fraction of the phospholipids which are destined to become part of the
NE are synthesized and incorporated into the organelle during early .
Rate of Precursor Pool Equilibration
To more rigorously interpret the label dilution data described
throughout the previous experiments (Tables IV and V and Figures 9
and 13), I examined the kinetics of precursor pool equilibration in
synchronized cultures which were manipulated in a fashion identical
to that already reported in Tables IV and V and Figures 9 and 13.
Such data is, of course, essential if I am to make more precise
estimates of the amount of NE peptide and phospholipid synthesis (and
subsequently dilution of pre-existing label) which occurs during the
G^-M-Gi transition in our synchronized cultures.
In the experiments presented in Figure 15, CHO cells were
grown for 5 generations in medium supplemented with 0.2pCi/ml [ H]
leucine (i.e. until the peptides in the cell had reached a constant
specific activity) and then synchronized as described in Materials
and Methods in the presence of 0.2pCi/ml [ H] leucine. Five hours


BIOGRAPHICAL SKETCH
Gregory E. Conner was born on December 21, 1950 in Jacksonville,
Florida, where he received his primary and secondary education in
parochial schools. In June of 1972 he received a Bachelor of Arts
degree from Vanderbilt University in Nashville, Tennessee. In
September of 1973 the author began graduate studies in the Department
of Biochemistry and Molecular Biology at the University of Florida.
Upon completion of these studies he will assume a post-doctoral
position at the Rockefeller University, New York, New York.
The author is married to the former Daphne Ann Wales of
Jacksonville, Florida.
88


76
8 hr in Figure 7). Cells were synchronized as described in Materials
and Methods and 5 hr after release from HU, an aliquot of cells was
removed, and prepared for sectioning as described in Materials and
Methods. After 75% of the cells had completed division (8 hr after
HU removal) another aliquot of cells was removed and prepared for
sectioning. Thick sections were taken from both cell samples,
stained, and photographed as described in Materials and Methods.
Measurements of the surface area of individual nuclei were made by
planimetry (Weibel, 1969). The nuclei in both cell populations were
2
found to have a mean nuclear surface area of 'vlOOp If we assume
that the mean nuclear surface area after the cells have completed
mitosis represents the mean surface area of "daughter" nuclei only,
then the magnitude of the G-j (8 hr after release from HU) nuclear
surface area relative to G,, nuclear surface area will reflect what
increase, if any, has occurred during the G^-M-G. transition. If no
increase in NE occurred during the G^-M-G^ transition, this would be
reflected by a mean nuclear surface area in G^ which is exactly one-
half of the G^ mean nuclear surface area. My measurements suggest
that the total nuclear surface area has doubled during the G2M-G^
transition (G^ and G., mean nuclear surface areas are approximately
equal). If such a doubling in nuclear surface area was due entirely
to NE synthesis (i.e. no stretching or expansion of available NE),
I would expect to see a 50% dilution of NE protein during the G^-M-G^
transition. My label dilution experiments have indicated that the G-j
NE peptides have a specific activity which is 60% of the G^ NE while
the G.| NE phospholipids have a specific activity which is 50% that
of the G,, NE. In my opinion both my labeling data and my surface
area data are, within the limits of resolution, complementary.


81
M and then stored in some cellular compartment for use in restruc
turing the NE after division. The most likely organelle for such a
"storage function" would be the ER. Testing such a possibility must
await the availability of NE specific probes which can be applied
to the cells at various phases of the cell cycle to determine whether
"NE specific" components exist in other cellular organelles prior
to or during M.
The same problems and interpretation just discussed with regard
to NE peptides apply to those experiments which indicate that 50% of
the early G-j NE phospholipid pre-existed M. This apparently higher
rate of NE phospholipid biosynthesis relative to NE protein bio
synthesis over the same G^-M-G^ transition may reflect a high turn
over has been reported in other tissue culture cells (Cunningham,
1972).
I feel that my data can be interpreted to rule out complete
de novo synthesis of the NE during mitotic breakdown and reformation.
I also feel that the majority of the very early G1 NE components
pre-exist mitosis. A question which should now be asked is what is
the exact fate of NE peptides during mitosis. This can be ascer
tained only by applying probes (e.g. antibodies) specific for NE
peptides to small populations of mitotic cells. Such probes should
allow identification of the precise location of NE peptides during
M and thus determine the "storage" and reutilization of such com
ponents.


27
Figure 5.
Electron Micrograph of the
Purified NE Fraction
The NE fraction was fixed, embedded, and stained
as described in the Materials and Methods
section (x31,850). Regions of double membranes
Cf) can be seen as well as pores in tranverse
(-j*]') and tangential section (f'f'p.


CHAPTER I
INTRODUCTION
The nuclear envelope (NE) is a complex subcellular organelle
which exists at the nuclear periphery and separates the genetic
machinery of the eucaryotic cell from other cellular components. During
mitosis in many plant and animal cells, the NE becomes morphologically
indistinct and reappears after daughter chromosome separation is com
plete. The studies which will be described in this dissertation are
directed towards understanding the mechanism(s) by which this large
membranous organelle disassembles and restructures during mitosis.
Before presentation of these studies, I will briefly discuss the cur
rently available information concerning NE structure, composition and
breakdown and reformation during mitosis.
NE Structure
The structure of the NE has been probed in great detail by many
light and electron microscopic (EM) studies (for reviews see Feldherr,
1972; Kay and Johnston, 1973; Kessel, 1973; Franke, 1974; and Franke
and Scheer, 1974). The NE (shown diagramatically in Figure 1) is com
posed primarily of two lipid bilayer membranes which contain pore
structures. Electron microscopic observation suggests that these
bilayer membranes are continuous at the pore structures leaving apparent
gaps when viewed in transverse sections. These pore complexes do con
tain internal structure and may be involved in nuclear-cytoplasmic
exchange of molecules (Feldherr, 1972). The outer surface of the outer
1


52


ACKNOWLEDGEMENTS
I would like to thank Dr. Kenneth D. Noonan, my graduate
supervisor, for his endless support and encouragement during the
course of my graduate studies.
I would also like to thank the members of my supervisory
committee, Drs. C. M. Allen, C. M. Feldherr, E. M. Hoffman, and
T. W. O'Brien for their help and advice throughout this research
project. I am particularly grateful to Dr. Feldherr for his assis
tance in obtaining the various micrographs. I would also like to
express my thanks to Dr. Allen for his assistance with thin layer
chromatography.


30
the nuclei and NE's, I have examined the enzymatic activities and
chemical content of both fractions.
Enzyme Activity
The activity of 5'-nucleotidase was examined in homogenate,
nuclei, and NE fractions in order to evaluate possible plasma membrane
contamination of the purified NE fraction. Table II clearly shows
that little plasma membrane (as assayed by 5'-nucleotidase) is found
associated with either the nuclei or NE. Similarly, assays of
succinate dehydrogenase activity (as a marker for mitochondrial con
tamination) indicated that neither the nuclear nor the NE fraction
has significant quantities of this enzyme associated with them
(Table II). Cytochrome c oxidase assays of homogenate, nuclei, and
NE indicated that this enzyme was a very minor component of the NE
fraction (Table II). In comparison to values obtained for purified
mitochondria, the NE fraction contains extremely low levels of this
enzyme which could reflect either minor mitochondrial contamination
(Jarasch and Franke, 1974) or that cytochrome c oxidase is a normal
component of the NE (Berezney et al., 1972).
Glucose-6-phosphatase activity, frequently reported to be a
marker of the ER (Kasper, 1974), has been cytochemically identified
in the NE of liver and purified in some NE fractions isolated from
this tissue (see Introduction). Assays of the glucose-6-phosphatase
activities of homogenate, nuclei, and NE fractions from CHO cells
(Table II) indicate that a 2 fold purification of this activity is
obtained in nuclei and NE relative to the homogenate. The lack of
further purification of this enzyme in the NE fraction relative to


61
synthesized during the G^-M^ transition. Reutilization of label
is not considered in these data but will be discussed below.
32
Dilution of [ P] Orthophosphate Labeled NE Phospholipid
Since choline labels a specific phospholipid which could con
ceivably behave differently than the majority of the NE phospholipid,
I felt it important to determine the dilution of total NE phospho
lipid. To do this I chose to label cells to constant specific
32
activity with [ P] orthophosphate. In these experiments CHO cell
cultures were maintained and synchronized in complete F-10 medium
32
supplemented with 0.4pCi/ml [ P] orthophosphate. As in the previous
radiolabel dilution experiments (Tables IV and V, Figure 9), the
culture, labeled to constant specific activity with [ P], was
harvested approximately 6.5 hr after release from HU (at the
transition) and washed twice with PBS. One aliquot of the washed cells
was immediately used for preparation of NE while the remaining cells
32
were returned to complete medium lacking [ P] and allowed to proceed
through M. Each hour after the cells were returned to unlabeled media,
a percentage of the cells was harvested, homogenate prepared, and NE
isolated. After the last isolation was completed (11.5 hr after
release from HU, Figure 13), phospholipid was extracted into chloro
form methanol, phosphorous anlaysis was performed on each fraction,
and the NE phospholipid specific activity (cpm/ug lipid phosphorus)
determined. Figure 13 describes the time course of the change in
phospholipid specific activity in cell homogenate and the NE fraction.
Clearly the specific activity of both the NE and homogenate remains
relatively constant while the majority of the cells are in late G,,


37
medium containing 10% calf serum, 5% fetal calf serum and ImM
hydroxyurea (HU). Ten hours after the addition of the medium
supplemented with HU (when the majority of the cells were arrested
at the G^/S boundary), the cultures were re-harvested and resuspended
in fresh, complete medium. Immediately upon resuspension in fresh
medium the cells began to traverse the cell cycle. Attempts to
synchronize other CHO subclones using this technique were not
sucessful.
In order to make comparisons between experiments, I have plotted
synchronization curves as fraction of cells divided (N-N0/N0) versus
time. N0 is the number of cells before division and N is the number
of cells at any given time during division.
Labeling of Cell Cultures
Labeling of cell cultures in preparation for isotope dilution
experiments was performed by maintaining cells for 5 generations
prior to synchronization in a particular radioactive precursor (at
the specific activity indicated) and then synchronizing the cultures
3 3
in the presence of either 0.2pCi/ml [ H] leucine, O.lpCi/ml [ H]
32
choline, or 0.4uCi/ml [ P] orthophosphate. In order to determine
the dilution of radiolabel which occurred during the mitotic phase
of the cell cycle, the labeled cell cultures were harvested
immediately prior to entrance into M (i.e. in very late G,,) and
washed twice in PBS, pH 7.2. One aliquot of cells was used to
prepare NE while the remaining cells were returned to culture in
fresh media containing no radioactive precursor. After the cells had
completed M, this aliquot was used to prepare the G^ NE's.


Carbohydrates have been detected in isolated NE fractions by
chemical analysis. Kashnig and Kasper (1969) have reported that rat
liver NE contains 3%-4% carbohydrate which is made up of mannose,
glucose, and galactose. These results have been confirmed by
Franke (1977).
The enzyme composition of isolated NE has been reported to be
different by different workers although in most instances it appears
to closely resemble that of the microsomal membranes. Cytochemically
glucose-6-phosphatase (Orrenius and Ericsson, 1966; Rosen, 1969, 1970;
2+
Leskes et al., 1971) and Mg dependent ATPase (Klein and Afzelius,
1966; Yasuzumi and Tsubo, 1966) have been localized to the NE of a
variety of mammalian cell types. Typically these enzymes are present
and occasionally purified in isolated NE fractions (Franke, 1974).
The enzymatic resemblance of the NE and microsomes is strengthened by
the presence of NADH-cytochrome c reductase in NE. This activity is
not, however, increased by application of drugs (e.g. phenobarbital)
as is the enzymatic activity in microsomes (Kasper, 1971). NADPH-
cytochrome c reductase (another constituent of microsomal membranes)
has also been reported by many authors to be present in isolated NE of
liver and in NE of avian erythrocytes (Zentgraff et al., 1971), a cell
which is practically devoid of endoplasmic reticulum. The lack of
drug induction of NE NADH-cytochrome c reductase and the presence
of NE NADPH-cytochrome c reductase in ER depleted cells (avian erythro
cytes) strongly suggests that these enzymes also do not represent ER
contamination of the isolated NE.


25


38
3
Pulse label experiments with [ H] leucine were performed by
growing and synchronizing cultures in the absence of radiolabel.
Following release of the cells from HU, the cultures were resuspended
in media containing 3.3pg/liter leucine (25% of the leucine normally
present in F-10 medium). One hour before each sequential NE iso
lation, an aliquot of the culture was removed from the synchronized
stock culture and [3H] leucine was added to a final concentration
of 0.2pCi/ml [3H] leucine.
32
Pulse labeling of cultures with [ P] orthophosphate was per-
3
formed as in [ H] leucine pulses except that media added after
release of the cells from the HU block was complete F-10 with no
32
dilution of any component. [ P] Orthophosphate was added to a final
concentration of 0.8pCi/ml.
Isolation of Nuclei and NE
Preparation of nuclei and NE was described in detail in
Chapter II.
Determination of Specific Activities
3
The specific activity of [ H] leucine labeled cell components
was determined by liquid scintillation counting (LSC) and protein
assay according to Lowry et al. (1951) after either solubilization
of the cell component in 10% sodium dodecyl sulfate (SDS), lOmM
Tris (pH 8.5), and 1% g-mercaptoethanol ($ME) and dialysis for 36 hr
against 2% SDS, 62.5mM Tris (pH 6.8), 10% glycerol, 0.1% BME and
O.lmM phenylmethylsulfonyl fluoride (PMSF) or following precipitation
of the isolated component with 10% trichloroacetic acid (TCA). The
specific activity of [ P] or [ H] choline labeled phospholipid was


3
Figure 11. Incorporation of [ H] Leucine into NE Protein
A suspension culture was grown to stationary
phase and synchronized as described in Materials
and Methods. The culture was released from HU
blockade into media containing 25% of the normal
F-10 leucine concentration. One hour before
synchronous division (i.e. 5.25 hr after removal
from HU), an aliquot of cells was removed, pulsed
with 0.2yCi/ml [^H] leucine for one hour and
harvested for NE isolation. Each hour thereafter,
an additional aliquot of cells was pulsed for 1 hr
with 0.2yCi/ml [^H] leucine, NE isolated, and the
specific activity determined on each NE and
homogenate fraction. Fraction of cells divided
was determined from 4 replicate countings in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0-0; homogenate specific
activity A-A ; fraction dividedn-Q.


71
calculation results in a mean value equal to 36% (average of two
3
experiments) of the [ H] leucine found in the soluble pool of cells
prior to removal of label. This mean value represents the percentage
of [ H] leucine in the soluble pool of cells dividing in the absence
of label relative to those cells (before PBS washes) whose cytoplasm
had reached equilibrium with [ H] leucine in the medium. Since the
leucine pool size has been shown to remain constant in HeLa cells
during this phase of the cell cycle (Robbins and Scharff, 1966), I
have concluded that the protein synthesized following removal of
[ H] leucine from the medium, is synthesized with a specific activity
that is, on the average, 36% of the specific activity of proteins
3
synthesized when [ H] leucine was maintained in the culture.
Using the formula below (which is derived in the appendix), I
have estimated the amount of early G-j NE protein synthesized during
the G^-M-G^ transition. I have based these calculations on the ratio
of specific activities of G2/M NE to M/G^ NE presented in Table I.
P =
SAG1 SAG2
SA' SA
G2
where: p = proportion of G^ NE synthesized during G^-M-G^,
SAgi = specific activity of H/G^-NE
SAg2 = specific activity of G^/H-NE
SA' = relative specific activity of pool after label removal
Setting SAG2 equal to unity gives SAG1 a value of 0.74 (Table IV).
Substituting these values into the equation we have:
= (0-74) 1.0
p (0.36) 1.0
= 0.41


41
Results
Cell Synchrony
The relative synchrony obtained by the technique outlined in
3
the Materials and Methods section is presented in Figure 7. [ H]
Thymidine incorporation into DNA indicated that following release
from HU, DNA synthesis was initiated immediately and that the syn
thetic phase (S pahse of the cell cycle) lasted approximately 5 hr.
A 5 hr S phase is in good agreement with the value previously obtained
by Tobey and Crissman (1972) using these same cells. Concurrent with
cessation of DNA synthesis (i.e. 5 hr after release from HU),
mitotic cells, defined by metaphase figures appeared (Figure 7). The
mitotic phase of the cell cycle was complete in approximately 4-5 hr
(i.e. occurred 5-10 hr after release from HU). During this time
frame the cell population increased by approximately 80% (Figure 7).
It must be pointed out that from Figure 7 it is apparent that the G,,
phase of the CHO cell cycle synchronized by the technique outlined
in Materials and Methods is very short. The fact that mitotic
figures begin appearing as soon as DNA synthesis is terminated
(Figure 7) does not allow accurate measurement of the G^ phase of the
cell cycle. It should also be noted that the maximum mitotic index
at any time after release from HU is 25-30% of the total cell popula
tion. Thus, during the 4-5 hr mitotic phase of these synchronized
cells a proportion of the cells are either in the preceding G,, or
have progressed into the G^ phase and are therefore coexisting with
cells in mitosis. Since I well recognize that I do not have 100% of
the cells in mitosis at any one time, I refer to the 4-5 hr time
period during which the cell population divides as the G^-M-G^


3
Figure 9. Dilution of [ H] Leucine Labeled NE Protein
A suspension culture was grown to stationary
phase in the presence of 0.2pCi/ml [3h] leucine
and synchronized as described in Materials and
Methods in 0.2pCi/ml [3h] leucine. Six and one-
half hours after release from HU NE were prepared
from an aliquot of cells. The remaining cells
were washed twice with PBS and returned to
culture in complete medium lacking [^H] leucine.
Nuclear envelopes were isolated each hour after
removal of [3h] leucine and specific activities
of each fraction were determined as described
in Materials and Methods. Fraction of cells divided
was determined from 4 replicate countings in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0-0; fraction divided


85
Kashnig, D. M. and C. B. Kasper (1969) J. Biol. Chem. 244:3786.
Kasper, C. B. (1971) J. Biol. Chem. 246:577.
Kasper, C. B. (1974) In: The Cel 1 Nucleus, H. Busch, ed., Academic
Press, Inc., New York, 1:349.
Kay, R. R. and I. R. Johnston (1973) Subcell. Biochem. 2:127.
Kay, R. R., D. Fraser, and I. R. Johnston (1972) Eur. J. Biochem.
30:145.
Keenan, T. W., R. Berezney, L. K. Funk, T. L. Crane (1970) Biochem.
Biophys. Acta 203:547.
Keenan, T. W., R. Berezney and T. L. Crane (1972) Lipids 7:212.
Kessel, R. G. (1973) In: Recent Progress in Surface and Membrane
Science, J. F. Daniel 1 i, A. E. Riddiford and M. D. Rosenberg, eds.,
Academic Press, New York 6:243.
Khandwala, A. S. and C. B. Kasper (1971) J. Biol. Chem. 246:6242.
Klein, R. L. and B. A. Afzelius (1966) Nature (London) 212:609.
Kleinig, H. (1970) J. Cell Biol. 46:396.
Laemmli, U. K. (1970) Nature (London) 227:640.
Leskes, A., P. Siekevitz, and G. E. Palade (1971) J. Cell Biol.
49:264.
Lowry, D. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall (1951)
J. Biol. Chem. 193:265.
Maruta, H. and L. Goldstein (1975) J. Cell Biol. 65:631.
Matsura, T. and K. Ueda (1972) Arch. Biochem. Biophys. 150:440.
Maul, G. G. (1977) J. Cell Biol. 74:492.
Maul, G. G., H. M. Maul, J. E. Scogna, M. W. Lieberman, G. S. Stein,
B. Hsu, and T. W. Borun (1972) J. Cell Biol. 55:433.
McClure, J. A. and K. D. Noonan (1978) Submitted to Exp. Cell Res.
Mitchison, J. M. (1971) The Biology of the Cell Cycle, University
Printing, Cambridge.
Monneron, A. and W. Bernhard (1969) J. Ultrastruct. Res. 27:266.


10
exists between completion of cell division and initiation of DNA syn
thesis, phase is defined as that time segment which' exists between
completion of DNA synthesis and initiation of cell division.
A variety of techniques are available which will induce individ
ual cells of a culture to traverse the cell cycle in unison. These
synchronization techniques have been employed by cell biologists to
study the biochemical and morphological events which characterize
particular segments of this cycle and which are "hidden" by the random
nature of an untreated cell culture.
Mitosis is perhaps the most carefully studied morphological event
of the cell cycle. The sequence of events of mitosis is traditionally
divided into several phases based on early morphological descriptions.
Briefly, mitosis consists of four phases: prophase, metaphase, anaphase,
and telophase. The onset of prophase is marked by chromosome conden
sation at the inner nuclear membrane. The nuclear envelope becomes
very irregular, taking on a "wave-like" appearance in the region of the
centrioles. Centriole movement to opposite sides of the nucleus (i.e.
the poles) and chromosome separation occur very early in prophase.
The nuclear envelope begins to dissociate at the poles and microtubules
of the forming spindle apparatus enter into the nucleoplasm. The
nuclear envelope breakdown proceeds toward the equatorial plane and in
late prophase the condensed chromosomes begin to assemble in the com
pleted spindle (metaphase). At this point the NE is completely dis
rupted. After a short pause the chromosomes separate at the centromere
into daughter chromatids and the chromatids begin moving toward the
poles (anaphase). The nuclear envelope begins to reform as this


15
sucrose-TM. Material caught on the 65% sucrose-TM cushion was removed,
diluted to 10% sucrose with distilled water, and pelleted in an HB-4
rotor at 1020 x gmax for 5 min. This pellet is designated the purified
nuclear fraction.
In order to bring about nuclear lysis the purified nuclei were
resuspended in 1 ml of lOmM Tris (pH 8.5), containing O.lmM MgCl^.
After 20 min in lOmM Tris, O.lmM MgCl,,, DNase I and RNase A were each
added to a final concentration of 100ug/ml and the incubation continued
for 20 min at 22C. Following lysis (which could be monitored with
phase optics) four volumes of 60% (w/w) sucrose, 50mM Tris (pH 7.6),
500mM MgCl,, were added to the lysed nuclei. The solutions were
thoroughly mixed and the suspension placed in the bottom of a Beckman
SW41 centrifuge tube. A linear 20%-45% (w/w) sucrose gradient con
taining 50mM Tris (pH 7.6) and 500mM MgCl2 was formed on top of the
sample and the gradients spun for 2 hr at 153,244 x gmax in a Beckman
SW41 rotor. Nuclear envelopes banded at 1.19 g/cc (35% sucrose in
500mM MgCl,,). The purified NE's were diluted with 50mM Tris (pH 7.6)
500mM MgCl,, and then pelleted in a Beckman fixed angle 40 rotor for
30 min at 130,766 x gn]ax- The NE's were subsequently resuspended in
10% sodium dodecyl sulfate (SDS), lOmM Tris (pH 8.5), 1% B-Mercapto-
ethanol if they were to be analyzed by electrophoresis, or in 10%
sucrose, lOmM Tris (pH 7.6), 0.5mM MgCl2 if chemical or enzymatic
analysis was to be performed on the sample.
Isolation of Plasma Membrane
Plasma membrane was isolated by a slight modification (McClure
and Noonan, 1978) of the aqueous two phase polymer technique of Brunette
and Till (1971).


14
Isolation of Nuclei and NE
A flow chart of this procedure is presented in Figure 2. All
procedures were carried out at 0C unless otherwise noted. For each
Q
NE preparation 3-4x10 cells were harvested, washed twice with phos
phate buffered saline (PBS, pH 7.2), and resuspended at 2 4 x 107
cells/ml in a homogenization buffer containing lOmM Tris (pH 7.6),
lOmM KC1, and lOmM EDTA. The cells were allowed to swell for 15 min
and then lysed (in 15 ml batches) by 5-7 strokes in a Dounce homogen-
izer fitted with a tight pestle. Homogenization was continually
monitored by phase contrast microscopy to assure maximum cell lysis
with minimum nuclear breakage.
Immediately following lysis, the homogenate obtained from each
batch of cells was diluted with 2 volumes of 65% (w/w) sucrose, 50mM
Tris (pH 7.6), 7.5mM MgCl^ and kept on ice until homogenization of
the entire cell sample was completed. The diluted homogenate was cen
trifuged in a Sorvall HB-4 rotor for 30 min at 4,936 x g The
max
resultant crude nuclear pellet was resuspended in 24 ml of 65% (w/w)
sucrose, 50mM Tris (pH 7.6), 2.5mM MgCl^ (65% sucrose-TM). Aliquots
of the crude nuclear pellet were overlayed with 6 ml of 60% sucrose-TM,
12 ml of 55% sucrose-TM, 6 ml of 50% sucrose-TM and 7 ml of 30% sucrose-
TM to form a discontinuous sucrose gradient. The gradient(s) was
subsequently spun for 1 hr at 72,100 x g in a Beckman SW27 rotor.
max
Nuclei banding at the 65%-60% and 60%-55% sucrose interfaces were
removed, combined, and diluted to 50% sucrose with TM buffer. Using
an SW27 rotor (72,100 x g for 1 hr) these nuclei were sedimented
max
through another discontinuous gradient containing 12 ml of 55% sucrose-
TM, 3 ml of 60% sucrose-TM and collected onto a cushion of 3 ml 65%


48
3
synchronized in the presence of [ H] leucine as described for the
experiments presented in Table IV. Six and one-half hours after
release from HU (Figure 9), cells were harvested and washed twice
in PBS. One aliquot of cells was used immediately for NE isolation
(G^/M NE), while the remaining cells were returned to culture in
complete medium lacking [ H] leucine. Each hour thereafter, for the
next 6 hr, an aliquot of cells was harvested, NE isolated and the
specific activity of NE protein was determined. The specific
activity of the NE's at each time point is displayed in Figure 9.
Clearly, NE's which are in the late phase of the cycle (8.5 hr
after release from HU) have lost 10% of the label associated with
the envelopes suggesting that during very late G^ labeled components
of the NE are rapidly diluted with unlabeled components. As the
NE's enter M there appears to be a sharp decrease in the rate of
dilution and then as NE's enter G-j a resumption of label dilution
occurs (Figure 9). Dilution of labeled NE peptides is greatly
reduced during that period of time in which the cell population is
dividing when compared to the dilution observed in NEs isolated from
cells in the G^ and G^ phases of the cell cycle.
In order to determine whether a particular peptide or group of
NE peptides were preferentially lost during the G^-M-G^ transition,
3
[ H] leucine-labeled NE, isolated during the experiments described in
Table IV were solubilized in SDS, labeled peptides and glycopeptides
separated on a discontinuous SDS-PAGE, and the profile examined by
fluorography. Figure 10 contains both the coomasie blue stained
profile of the SDS-PAGE and the fluorogram obtained from the labeled


56
after release from HU (by which time approximately 40% of the cells
in the population have divided). However between 9 and 10 hr after
release from HU when the majority of the cell population is in the
early portion of the G-| phase of the cell cycle (Figure 11), incor-
3
poration of [ H] leucine into the NE's increased 1.25 times that
3
seen in M and by 11 hr after release, incorporation of [ H] leucine
into the NE is 1.56 that seen in M. Taken together, the data in
Figure 9 and Figure 11 suggest that the majority of NE synthesis (and
consequently dilution of pre-existing label) occurs in late G,, and
early to mid G^, with NE specific synthesis in M being low relative
to either of these time periods.
Determination of acid soluble [ H] leucine counts (cpm/mg protein)
in the whole cell homogenate at each time point clearly demonstrated
that the increase in specific activity of NE and homogenate protein
seen in G^ was not due to increased transport of labeled precursor
but rather was due to enhanced de novo biosynthesis.
To determine whether specific peptides of the NE might be
preferentially synthesized during the G,,-M-G.| segment of the cell cycle
NE fractions, obtained from the time points taken in Figure 11, were
solubilized in SDS and the peptide and glycopeptide composition dis
played by coomasie blue staining and fluorography on a 7.5 12.5%
discontinuous SDS-PAGE. The fluorogram presented in Figure 12A
indicates that, at the limits of detection by one dimensional SDS gel
electrophoresis and fluorography, all NE peptides are synthesized at
some point in the G^-M-G-j transition. Comparison of the coomasie
blue stained profile (Figure 12B) with the intensities of the individ
ual bands in the exposed fluorogram (Figure 12A) suggests that the


Spin 1020 x g
5 min
max
Supernatant pellet
Supernatant pellet
(DISCARD)
Purified Nuclear Fraction
Lyse in lOmM Tris (pH 8.5), O.lmM MgCl2
nuclease digest, dilute with 60% sucrose,
50mM Tris (pH 7.6), 500mM MgClz and place
at the bottom of a SW41 gradient.
Spin 153,244 x gmax, 2 hr
Remove NE (band at 1.19 g/cc). Dilute
with 50mM Tris (pH 7.6), 500mM MgC^.
Spin 130,766 x 9max> 30 min
Purified NE Fraction
Figure 2. Flow Diagram of Nuclei and NE Isolation Procedure
rv>
o


22
nuclei. Breakage of the nuclei at early stages in the isolation pro
cedure inevitably results in nuclear clumping with concomitant
entrapment of cytoplasmic and nucleoplasmic contaminants, (c) Homog
enization, resuspension, and mixing must be carefully monitored to
insure oneself that the nuclei are adequately dispersed. Failure to
achieve a relatively homogeneous mixture of nuclei inevitably results
in failure of the subsequent centrifugation steps to remove cytoplasmic
contaminants.
Morphology of the Purified Nuclear Fraction
As can be seen in the phase contrast micrograph presented in
Figure 3, nuclei purified via the procedure outlined in Figure 2 are
intact, contain distinct nucleoli, and show few cytoplasmic "tags."
Electron micrographs of the purified nuclear fraction show no obvious
contamination of the nuclei with mitochondria, RER, vesicles or large
sheets of plasma membrane (Figure 4A). Although rough or smooth
microsomes are not immediately detectable in the nuclear fraction,
it must be pointed out that the outer nuclear membranes of some nuclei
are blebbed. It is possible that these blebs could represent micro
somal contamination. However it is more likely that the blebs simply
represent swelling of the outer nuclear membrane. A higher magnifi
cation micrograph (Figure 4B) of nuclei isolated in the "purified
nuclear pellet" (Figure 2) clearly demonstrates that both the inner
(arrow) and outer (double arrow) nuclear membranes are present on
the isolated nuclei and that very few ribosomes are attached to the
outer membrane. Furthermore, the isolated nuclei have obvious pores
(triple arrow) associated with them.


5
Association of Chromatin with NE
Examination of the NE by EM has demonstrated a close association
of heterochromatin with the inner side of the inner nuclear membrane.
Based on technical difficulties associated with removing trace amounts
of DNA from purified NE, several authors have suggested that the asso
ciation of DNA with the NE may play a functional role in DNA replica
tion (e.g. O'Brien et al., 1972). Currently no solid evidence is
available which can be used to support this contention in vertebrate
cells (Franke, 1974).
Isolation and Composition of NE
Subcellular fractionation has proved to be one of the most pro
ductive techniques employed by cell biologists.to probe the composition,
function, and biosynthesis of various components of the cell. Many
procedures for isolation of nuclei and NE from whole tissues have been
reported (for reviews see Franke, 1974; Kasper, 1974). To our know
ledge, however, no large scale procedure for isolation of biochemically
characterized NE from cultured cells has been described. Procedures
which purport to isolate structural components from nuclei of cultured
cells (the so-called nuclear matrix [Hodge et al., 1977] or nuclear
ghost [Riley et al., 1975]) have been reported. These isolation
methods, however, are relatively harsh removing most of the lipid
bilayers of the nuclear membranes. These procedures appear to isolate
a nonmembranous portion of the NE which, it has been suggested, is
comprised of the proteinaceous lamina and pore complexes (Dwyer and
Blobel, 1976).
Isolation Procedures
t
Isolation of NE is attained by disruption of purified nuclei and
subsequent centrifugal separation of membranous components from the


9
The cytochrome oxidase content of NE remains a controversial issue.
Several authors have reported the presence of cytochrome oxidase or
cytochrome aa^ spectrum in isolated NE (Zbarsky et al., 1969; Berezney
et al., 1972; Matsuura and Ueda, 1972; Jarasch and Franke, 1974).
Jarasch and Franke (1974) interpret their results as mitochondrial con
tamination while Berezney et al. (1972) suggest that cytochrome oxidase
is a NE enzyme. Extensive discussions of this issue can be found in
Jarasch and Franke (1974) and Berezney et al. (1972).
At the present time, enzymatic analysis can not accurately deter
mine NE enrichment or contamination by mitochondria or microsomes. Only
plasma membrane contamination can be accurately quantitated by use of
enzymatic markers because several plasma membrane specific enzymes
(e.g. Na+, K+, stimulated ATPase and 5' nucleotidase) have not been
reported to be present in any NE fraction. When evaluating the enrich
ment or purity of NE fractions, it is necessary to rely heavily on the
presence of NE specific morphological features (pore structures and
double membranes) and the lack of other organellar structures such as
those of mitochondria or unbroken nuclei.
Morphological Changes in the NE
During Cell Cycle
Continuously growing cells proceed through a cycle of biochemical
and morphological events which reproducibly repeat in a fixed order
after each cell division. The events of this cell cycle have been
extensively studied (for review see Mitchison, 1971). The cell cycle
can be divided into four segments -- G-| phase, S (DNA synthetic) phase,
phase, and mitosis. G. phase is defined as that time segment which


Table IV
Dilution of [JH] Leucine Labeled NE Protein
Specific Activities of NE Protein (cpm/mg protein)
G,/M M/G, Ratio (M/G,:G,/M)
5.3 x 105 3.9
9.3 x 105 6.7
6.3 x 105 4.2
2.7 x 105 2.1
1.4 x 105 1.1
105
0.74
105
0.72
105
0.67
105
0.78
105
0.79
x = 0.74
SD = t 0.05
3
Cells were grown and synchronized in media containing 0.2uCi/ml [ H] leucine and NE
isolated from half of the cell population immediately before synchronous division (G2/M NE).
Four hours later (when n,80% of the cells had completed M) NE were isolated from the other
half of the cell population which had proceeded through M in the absence of [^H] leucine (M/G-| NE).
Specific activities were determined as described in Materials and Methods.


BIBLIOGRAPHY
Aaronson, R. P. and G. Blobel (1974) J. Cell Biol. 62:746.
Berezney, R. and D. S. Coffey (1974) Biochem. Biophys. Res. Commun.
60:1410.
Berezney, R., L. K. Funk and F. L. Crane (1970) Biochim. Biophys.
Acta 203:531.
Berezney, R., L. K. Macaulay, and F. L. Crane (1972) J. Biol. Chem.
247:5549.
Bonner, W. M. and R. A. Laskey (1974) Eur. J. Biochem. 46:83.
Bornens, M. (1968) C. R. Acad. Sci. Ser. 0. 266:270.
Bornens, M. (1973) Nature (London) 244:28.
Bornens, M. (1978) J. Cell Biol. 76:191.
Brinkley, B. R., E. Stubblefield, and T. C. Hsu (1967) 0. Ultra-
struct. Res. 19:1
Brunette, D. M. and 0. E. Till (1971) 0. Membrane Biol. 5:215.
Burton, K. (1956) Biochem. J. 62:315.
Carter, D. B., P. H. Efird, C.-B. Chae (1976) Biochemistry 15:2603.
Chauveau,J., Y. Moule, and C. Rouiller (1956) Exp. Cell Res. 11:317.
Chen, P. S., T. Y. Toribara, and H. Warner (1956) Anal. Chem. 28:1756.
Comes, P. and W. W. Franke (1970) Z. Zellforsch. 107:240.
Cunningham, D. D. (1972) J. Biol. Chem. 247:2161.
Dhainaut, A. (1970) J. Microsc. 9:99.
Dwyer, N. and G. Blobel (1976) J. Cell Biol. 70:581.
Erlandson, R. A. and E. De Harven (1971) J. Cell Sci. 8:353.
Fawcett, D. W. (1966) Amer. J. Anat. 119:129.
83


28
were isolating intact NE's, nuclear pores can be seen in both trans
verse (double arrow) and tangential section (triple arrow). Clearly
the presence of pores and double membranes in this purified fraction
supports its identify as a NE fraction.
Few, if any, vesicles are apparent in this preparation (Figure 5),
suggesting little or no microsomal contamination. Similarly no mito
chondria or unbroken nuclei are observed in this "purified NE fraction."
Recovery of Isolated Fractions
Typical recoveries of protein, phospholipid and ONA obtained
during preparation of the NE are presented in Table I. Approximately
14% and 30% of the homogenate protein and DNA, respectively, are
recovered in the nuclear fraction. Based on the recovery of DNA it
appears that 30% of the total starting nuclei are isolated in the
purified nuclear fraction" (Figure 2). My work suggests that ^70%
of the starting nuclei are lost during the repeated centrifugations
(Figure 2) which I find to be necessary for preparing clean, intact
nuclei.
As can be seen in Table I the NE fraction contains 8% of the
starting nuclear protein and 50% of the starting nuclear phospholipid
while only 0.3% of the nuclear DNA is recovered in this fraction. If
one assumes that all nuclear phospholipid resides in the nuclear
membranes (Franke, 1974), one can estimate that approximately 50% of
the starting NE are isolated in the "purified NE fraction" (Figure 2).
Characterization and Purity of Nuclear and NE Fractions
In order to further confirm the relative purity of the isolated
nuclear and NE fractions, as well as to determine the composition of


CHAPTER IV
DISCUSSION
In Chapter II of this dissertation I have described a procedure
for the isolation of highly enriched nuclear and NE fractions from
CHO cells. The nuclei and NE fractions obtained by use of this
procedure have been characterized by light and electron microscopy;
chemical and enzymatic assay; as well as SDS gel electrophoresis.
These characterizations have demonstrated that the nuclei and NE
fractions isolated are distinct from each other and that both frac
tions are distinct from the plasma membrane and whole cell homogen
ate.
Although electron microscopy and marker enzyme assays indicate
that contamination of the nuclei and NE fraction with other organ
elles is very low, the true enrichment of NE in the final pellet as
compared to the homogenate is impossible to determine since no
dependable enzymatic marker has been unequivocally localized to this
organelle in CHO cells. However, it must be again stressed that
the final NE fraction obtained by the procedures outlined in this
dissertation is morphologically similar to the intact NE found in
the cell in that the isolated NE's contain a double membrane bilayer
as well as the pore-lamina complex, thus strongly suggesting that
the isolated procedure described produces a highly enriched NE
fraction.
77


58


45
~ B a
-
12 3 4
Figure 8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells
Nuclear envelopes were isolated from cells in
logarithmic growth and from cells at various
stages of the cell cycle. Nuclear envelope
fractions were solubilized in SDS and electro
phoresis was performed as described in
Materials and Methods. Lane 1, log phase;
Lane 2, G2/M; Lane 3, M/G-j; Lane 4, G-|/S.


86
Monneron, A., G. Blobel, and G. E. Palade (1972) J. Cell Biol. 55:104.
Moses, M. J. (1964) In: Cytology and Cell Physiology, G. H. Bourne,
ed., Academic Press, Inc., New York, pg. 423.
Muramatsu, M. (1970) In: Methods in Cell Biology, Vol 4, D. M.
Prescott, Ed., Academic Press, Inc., New York, pg. 195.
Murray, R. G., A. S. Murray, and A. Pizzo (1965) J. Cell Biol. 26:601.
O'Brien, R. L., A. B. Sanyal, and R. H. Stanton (1972) Exp. Cell
Res. 70:106.
Orrenius, S. and J. L. E. Ericsson (1966) J. Cell Biol. 31:243.
Patrizi, G. and M. Poger (1967) J. Illtrastruct. Res. 17:1277.
Porter, K. R. and R. 0. Machado (1960) J. Biophys. Biochem. Cytol.
7:167.
Riley, D. E. and J. M. Keller (1978) J. Cell Sci. 29:129.
Riley, D. E., J. M. Keller, and B. Byers (1975) Biochemistry 14:3005.
Robbins, E. and N. K. Gonatas (1964) J. Cell Biol. 21:429.
Robbins, W. and Scharff, M. D. (1966) In: Cell Synchrony, I. Cameron
and J. Padilla, eds., Academic Press, Inc., New York, pg. 353.
Rosen, S. 1.(1969) J. Anat. 105:579.
Rosen, S. I. (1970) Experientia 26:839.
Rouser, G. and S. Fleischer (1967) In: Methods in Enz.ymology,
R. N. Estabrook and M. E. Pallman, eds., Academic Press, Inc., New
York, 10:385.
Rouser, G., A. N. Siakotos.and S. Fleischer(1966) Lipids 1:85.
Sieber-Blum, M. and M. M. Burger (1977) Biochem. Biophys. Res.
Commun. 74:1.
Skipski, V. P. and Barclay, M. (1969) In: Methods in Enz.ymology,
J. M. Lowenstein, ed., Academic Press, Inc., New York, 14:530.
Smith, L. (1955) Meth. Biochem. Anal. 2:427.
Spector, A. A. (1972) In: Growth, Nutrition and Metabolism of Cells
in Culture, G. H. Rothblat and V. J. Cristofalo, eds., Academic
Press, Inc., New York, 1:257.
Spurr, A. R. (1969) J. Ultrastruct. Res. 26:31.
Stelly, N., B. J. Stevens, and J. Andr (1970) J. Microsc. 9:1015.


16
Chemical Assays
All protein determinations were performed according to the pro
cedure of Lowry et al. (1951). Phospholipid phosphorus (Rouser et al
1966) was measured in chloroform-methanol extracts (Rouser and
Fleischer, 1967) of various subcellular fractions. Phospholipid
content of the various fractions was extrapolated from the phosphorus
determination by multiplying the phosphorus value by a factor of 25.
Cholesterol was determined according to Glick et al. (1964). In
order to determine the RNA and DNA content of the NE fraction, cells
3
were grown for five generations in the presence of 0.5pCi/ml [ H]
thymidine or 0.4pCi/ml [ H] uridine prior to preparation of NE. The
specific radioactivity of DNA or RNA (cpm/pg nucleic acid) was deter
mined from the cell homogenate by liquid scintillation counting and
spectrophotometric assay. DNA content was determined by the diphenyl
amine assay (Burton, 1956) while RNA content was determined by the
orcinol (I-San Lin and Schjeide, 1969) assay of extracts prepared
according to Fleck and Munro (1962). The specific radioactivity
obtained for DNA and RNA was then used to determine the relative
nucleic acid content of nuclei and NE.
Enzyme Assays
G1ucose-6-phosphatase activity was determined according to
Franke et al. (1970) with the inorganic phosphate release being mea
sured according to Fiske and Subbarowe (1925). 5'-Nucleotidase was
assayed via the technique of Widnell and Unkless (1969) with the
inorganic phosphate released being measured by a modification of the
technique described by Chen et al. (1956). Succinate dehydrogenase


72
Thus when one takes into account the fact that the soluble pool
3 3
is not completely depleted of [ H] leucine after removal of [ H]
leucine from the media, one calculates that''-40% of the M/G-j-NE
protein was synthesized during the G2-M-G1 transition. By simple sub
traction then ^60% of the M/G1 NE protein must have pre-existed M.
I have used the same experimental design and logic to examine
32
the dilution of the [ P] labeled phosphatidic acid pool in order to
more precisely estimate NE phospholipid biosynthesis during the
G^-M-G-j transition. Phosphatidic acid was chosen since it is a
precursor of phospholipids and is not shunted into other pathways
(Howard and Howard, 1974; Spector, 1972).
In these experiments (Figure 16) CHO cells were grown for 5
32
generations in 0.4 pCi/ml [ P] and then synchronized as described
32
in Materials and Methods in 0.4 pCi/ml [ P] orthophosphate. Six
hours after release from HU (Figure 16) an aliquot of cells was
harvested and immediately extracted with chloroform-methanol to give
me a base value for phosphatidic acid specific activity. The
remaining cells were also harvested, washed twice in PBS and another
aliquot of cell population extracted with chloroform-methanol
(Figure 16, 6.5 hr after HU removal). The remaining cells were then
returned to media containing 60 mg/liter cold phosphate but free of
32
[ P]. As shown in Figure 16, aliquots were harvested from the cell
population each hour through the G^-M-G^ transition and extracted
with chloroform-methanol. After all samples had been extracted, each
was Folch backwashed (Rouser and Fleischer, 1967), filtered, and
concentrated for application to thin layer chromatography plates.


LIST OF FIGURES
1. Diagrammatic Representation of NE
Structure 2
2. Flow Diagram of Nuclei and NE Isolation
Procedures 19
3. Phase Contrast Micrograph of the Purified
Nuclear Fraction 23
4. Electron Micrograph of the Purified Nuclear
Fraction 25
5. Electron Micrograph of the Purified NE
Fraction 27
6. Coomasie Blue Stained Profile of Sub-
cellular Fractions 35
7. Cell Cycle Parameters in Synchronized
CHO Cells 42
8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells 45
9. Dilution of t^H] Leucine Labeled NE
Protein 50
10. SDS Gel Electrophoresis of NE Peptides
Isolated after Removal of [3H] Leucine
at the G2/M Boundary 52
11. Incorporation of [3H] Leucine into
NE Protein 55
12. SDS Gel Electrophoresis of [^H] Leucine
Pulse Labeled NE Peptides 58
13. Dilution of [^P] Labeled NE Phospholipid 63
32
14. Incorporation of [ P] Labeled NE Phos
pholipid 67
15. [^H] Leucine Pool Dilution 70
16. [^P] Phosphatidic Acid Pool Dilution 74
vi


78
The NE fraction isolated from CHO cells is composed primarily
of protein and phospholipid having 200pg phospholipid/mg protein.
This phospholipid/protein ratio suggests that, in comparison to other
isolated cellular membranes (Fleischer and Kervina, 1974), the isolated
NE fraction is relatively protein rich. This finding may derive
from the fact that the isolation procedure described, isolates both
the nuclear membrane and the so-called proteinaceous lamina. The
inclusion of the proteinaceous lamina in the NE fraction would be
expected to increase the protein to lipid ratio.
Examination of the peptide composition of the NE fraction by
SDS gel electrophoresis demonstrates that the major coomasie blue
staining peptides of the NE fraction chromatograph with molecular
weights between 55,000 and 75,000 daltons. These peptides are
undoubtedly related to the pore-lamina and nuclear matrix proteins
identified in rat liver (Dwyer and Blobel, 1976) and HeLa cell
(Hodge et al., 1977) NE's. It is of interest to note that the
majority of the remaining NE peptides chromatograph with a molecular
weight greater than 75,000 daltons. Relatively few coomasie blue
staining bands are found to run at molecular weights below 50,000
daltons and no peptides appear to co-migrate with the histones seen
in the nuclear fraction.
It can not be emphasized too much that although the isolation
technique described required careful handling of material during the
preparation of the nuclei and NE, it does produce material in
sufficient quantity and of sufficient purity to permit biochemical
studies directed at elucidating the molecular basis of both NE
structure and biosynthesis.


80
some NE components are specifically synthesized in the G,,, M, or G-|
phases of the cell cycle. However, my data do indicate that at some
point in the G^-M-G^ transition all of the NE peptides separable on
3
a one-dimensional SDS-PAGE are labeled with [ H] leucine suggesting
that no specific component(s) is carried through the transition while
another component(s) is degraded and totally resynthesized during
the G2-M-G-| transition.
Measurement of nuclear surface area in synchronized populations
of cells in G^ and G^ indicated that there was no significant differ
ence in the average surface area of the two nuclear populations.
These data suggest that the total nuclear surface has increased
n-2 fold between G,, and early G^. If one assumes that increases in
surface area are directly correlated to NE biosynthesis, these data
predict that "label dilution" experiments would detect an^SOJ!
dilution of NE protein during the time period examined. This is in
good agreement with our calculated results of 40% dilution and 60%
reutilization of pre-existing peptides.
The 60% of the early G^-NE protein which the data suggest
pre-existed M, when taken together with the data suggesting a burst
of early G^ NE synthesis does strongly imply that a majority of
G.| NE proteins come from pre-existing cellular components. Thus,
in my opinion, these data unequivocably rule out "complete" de novo
synthesis of NE protein as being responsible for mitotic reassembly.
Unfortunately I cannot state unequivocally that the early G1 NE
protein which pre-existed M resided solely in the G,, NE. The
possibility clearly exists that G, NE components are made prior to


64
and M and then begins to drop as the cells enter After 80% of
the cells have completed division (Figure 13, 11.5 hr after release
from HU), the specific activity of the NE is approximately 65% of
the NE phospholipid specific activity before M (Figure 13 and Table V).
The simplest explanation for these data is that 65% of the phospho
lipid present in NE 12 hr after release from HU (i.e. early see
Figure 13) was present in the cell prior to M.
Interestingly the specific activity of the NE phospholipid
begins to decrease after 20-30% of the cells have entered the G^ phase
of the cell cycle whereas the specific activity of the whole cell
phospholipid drops only after ^80% of the cells have completed M
(i.e. the majority of the cells are in early to mid G^. The data
in Figure 13 clearly suggest that synthesis of phospholipid to be
incorporated into NE and other cellular phospholipid increases as the
cel 1s enter G^.
32
Incorporation of [ P] Orthophosphate into NE Phospholipid
In order to confirm that the dilution of NE phospholipid specific
activity, seen in Table V and Figure 13, is due to synthesis of new
phospholipid, which is, in turn incorporated into NE, cell cultures
were grown to stationary phase and then synchronized as described
previously in complete medium containing no label. Seven hours after
release from HU (i.e. at the G^/M transition, Figure 14), an aliquot
of cells was removed from the stock culture and pulsed for 1 hr with
32 32
0.8pCi/ml [ P] orthophosphate. After one hour in [ P], the labeled
culture was harvested and both NE and homogenate prepared. Similarly
treated aliquots of cells were pulsed for 1 hr with [ P] over the


Table I
Recovery of Protein, DNA, and Phospholipid in Subcellular Fractions
Fraction
1 of Total
Cellular DNA
1 of Total
Cellular Protein
% of Total
Nuclear DNA
% of Total
Nuclear Protein
% of Total
Nuclear Phospholipid
Homogenate
100
100



Nuclei
30
14
100
100
100
NE
0.1
1
0.3
8
50
Recoveries were
determined by assay
of individual
fractions sampled during the isolation
procedure.
ro


Table III
Chemical Content of Subcellular Fractions
pg DNA/mg pg RNA/mg pg Phospholipid/mg pg Cholesterol/mg
Fraction Protein Protein Protein Protein
Homogenate
90
220
91

Nuclei
200
90
30

NE
10
20
200
20
PM
117
Individual fractions were sampled during the isolation procedure and assayed for protein,
DNA, RNA, phospholipid or cholesterol as described in Materials and Methods. A plasma membrane
enriched fraction was prepared from CHO cells via the two-phase aqueous polymer technique of
Brunette and Till (1971).
CO
CO


84
Feldherr, C. M. (1972) In: Advances in Cell and Molecular Biology,
E. 0. Dupraw, ed., Academic Press, Inc., New York 2:273.
Feldherr, C. M., P. A. Richmond, and K. D. Noonan (1977) Exp. Cell
Res. 107:439.
Fiske, C. H. and Y. Subbarowe (1925) J. Biol. Chem. 66:375.
Fleck, A. and H. N. Munro (1962) Biochim. Biophys. Acta 55:571.
Fleischer, S. and M. Kervina (1974) In: Methods in Enz.ymolog.y,
S. Fleischer and L. Packer, eds., Academic Press, Inc., New York.
31:6.
Franke, W. W. (1967) Z. Zellforsch. Mikrosk. Anat. 80:585.
Franke, W. W. (1974) In: International Review of Cytology, G. H.
Bourne and J. F. Daniel 1 i, eds., Academic Press, Inc., New York.
Suppl. 4:71.
Franke, W. W. (1977) In: Biochemistry of the Cell Nucleus,
P. B. Garlord and A. P. Mathias, eds., The Biochemical Society,
London, 125.
Franke, W. W. and U. Scheer (1974) In: The Cell Nucleus, H. Busch,
ed., Academic Press, Inc., New York, 1:219.
Franke, W. W., B. Deumling, B. Ermen, E. 0. Jarasch, and H. Kleinig
(1970) J. Cell Biol. 46:379.
Glick, D., B. F. Fell, and K. Sjolin (1964) Anal. Chem. 36:1119.
Hildebrand, C. E. and R. T. Okinaka (1976) Anal. Biochem. 75:290.
Hodge, L. D., P. Mancini, F. M. Davis, and P. Heywood (1977)
J. Cell Biol. 72:194.
Howard, B. V. and W. J. Howard (1974) In: Advances in Lipid Research,
R. Paoletti and D. Kritchevsky, eds., Academic Press, Inc., New
York, 12:52.
I-San Lin, R. and 0. A. Schjeide (1969) Anal. Biochem. 27:473.
Jarasch, E-D. and W. W. Franke (1974) J. Biol. Chem. 249:7245.
Jones, 0. P. (1960) Nature (London) 226:717.
Kalifat, S. R., M. Bouteille, and J. Delarue (1967) J. Microsc.
6:1019.
Kartenbeck, J., H. W. Zentgraff.U. Scheer, and W. W. Franke (1971)
Ergeb. A. Entwicklungsgeesch. 45:1.


18
buffer is essential since we have found that NE's dissolved in sample
buffer lacking PMSF are subject to extensive proteolysis, possibly
as the result of the co-purification of an intrinsic protease with
the purified envelopes (Carter et al., 1976).
Following electrophoretic separation of the membrane peptides
and glycopeptides, the slabs were fixed for 30 min in 10% trichloro
acetic acid and then allowed to equilibrate overnight in 25% ethanol,
8% acetic acid. The following day the gels were stained with Coomasie
brilliant blue according to Weber and Osborn (1969) and finally
destained in 25% ethanol, 8% acetic acid.
Results
Isolation of Nuclei
The primary goal which I set for myself prior to isolating NE's
was that the purified NE's contain both the inner and outer membrane
bilayer as well as the pore complex and be free of contaminating cyto
plasm or nucleoplasm. In order to achieve this goal, a nuclear
fraction had to be isolated which itself contained both the inner and
outer nuclear membranes and was free of substantial cytoplasmic con
tamination. The nuclear isolation procedure which I have employed in
my work has been described in detail in the Materials and Methods
section and is presented diagrammatically in Figure 2.
In many of the nuclear isolation procedures previously published
workers have used nonionic detergents to remove cytoplasmic components
(Muramatsu, 1970) from the nuclei. Unfortunately the use of such
detergents on isolated nuclei from rat liver has been shown to remove
almost all of the nuclear membrane phospholipid and much of the protein


ABBREVIATIONS USED
3ME
B-Mercaptoethanol
CHO
Chinese hamster ovary
DNase I
Deoxubonuclease I
EDTA
ethylene diaminetetraacetic acid
EM
electron microscopy
ER
endoplasmic reticulum
HU
hydroxyurea
LSC
liquid scintillation counting
M
mitosis
NE
nuclear envelope
PBS
phosphate buffered saline
PMSF
phenylmethylsulfonyl fluoride
RER
rough endoplasmic reticulum
RNase A
Ribonuclease A
SDS
sodium dodecyl sulfate
TM
50mM Tris, pH 7.6, 2.5mM MgCl2
vi i


REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS
By
GREGORY E. CONNER
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA

ACKNOWLEDGEMENTS
I would like to thank Dr. Kenneth D. Noonan, my graduate
supervisor, for his endless support and encouragement during the
course of my graduate studies.
I would also like to thank the members of my supervisory
committee, Drs. C. M. Allen, C. M. Feldherr, E. M. Hoffman, and
T. W. O'Brien for their help and advice throughout this research
project. I am particularly grateful to Dr. Feldherr for his assis¬
tance in obtaining the various micrographs. I would also like to
express my thanks to Dr. Allen for his assistance with thin layer
chromatography.

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS USED vii
ABSTRACT viii
CHAPTER I-INTRODUCTION 1
NE Structure 1
Membranes of the NE 3
Pore Structures 3
Proteinaceous Lamina 4
Association of Chromatin with NE 5
Isolation and Composition of NE 5
Isolation Procedures 5
Composition 7
Morphological Changes in the NE
During Cell Cycle 9
Objectives 12
CHAPTER II-ISOLATION AND CHARACTERIZATION 13
Materials and Methods 13
Materials 13
Cell Culture 13
Isolation of Nuclei and NE 14
Isolation of Plasma Membrane 15
Chemical Assays 16
Enzyme Assays 16
Electron Microscopy 17
SDS Polyacrylamide Disc Gel
Electrophoresis (PADGE) 17
Results 18
Isolation of Nuclei 18
Morphology of the Purified Nuclear
Fraction 22
Isolation of NE 26
Morphology of the Purified NE Fraction 26
Recovery of Isolated Fractions 28
Characterization and Purity of Nuclear
and NE Fractions 28
Enzyme Activity 30
Chemical Composition 32
Peptide and Glycopeptide Composition 34
CHAPTER 11¡-REFORMATION OF THE NUCLEAR ENVELOPE
DURING MITOSIS 36
Materials and Methods 36
Materials 36
Cell Culture and Synchrony 36

Labeling of Cell Cultures 37
Isolation of Nuclei and NE 38
Determination of Specific Activities 38
Determination of Precursor Pool
Equilibration Rates 39
Measurement of Nuclear Surface Areas 40
Polyacrylamide Gel Electrophoresis 40
Results 41
Cell Synchrony 41
Peptide Composition of the NE during
Different Phases of the Cell Cycle 43
Dilution of [3H] Leucine Labeled NE 44
Incorporation of the [3H] Leucine
into NE Protein 53
Dilution of [3h] Choline Labeled NE 59
Dilution of [32p] Orthophosphate
Labeled NE Phospholipid 61
Incorporation of [32p] Orthophosphate
into NE Phospholipid 64
Rate of Precursor Pool Equilibration 65
Changes in Nuclear Surface Area 75
CHAPTER IV-DISCUSSION 77
APPENDIX—DERIVATION OF THE EQUATION FOR CALCULATING
THE PROPORTION OF M/G-i NE SYNTHESIZED DE NOVO
DURING THE G2-M-G-] TRANSITION 82
BIBLIOGRAPHY 83
BIOGRAPHICAL SKETCH 88
IV

LIST OF TABLES
I. Recovery of Protein, DNA, and
Phospholipid in Subcellular Fractions 29
II. Enzyme Activities of Subcellular
Fractions 31
III. Chemical Content of Subcellular Fractions 33
IV. Dilution of [^H] Leucine Labeled NE
Protein 47
V. Dilution of Labeled NE Phospholipids 60
v

LIST OF FIGURES
1. Diagrammatic Representation of NE
Structure 2
2. Flow Diagram of Nuclei and NE Isolation
Procedures 19
3. Phase Contrast Micrograph of the Purified
Nuclear Fraction 23
4. Electron Micrograph of the Purified Nuclear
Fraction 25
5. Electron Micrograph of the Purified NE
Fraction 27
6. Coomasie Blue Stained Profile of Sub-
cellular Fractions 35
7. Cell Cycle Parameters in Synchronized
CHO Cells 42
8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells 45
9. Dilution of t^H] Leucine Labeled NE
Protein 50
10. SDS Gel Electrophoresis of NE Peptides
Isolated after Removal of [3H] Leucine
at the G2/M Boundary 52
11. Incorporation of [3H] Leucine into
NE Protein 55
12. SDS Gel Electrophoresis of [^H] Leucine
Pulse Labeled NE Peptides 58
13. Dilution of [^P] Labeled NE Phospholipid 63
32
14. Incorporation of [ P] Labeled NE Phos¬
pholipid 67
15. [^H] Leucine Pool Dilution 70
16. [^P] Phosphatidic Acid Pool Dilution 74
vi

ABBREVIATIONS USED
3ME
B-Mercaptoethanol
CHO
Chinese hamster ovary
DNase I
Deoxubonuclease I
EDTA
ethylene diaminetetraacetic acid
EM
electron microscopy
ER
endoplasmic reticulum
HU
hydroxyurea
LSC
liquid scintillation counting
M
mitosis
NE
nuclear envelope
PBS
phosphate buffered saline
PMSF
phenylmethylsulfonyl fluoride
RER
rough endoplasmic reticulum
RNase A
Ribonuclease A
SDS
sodium dodecyl sulfate
TM
50mM Tris, pH 7.6, 2.5mM MgCl2
vi i

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS
By
Gregory E. Conner
August 1978
Chairman: Kenneth D. Noonan
Major Department: Biochemistry
A technique for isolating nuclei and nuclear envelopes (NE) from
Chinese hamster ovary (CHO) cells has been developed. This technique
does not depend on the use of detergents to solubilize contaminating
chromatin. In this procedure NE are prepared from purified nuclei by
nuclease digestion and subsequent high salt-sucrose gradient centrifu¬
gation. The nuclei and NE fractions are free of significant contamina¬
tion by other subcellular organelles as judged by electron microscopy
and enzymatic analysis. Electron microscopic observation of the puri¬
fied NE clearly demonstrates that the isolated material retains both a
double bilayer and the pore complexes observed in intact nuclei,
strongly suggesting that the isolation procedure described in this
dissertation permits the recovery of "intact" NE. Biochemical analysis
of the isolated nuclei and NE shows the NE fraction to be composed
primarily of protein and phospholipid, while containing only small
amounts of DNA and RNA. Examination of the peptide composition of the
NE fraction by SDS polyacrylamide gel electrophoresis reveals a very
complex coomasie blue staining profile with prominent bands in the
55,000 to 75,000 dalton molecular weight range.

Employing this isolation technique I have examined the breakdown
and reformation of the NE during a limited stage (late G^, M, and early
G.|) of the replicative cycle in synchronized populations of CHO cells.
3 3
Using [ H] leucine as a precursor for protein and either [ H] choline
32
or [ P] orthophosphate as precursors for phospholipid, I have shown
that a minimum of 60'/ of the early G1, NE protein and a minimum of 50%
of the early NE phospholipid were present during the preceeding G,,
phase of the cell cycle and were reutilized in the reformation of the
NE which occurs during late M and early G-j. Pulse label studies
employing [ H] leucine or [ P] orthophosphate indicate that a burst
of NE biosynthesis occurs in early G^. Autoradiographic examination
of NE peptides isolated during these pulse label and label dilution
studies shows neither preferential loss nor preferential biosynthesis
of specific NE peptides during the G^ to M or M to G^ transition. My
evidence suggests that all the peptides of the NE, which I can resolve
in one dimensional gel electrophoresis, are synthesized during this
portion of the cell cycle. Examination of NE peptides by one dimension¬
al gel electrophoresis does not highlight any reproducible changes in
NE peptide composition which can be correlated with specific phase of
the cell cycle.
IX

CHAPTER I
INTRODUCTION
The nuclear envelope (NE) is a complex subcellular organelle
which exists at the nuclear periphery and separates the genetic
machinery of the eucaryotic cell from other cellular components. During
mitosis in many plant and animal cells, the NE becomes morphologically
indistinct and reappears after daughter chromosome separation is com¬
plete. The studies which will be described in this dissertation are
directed towards understanding the mechanism(s) by which this large
membranous organelle disassembles and restructures during mitosis.
Before presentation of these studies, I will briefly discuss the cur¬
rently available information concerning NE structure, composition and
breakdown and reformation during mitosis.
NE Structure
The structure of the NE has been probed in great detail by many
light and electron microscopic (EM) studies (for reviews see Feldherr,
1972; Kay and Johnston, 1973; Kessel, 1973; Franke, 1974; and Franke
and Scheer, 1974). The NE (shown diagramatically in Figure 1) is com¬
posed primarily of two lipid bilayer membranes which contain pore
structures. Electron microscopic observation suggests that these
bilayer membranes are continuous at the pore structures leaving apparent
gaps when viewed in transverse sections. These pore complexes do con¬
tain internal structure and may be involved in nuclear-cytoplasmic
exchange of molecules (Feldherr, 1972). The outer surface of the outer
1

PS
Figure 1. Diagrammatic representation of NE structure.
ONM - outer nuclear membrane; INM - inner
nuclear membrane; PS - pore structure; PL -
proteinaceous lamina; HC - heterochromatin.

3
nuclear membrane is often studded with ribosomes in a manner similar to
rough endoplasmic reticulum (RER) and occasionally has been observed to
be continuous with RER (Watson, 1955). The inner aspect of the inner
nuclear membrane appears to be in close association with a proteinaceous
lamina (Fawcett, 1966) and to be intimately associated with hetero¬
chromatin (i.e. dense inactive chromatin).
Membranes of the NE
The membranes of the NE, when visualized by EM, are most fre-
O
quently found to be 60-S0 A in thickness. The two membranes are normally
O
separated by 100-700 A except at the pore structures where they appear
to be joined, thus forming a perinuclear cisternum. The frequent local¬
ization of ribosomes and the occasional continuity of the outer membrane
with RER indicates the strong morphological similarity between membranes „
of the NE and RER. As will be discussed later, the enzymatic and bio¬
chemical composition of the NE serves to strengthen the resemblance of
these two membranous organelles. Currently no data are available which
conclusively demonstrate that components of the NE arise from RER or
that components of the RER arise from those of the NE.
Recent studies by Feldherr et al. (1977) and Virtanen (1977,1978)
and Virtanen and Wartiovaaraâ– (1976) suggest that the nuclear membranes
possess a property of sidedness similar to that of other cellular mem¬
branes. Specifically these authors have reported that the carbohydrate
containing portions of NE glycopeptides are located on only one side,
the cisternal side, of each nuclear membrane.
Pore Structures
When pore structures of the NE are visualized by EM in transverse
c o
sections, they appear as gaps (150 A - 700 A wide) at which the nuclear

membranes seem to be fused (Figure 1). When visualized tangentially,
the pore structures appear as complexes of 8 or 9 granules arranged as
an annulus. The central zone of the pore structure is less electron
dense than the surrounding annular material and may coincide with the
gaps seen in transverse sections. The annular pore complexes have
o
an overall diameter of 1000 - 2000 A suggesting that the pore structure
extends beyond the gaps visualized in transverse sections possibly
overlapping part of the nuclear membranes. Electron dense granules
can occasionally be seen in the interior of the pores. Some data have
been obtained which suggest that these granules are material in transit
between nucleus and cytoplasm. Many authors have suggested that the
pore complexes function in the control of nucleo-cytoplasmic communi¬
cation (for review see Feldherr, 1972).
Proteinaceous Lamina
In some cell types the inner aspect of the inner nuclear membrane
appears to be associated with an electron dense material. This layer,
variously called the dense lamella (Kalifat et al., 1967), zonula
nucleum limitans (Patrizi and Poger, 1967), and the fibrous lamina
O O
(Fawcett, 1966), ranges in thickness from 2800 A in amoeba to 150 - 200 A
in vertebrate cell types. This layer, apparently proteinaceous in com¬
position (Stelly et al., 1970), appears to be tightly associated with
the interphase NE, in that it may copurify with NE during subcellular
isolation procedures. The exact function of this proteinaceous lamina
has not been determined, however Stelly et al. (1970) have suggested
that it is responsible for the shape and rigidity of the nucleus.

5
Association of Chromatin with NE
Examination of the NE by EM has demonstrated a close association
of heterochromatin with the inner side of the inner nuclear membrane.
Based on technical difficulties associated with removing trace amounts
of DNA from purified NE, several authors have suggested that the asso¬
ciation of DNA with the NE may play a functional role in DNA replica¬
tion (e.g. O'Brien et al., 1972). Currently no solid evidence is
available which can be used to support this contention in vertebrate
cells (Franke, 1974).
Isolation and Composition of NE
Subcellular fractionation has proved to be one of the most pro¬
ductive techniques employed by cell biologists.to probe the composition,
function, and biosynthesis of various components of the cell. Many
procedures for isolation of nuclei and NE from whole tissues have been
reported (for reviews see Franke, 1974; Kasper, 1974). To our know¬
ledge, however, no large scale procedure for isolation of biochemically
characterized NE from cultured cells has been described. Procedures
which purport to isolate structural components from nuclei of cultured
cells (the so-called nuclear matrix [Hodge et al., 1977] or nuclear
ghost [Riley et al., 1975]) have been reported. These isolation
methods, however, are relatively harsh removing most of the lipid
bilayers of the nuclear membranes. These procedures appear to isolate
a nonmembranous portion of the NE which, it has been suggested, is
comprised of the proteinaceous lamina and pore complexes (Dwyer and
Blobel, 1976).
Isolation Procedures
t
Isolation of NE is attained by disruption of purified nuclei and
subsequent centrifugal separation of membranous components from the

6
remaining elements of the nucleus (e.g. nucleoli, chromatin, and soluble
proteins). Because separation of NE from other nuclear components is
usually dependent on the membranous characteristic of this organelle,
the purified nuclei from which NE are to be isolated, must be prepared
in a fashion which retains both nuclear membranes and yet removes other
contaminating organelles of the cell. Such nuclear isolation procedures
have been described and are usually modifications of the technique of
Chauveau et al. (1956). The lack of a well characterized preparation
of NE from cultured cells is most probably due to difficulties in iso¬
lation of large quantities of "clean" nuclei which contain both nuclear
membranes. The use of detergents to remove cytoplasmic contamination
from nuclei most likely strips the nuclei of the lipid bilayers of the
nuclear membranes.
Disruption of nuclei for isolation of NE is usually accomplished
by mechanical and/or chemical treatments. The most frequently used
mechanical techniques are sonication (e.g. Franke, 1967; Kashnig and
Kasper, 1969) and hypotonic shock (e.g. Zbarsky et al., 1969; Kartenbeck
et al., 1971). High concentrations of MgC^ (Berezney et al., 1970;
Monneron et al., 1972), sodium citrate (Bornens, 1968; Kashnig and
Kasper, 1969), NaCl and KC1 (Franke et al., 1970; Matsuura and Ueda,
1972), and heparin (Bornens, 1973,1978) have been shown to disrupt
nuclei and aid in solubilization of chromatin during NE isolation.
Deoxyribonuclease I (DNase 1) has often been utilized to reduce the
viscosity of ruptured nuclei and to reduce the level of DNA contamina¬
tion in isolated NE fractions (e.g. Berezney et al., 1970; Kay et al.,
1972).

7
After disruption of nuclei and dispersion of chromatin, differ¬
ential or isopynic centrifugation is then employed to separate NE from
the remaining nuclear components (Kasper, 1974).
Composition
Franke (1974) and Kasper (1974) have extensively reviewed the
chemical and enzymatic composition of NE fractions isolated from
tissue. The NE is comprised primarily of protein and phospholipid and
has generally been described as a protein rich organelle. Reported
protein values generally vary between 60% and 75% of the total membrane
mass while phospholipid values range from 20% to 30% of the total
membrane mass (see Kasper, 1974). Phosphatidyl choline is the pre¬
dominant lipid species of NE followed by phosphatidyl ethanolmine,
phosphatidyl serine and phosphatidyl inositol (Keenan et al., 1970,
1972; Khandwala and Kasper, 1971). Some neutral lipids such as
cholesterol and free fatty acids have also been reported in NE
fractions (Kleinig, 1970; Keenan et al., 1970, 1972).
Almost all isolated NE fractions contain small quantities of
DNA and RNA which vary in amount depending on the preparative pro¬
cedure. Those procedures which do not strip the outer membrane of
ribosomes normally result in RNA values which are higher than those
obtained after NE isolation procedures which employ citrate or
MgClg- Small residual amounts of RNA associated with isolated NE
may reflect a functional component of the pores (Monneron and Bernhard,
1969; Dhainaut, 1970). The extent of DNase I treatment and the con¬
centration of salt used in NE isolation procedures appear to affect
the quantity of DNA associated with the NE fraction.

Carbohydrates have been detected in isolated NE fractions by
chemical analysis. Kashnig and Kasper (1969) have reported that rat
liver NE contains 3%-4% carbohydrate which is made up of mannose,
glucose, and galactose. These results have been confirmed by
Franke (1977).
The enzyme composition of isolated NE has been reported to be
different by different workers although in most instances it appears
to closely resemble that of the microsomal membranes. Cytochemically
glucose-6-phosphatase (Orrenius and Ericsson, 1966; Rosen, 1969, 1970;
2+
Leskes et al., 1971) and Mg dependent ATPase (Klein and Afzelius,
1966; Yasuzumi and Tsubo, 1966) have been localized to the NE of a
variety of mammalian cell types. Typically these enzymes are present
and occasionally purified in isolated NE fractions (Franke, 1974).
The enzymatic resemblance of the NE and microsomes is strengthened by
the presence of NADH-cytochrome c reductase in NE. This activity is
not, however, increased by application of drugs (e.g. phenobarbital)
as is the enzymatic activity in microsomes (Kasper, 1971). NADPH-
cytochrome c reductase (another constituent of microsomal membranes)
has also been reported by many authors to be present in isolated NE of
liver and in NE of avian erythrocytes (Zentgraff et al., 1971), a cell
which is practically devoid of endoplasmic reticulum. The lack of
drug induction of NE NADH-cytochrome c reductase and the presence
of NE NADPH-cytochrome c reductase in ER depleted cells (avian erythro¬
cytes) strongly suggests that these enzymes also do not represent ER
contamination of the isolated NE.

9
The cytochrome oxidase content of NE remains a controversial issue.
Several authors have reported the presence of cytochrome oxidase or
cytochrome aa^ spectrum in isolated NE (Zbarsky et al., 1969; Berezney
et al., 1972; Matsuura and Ueda, 1972; Jarasch and Franke, 1974).
Jarasch and Franke (1974) interpret their results as mitochondrial con¬
tamination while Berezney et al. (1972) suggest that cytochrome oxidase
is a NE enzyme. Extensive discussions of this issue can be found in
Jarasch and Franke (1974) and Berezney et al. (1972).
At the present time, enzymatic analysis can not accurately deter¬
mine NE enrichment or contamination by mitochondria or microsomes. Only
plasma membrane contamination can be accurately quantitated by use of
enzymatic markers because several plasma membrane specific enzymes
(e.g. Na+, K+, stimulated ATPase and 5' nucleotidase) have not been
reported to be present in any NE fraction. When evaluating the enrich¬
ment or purity of NE fractions, it is necessary to rely heavily on the
presence of NE specific morphological features (pore structures and
double membranes) and the lack of other organellar structures such as
those of mitochondria or unbroken nuclei.
Morphological Changes in the NE
During Cell Cycle
Continuously growing cells proceed through a cycle of biochemical
and morphological events which reproducibly repeat in a fixed order
after each cell division. The events of this cell cycle have been
extensively studied (for review see Mitchison, 1971). The cell cycle
can be divided into four segments -- G-| phase, S (DNA synthetic) phase,
phase, and mitosis. G. phase is defined as that time segment which

10
exists between completion of cell division and initiation of DNA syn¬
thesis, phase is defined as that time segment which' exists between
completion of DNA synthesis and initiation of cell division.
A variety of techniques are available which will induce individ¬
ual cells of a culture to traverse the cell cycle in unison. These
synchronization techniques have been employed by cell biologists to
study the biochemical and morphological events which characterize
particular segments of this cycle and which are "hidden" by the random
nature of an untreated cell culture.
Mitosis is perhaps the most carefully studied morphological event
of the cell cycle. The sequence of events of mitosis is traditionally
divided into several phases based on early morphological descriptions.
Briefly, mitosis consists of four phases: prophase, metaphase, anaphase,
and telophase. The onset of prophase is marked by chromosome conden¬
sation at the inner nuclear membrane. The nuclear envelope becomes
very irregular, taking on a "wave-like" appearance in the region of the
centrioles. Centriole movement to opposite sides of the nucleus (i.e.
the poles) and chromosome separation occur very early in prophase.
The nuclear envelope begins to dissociate at the poles and microtubules
of the forming spindle apparatus enter into the nucleoplasm. The
nuclear envelope breakdown proceeds toward the equatorial plane and in
late prophase the condensed chromosomes begin to assemble in the com¬
pleted spindle (metaphase). At this point the NE is completely dis¬
rupted. After a short pause the chromosomes separate at the centromere
into daughter chromatids and the chromatids begin moving toward the
poles (anaphase). The nuclear envelope begins to reform as this

11
movement begins. Rows of elongated membranous cisternae appear at
the polar aspect of the spindle. As the chromatids begin to fuse into
a chromatin mass, the row of cisternae becomes juxtaposed and extends
further toward the equatorial constriction (cytokinesis and telophase).
The NE is completely reconstructed before the finish of cytokinesis
which marks the end of the mitotic cycle.
Unfortunately very little is known concerning the fate of the
NE during mitotic breakdown nor is there good evidence relating to the
origin of the components which reform the NE in late M and early .
Similarly the mechanism(s) whereby the NE is disassembled at the mitosis
and reformed in late mitosis have not been elucidated.
It has been suggested by a number of workers (Porter and Machado,
1960; Moses, 1964; Robbins and Sonatas, 1964; Murray et a!., 1965;
Brinkley et a!., 1967) that following NE breakdown, fragments of NE
mingle with and become indistinguishable from the enaoplasmic reticulum
(ER). Furthermore, it has been argued that components of the ER are
utilized in reformation of tne G-j-NE (Porter and Machado, 1960; Moses,
1964). Other authors have suggested that the NE, or components of the
NE, persist through mitosis as distinct entities, biochemically differ¬
ent from the ER, and that these components are specifically reutilized
to reform NE at the completion of M (Erlandson and De Harven, 1971;
Maruta and Goldstein, 1975; Maul, 1977). Finally some workers in the
field have suggested that the NE which reappears at the end of M is a
product of de novo synthesis of all the NE components (e.g. Jones,
1960).
The multiplicity and diversity of hypotheses which have been pre¬
sented to explain NE breakdown and reformation during M are probably

12
due to the variety of animal and plant species in which the predom¬
inantly microscopic work was performed and to the fact that the evidence
available concerning NE breakdown and reformation consists almost
entirely of morphological studies which suffer from the difficulty of
positively identifying cytoplasmic components which might be NE frag¬
ments released during mitosis.
Objectives
The main objective of the studies presented in this dissertation
is to investigate the mechanism whereby the NE disassembles and reforms
during mitosis. To examine biochemical changes and the biosynthesis
of NE during the cell cycle it is necessary to develop a procedure to
use with cultured cells which would permit the isolation of "clean"
NE containing both lipid bilayers of the nuclear membranes as well as
the proteinaceous lamina.
In Chapter II of this dissertation I will report a preparation
technique which permits the isolation of nuclei from Chinese hamster
ovary (CHO) cells without the use of reagents (such as detergents) which
might disrupt membranes. The isolated nuclear fraction has been used
to purify sufficient quantities of NE to allow both biochemical and
EM studies of this organelle.
In Chapter III of this dissertation I will present a biochemical
investigation of the breakdown and reformation of the NE during mitosis
in CHO cells. Using radioactive precursors of protein and phospho¬
lipid I have studied the reutilization and biosynthesis of NE in the
late G^-M-early portion of the cell cycle.

13
CHAPTER II
ISOLATION AND CHARACTERIZATION
Materials and Methods
Materials
All tissue culture materials were purchased from Grand Island
Biological Company (Grand Island, New York). Tissue culture plastic-
ware was obtained from Corning Glass Works (Corning, N.Y.). Ultra-
3 3
pure sucrose, [methyl- H] thymidine, and [5- H] uridine were obtained
from Schwarz-Mann Division, Becton, Dickinson and Company (Orangeburg,
New York). Deoxyribonuclease I (DNase I) and ribonuclease A (RNase A)
were purchased from Worthington Biochemical Corporation (Freehold, New
Jersey) and were shown to be protease-free via the assay of Tomarelli
et al. (1949). [Methyl- H] choline chloride was obtained from Amer-
sham Searle Corporation (Arlington Heights, Illinois). All reagents
for electrophoresis were purchased from Bio-Rad Laboratories (Rockville
Centre, New York). All other reagents were obtained from Scientific
Products (Ocala, Florida).
Cell Culture
Chinese hamster ovary cells (originally obtained from Dr. Kenneth
Ley, Sandia Laboratories, Albuquerque, New Mexico) were maintained at
37°C in suspension culture in Ham's F-10 nutrient medium, supplemented
with 10% (v/v) calf serum and 5% (v/v) fetal calf serum. Cell density
5 S
was monitored daily and maintained between 1.2 x 10 - 4 x 10 cells/ml.

14
Isolation of Nuclei and NE
A flow chart of this procedure is presented in Figure 2. All
procedures were carried out at 0°C unless otherwise noted. For each
Q
NE preparation 3-4x10 cells were harvested, washed twice with phos¬
phate buffered saline (PBS, pH 7.2), and resuspended at 2 - 4 x 107
cells/ml in a homogenization buffer containing lOmM Tris (pH 7.6),
lOmM KC1, and lOmM EDTA. The cells were allowed to swell for 15 min
and then lysed (in 15 ml batches) by 5-7 strokes in a Dounce homogen-
izer fitted with a tight pestle. Homogenization was continually
monitored by phase contrast microscopy to assure maximum cell lysis
with minimum nuclear breakage.
Immediately following lysis, the homogenate obtained from each
batch of cells was diluted with 2 volumes of 65% (w/w) sucrose, 50mM
Tris (pH 7.6), 7.5mM MgCl^» and kept on ice until homogenization of
the entire cell sample was completed. The diluted homogenate was cen¬
trifuged in a Sorvall HB-4 rotor for 30 min at 4,936 x g . The
max
resultant crude nuclear pellet was resuspended in 24 ml of 65% (w/w)
sucrose, 50mM Tris (pH 7.6), 2.5mM MgCl^ (65% sucrose-TM). Aliquots
of the crude nuclear pellet were overlayed with 6 ml of 60% sucrose-TM,
12 ml of 55% sucrose-TM, 6 ml of 50% sucrose-TM and 7 ml of 30% sucrose-
TM to form a discontinuous sucrose gradient. The gradient(s) was
subsequently spun for 1 hr at 72,100 x g in a Beckman SW27 rotor.
max
Nuclei banding at the 65%-60% and 60%-55% sucrose interfaces were
removed, combined, and diluted to 50% sucrose with TM buffer. Using
an SW27 rotor (72,100 x g for 1 hr) these nuclei were sedimented
max
through another discontinuous gradient containing 12 ml of 55% sucrose-
TM, 3 ml of 60% sucrose-TM and collected onto a cushion of 3 ml 65%

15
sucrose-TM. Material caught on the 65% sucrose-TM cushion was removed,
diluted to 10% sucrose with distilled water, and pelleted in an HB-4
rotor at 1020 x gmax for 5 min. This pellet is designated the purified
nuclear fraction.
In order to bring about nuclear lysis the purified nuclei were
resuspended in 1 ml of lOmM Tris (pH 8.5), containing O.lmM MgCl^.
After 20 min in lOmM Tris, O.lmM MgCl,,, DNase I and RNase A were each
added to a final concentration of 100ug/ml and the incubation continued
for 20 min at 22°C. Following lysis (which could be monitored with
phase optics) four volumes of 60% (w/w) sucrose, 50mM Tris (pH 7.6),
500mM MgCl,, were added to the lysed nuclei. The solutions were
thoroughly mixed and the suspension placed in the bottom of a Beckman
SW41 centrifuge tube. A linear 20%-45% (w/w) sucrose gradient con¬
taining 50mM Tris (pH 7.6) and 500mM MgCl2 was formed on top of the
sample and the gradients spun for 2 hr at 153,244 x gmax in a Beckman
SW41 rotor. Nuclear envelopes banded at 1.19 g/cc (35% sucrose in
500mM MgCl,,). The purified NE's were diluted with 50mM Tris (pH 7.6)
500mM MgCl,, and then pelleted in a Beckman fixed angle 40 rotor for
30 min at 130,766 x gn]ax- The NE's were subsequently resuspended in
10% sodium dodecyl sulfate (SDS), lOmM Tris (pH 8.5), 1% B-Mercapto-
ethanol if they were to be analyzed by electrophoresis, or in 10%
sucrose, lOmM Tris (pH 7.6), 0.5mM MgCl2 if chemical or enzymatic
analysis was to be performed on the sample.
Isolation of Plasma Membrane
Plasma membrane was isolated by a slight modification (McClure
and Noonan, 1978) of the aqueous two phase polymer technique of Brunette
and Till (1971).

16
Chemical Assays
All protein determinations were performed according to the pro¬
cedure of Lowry et al. (1951). Phospholipid phosphorus (Rouser et al
1966) was measured in chloroform-methanol extracts (Rouser and
Fleischer, 1967) of various subcellular fractions. Phospholipid
content of the various fractions was extrapolated from the phosphorus
determination by multiplying the phosphorus value by a factor of 25.
Cholesterol was determined according to Glick et al. (1964). In
order to determine the RNA and DNA content of the NE fraction, cells
3
were grown for five generations in the presence of 0.5pCi/ml [ H]
thymidine or 0.4pCi/ml [ H] uridine prior to preparation of NE. The
specific radioactivity of DNA or RNA (cpm/pg nucleic acid) was deter¬
mined from the cell homogenate by liquid scintillation counting and
spectrophotometric assay. DNA content was determined by the diphenyl
amine assay (Burton, 1956) while RNA content was determined by the
orcinol (I-San Lin and Schjeide, 1969) assay of extracts prepared
according to Fleck and Munro (1962). The specific radioactivity
obtained for DNA and RNA was then used to determine the relative
nucleic acid content of nuclei and NE.
Enzyme Assays
G1ucose-6-phosphatase activity was determined according to
Franke et al. (1970) with the inorganic phosphate release being mea¬
sured according to Fiske and Subbarowe (1925). 5'-Nucleotidase was
assayed via the technique of Widnell and Unkless (1969) with the
inorganic phosphate released being measured by a modification of the
technique described by Chen et al. (1956). Succinate dehydrogenase

17
was assayed according to Veeger et al. (1969). Cytochrome c oxidase
was assayed according to Smith (1955).
Electron Microscopy
Nuclei and NE's were fixed (0°C, 2 hr) in 1.8% glutaraldehyde,
8% sucrose, 50mM phosphate (pH 7.6), and 2.5mM MgCl^. Both the nuclei
and NE fractions were postfixed in the same buffer containing 1%
osmium tetroxide (22°C, 1 hr). Samples were sequentially dehydrated
in increasing concentrations of ethanol and finally embedded in Spurr's
low viscosity medium (1969). Thin sections were stained with uranyl
acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965)
and then examined in an Hitachi AS8 electron microscope.
SDS Polyacrylamide Disc Gel Electrophoresis (PADGE)
All electrophoretic analyses were performed in 1.5mm thick slab
gels according to the procedure of Laemmli (1970). The gels used were
composed of a 5.6% acrylamide stacking gel overlaying a linear 7.5%
to 12.5% acrylamide gradient separation gel. The acrylamide ratio to
bis-acrylamide was maintained at 37.5:1 in both the stacking and
running gel. Purified NE's were solubilized by boiling at 100°C for
5 min in 10% SDS, lOmM Tris (pH 8.5), 1% B-mercaptoethanol (gME).
Plasma membrane was solubilized by resuspension and boiling in 2% SDS,
62.5mM Tris (pH 6.8), 10% glycerol, and 0.1% |3ME ("sample buffer").
Lysed nuclei, sampled after DNase I and RNase A digestion, and whole
cell homogenates were solubilized by boiling in one volume of 2x
"sample buffer." Each of the samples were then exhaustively dialyzed
against "sample buffer" containing 0.1% 6-mercaptoethanol and O.lmM
phenylmethylsulfinyl fluoride (PMSF). Inclusion of PMSF in the sample

18
buffer is essential since we have found that NE's dissolved in sample
buffer lacking PMSF are subject to extensive proteolysis, possibly
as the result of the co-purification of an intrinsic protease with
the purified envelopes (Carter et al., 1976).
Following electrophoretic separation of the membrane peptides
and glycopeptides, the slabs were fixed for 30 min in 10% trichloro¬
acetic acid and then allowed to equilibrate overnight in 25% ethanol,
8% acetic acid. The following day the gels were stained with Coomasie
brilliant blue according to Weber and Osborn (1969) and finally
destained in 25% ethanol, 8% acetic acid.
Results
Isolation of Nuclei
The primary goal which I set for myself prior to isolating NE's
was that the purified NE's contain both the inner and outer membrane
bilayer as well as the pore complex and be free of contaminating cyto¬
plasm or nucleoplasm. In order to achieve this goal, a nuclear
fraction had to be isolated which itself contained both the inner and
outer nuclear membranes and was free of substantial cytoplasmic con¬
tamination. The nuclear isolation procedure which I have employed in
my work has been described in detail in the Materials and Methods
section and is presented diagrammatically in Figure 2.
In many of the nuclear isolation procedures previously published
workers have used nonionic detergents to remove cytoplasmic components
(Muramatsu, 1970) from the nuclei. Unfortunately the use of such
detergents on isolated nuclei from rat liver has been shown to remove
almost all of the nuclear membrane phospholipid and much of the protein

Cells
J
homogenate
Supernatant pellet
gradient 60%-65% sucrose
(DISCARD) interfaces
Remainder of
gradient
(DISCARD)
60%-65% sucrose
interface
Dounce homogenize in hypotonic buffer
at 2-4 x 10' cells/ml. Dilute with
2 volumes 65% sucrose, 50mM Tris
(pH 7.6), 7.5 mM MgCl2.
Spin 4936 x gm . 30 min
max
Resuspend in 65% sucrose TM and place
at the bottom of a SW27 gradient.
Spin 72,100 x gmax, 1 hr
Remove nuclei from interfaces.
Dilute to 50% sucrose TM, and place
in a SW27 gradient.
Spin 72,100 x g , 1 hr
3max
Remove nuclei from 60%-65% sucrose inter¬
face, dilute with distilled water to 10%
sucrose

Spin 1020 x g
5 min
max’
Supernatant pellet
Supernatant pellet
(DISCARD)
Purified Nuclear Fraction
Lyse in lOmM Tris (pH 8.5), O.lmM MgCl2»
nuclease digest, dilute with 60% sucrose,
50mM Tris (pH 7.6), 500mM MgClz and place
at the bottom of a SW41 gradient.
Spin 153,244 x gmax, 2 hr
Remove NE (band at 1.19 g/cc). Dilute
with 50mM Tris (pH 7.6), 500mM MgC^.
Spin 130,766 x 9max> 30 min
Purified NE Fraction
Figure 2. Flow Diagram of Nuclei and NE Isolation Procedure
rv>
o

21
(Aaronson and Blobel, 1974). For this reason I avoided the use of any
detergents in my nuclear isolation procedure. Instead, I developed
homogenization conditions which minimized nuclear clumping and adhesion
of cytoplasmic material to the nuclei. Specifically I found that
immediate dilution of the cell homogenate with 65% (w/w) sucrose, 50mM
Tris (pH 7.6), 7.5mM MgCl2 (Figure 2) maintained the nuclei in a
dispersed state. Isolation of "clean" nuclei from such an homogenate
could then be readily accomplished by density gradient centrifugation
if the nuclei were maintained under the salt and pH conditions outlined
in Figure 2 and handled with care so as to avoid premature lysis.
In the procedure which I developed (Figure 2) the whole cell
homogenate is diluted with 65% sucrose in 50mM Tris (pH 7.6), 7.5mM
MgCl,, and spun for 30 min at 4,936 x g . This differential centri-
fugation serves to collect nuclei in a pellet while leaving most other
membranous organelles in the supernatant. This crude nuclear pellet
is subsequently subjected to two sequential centrifugations on
sucrose step gradients (Figure 2) in which purified nuclei band at
the 60-65% sucrose interface. The purified nuclei are subsequently
diluted with distilled water (to give a final sucrose concentration
of 10%) and then collected as a pellet by a 5 min, low speed, differ¬
ential centrifugation step (Figure 2).
Three precautions must be taken to ensure the isolation of a
"clean" nuclear fraction from CHO cells, (a) Homogenization must
be performed at pH 7.6 in the absence of divalent cations, (b)
Homogenization, resuspension, and mixing of fractions must be done
very carefully so as to avoid premature lysis or shearing of the

22
nuclei. Breakage of the nuclei at early stages in the isolation pro¬
cedure inevitably results in nuclear clumping with concomitant
entrapment of cytoplasmic and nucleoplasmic contaminants, (c) Homog¬
enization, resuspension, and mixing must be carefully monitored to
insure oneself that the nuclei are adequately dispersed. Failure to
achieve a relatively homogeneous mixture of nuclei inevitably results
in failure of the subsequent centrifugation steps to remove cytoplasmic
contaminants.
Morphology of the Purified Nuclear Fraction
As can be seen in the phase contrast micrograph presented in
Figure 3, nuclei purified via the procedure outlined in Figure 2 are
intact, contain distinct nucleoli, and show few cytoplasmic "tags."
Electron micrographs of the purified nuclear fraction show no obvious
contamination of the nuclei with mitochondria, RER, vesicles or large
sheets of plasma membrane (Figure 4A). Although rough or smooth
microsomes are not immediately detectable in the nuclear fraction,
it must be pointed out that the outer nuclear membranes of some nuclei
are blebbed. It is possible that these blebs could represent micro¬
somal contamination. However it is more likely that the blebs simply
represent swelling of the outer nuclear membrane. A higher magnifi¬
cation micrograph (Figure 4B) of nuclei isolated in the "purified
nuclear pellet" (Figure 2) clearly demonstrates that both the inner
(arrow) and outer (double arrow) nuclear membranes are present on
the isolated nuclei and that very few ribosomes are attached to the
outer membrane. Furthermore, the isolated nuclei have obvious pores
(triple arrow) associated with them.

23
Figure 3. Phase Contrast Micrograph of the Purified
Nuclear Fraction. (xl340)

Figure 4. Electron Micrograph of the Purified Nuclear Fraction
Purified nuclei were fixed, embedded, and stained as
described in Materials and Methods.
A. Representative section through "purified nuclear
fraction” (x3775).
B. High magnification EM of two randomly chosen nuclei
with the attention being directed to the nuclear
membranes (x24,350) (]]') outer bilayer; (T) inner
bilayer; CfTT) P°res-

25

26
Isolation of NE
The NE isolation procedure described in the Materials and Methods
section is a combination and modification of the procedures published
by Kay et al. (1972) and Monneron et al. (1972) for the isolation of
NE from rat liver nuclei.
As outlined in Figure 2, the NE isolation procedure begins with
the "purified nuclear fraction." In the isolation method used the
purified nuclei are lysed in lOmM Tris (pH 8.5) containing O.lmM MgC^.
Incubation of the nuclei in this hypotonic buffer for 20 min at 0°C
produces a very viscous solution which is subsequently incubated with
DNase I and RNase A for 20 min at 22°C. Addition of the respective
nucleases immediately reduces the viscosity of the lysed nuclei.
Following nuclease digestion, the material is diluted with 4 volumes
of 60% sucrose, 500mM MgCl2 (to remove residual chromatin [Monneron
et al., 1972]) in 50mM Tris (pH 7.6) and the mixture vortexed.
During the ensuing centrifugation (SW41 rotor at 153,244 x 9max>
2 hr), NE float up into the gradient leaving nucleic acid, soluble
proteins of the nucleoplasm, and unlysed nuclei in the dense load
zone. Under the centrifugation conditions used, the NE fraction bands
in the gradient at a density of approximately 1.19 g/cc. The NE are
subsequently removed from the gradient and pelleted (Figure 2).
Morphology of the Purified NE Fraction
Electron micrographs of sections through the purified NE frac¬
tion (Figure 5) demonstrate that most, if not all, of the isolated
nuclear membranes retain the double membrane sheets (single arrow)
characteristic of the NE. Furthermore, as would be expected if one

27
Figure 5.
Electron Micrograph of the
Purified NE Fraction
The NE fraction was fixed, embedded, and stained
as described in the Materials and Methods
section (x31,850). Regions of double membranes
Cf) can be seen as well as pores in tranverse
(-j*]') and tangential section (f'f'p.

28
were isolating intact NE's, nuclear pores can be seen in both trans¬
verse (double arrow) and tangential section (triple arrow). Clearly
the presence of pores and double membranes in this purified fraction
supports its identify as a NE fraction.
Few, if any, vesicles are apparent in this preparation (Figure 5),
suggesting little or no microsomal contamination. Similarly no mito¬
chondria or unbroken nuclei are observed in this "purified NE fraction."
Recovery of Isolated Fractions
Typical recoveries of protein, phospholipid and ONA obtained
during preparation of the NE are presented in Table I. Approximately
14% and 30% of the homogenate protein and DNA, respectively, are
recovered in the nuclear fraction. Based on the recovery of DNA it
appears that 30% of the total starting nuclei are isolated in the
“purified nuclear fraction" (Figure 2). My work suggests that ^70%
of the starting nuclei are lost during the repeated centrifugations
(Figure 2) which I find to be necessary for preparing clean, intact
nuclei.
As can be seen in Table I the NE fraction contains 8% of the
starting nuclear protein and 50% of the starting nuclear phospholipid
while only 0.3% of the nuclear DNA is recovered in this fraction. If
one assumes that all nuclear phospholipid resides in the nuclear
membranes (Franke, 1974), one can estimate that approximately 50% of
the starting NE are isolated in the "purified NE fraction" (Figure 2).
Characterization and Purity of Nuclear and NE Fractions
In order to further confirm the relative purity of the isolated
nuclear and NE fractions, as well as to determine the composition of

Table I
Recovery of Protein, DNA, and Phospholipid in Subcellular Fractions
Fraction
1 of Total
Cellular DNA
1 of Total
Cellular Protein
% of Total
Nuclear DNA
% of Total
Nuclear Protein
% of Total
Nuclear Phospholipid
Homogenate
100
100
—
—
—
Nuclei
30
14
100
100
100
NE
0.1
1
0.3
8
50
Recoveries were
determined by assay
of individual
fractions sampled during the isolation
procedure.
ro

30
the nuclei and NE's, I have examined the enzymatic activities and
chemical content of both fractions.
Enzyme Activity
The activity of 5'-nucleotidase was examined in homogenate,
nuclei, and NE fractions in order to evaluate possible plasma membrane
contamination of the purified NE fraction. Table II clearly shows
that little plasma membrane (as assayed by 5'-nucleotidase) is found
associated with either the nuclei or NE. Similarly, assays of
succinate dehydrogenase activity (as a marker for mitochondrial con¬
tamination) indicated that neither the nuclear nor the NE fraction
has significant quantities of this enzyme associated with them
(Table II). Cytochrome c oxidase assays of homogenate, nuclei, and
NE indicated that this enzyme was a very minor component of the NE
fraction (Table II). In comparison to values obtained for purified
mitochondria, the NE fraction contains extremely low levels of this
enzyme which could reflect either minor mitochondrial contamination
(Jarasch and Franke, 1974) or that cytochrome c oxidase is a normal
component of the NE (Berezney et al., 1972).
Glucose-6-phosphatase activity, frequently reported to be a
marker of the ER (Kasper, 1974), has been cytochemically identified
in the NE of liver and purified in some NE fractions isolated from
this tissue (see Introduction). Assays of the glucose-6-phosphatase
activities of homogenate, nuclei, and NE fractions from CHO cells
(Table II) indicate that a 2 fold purification of this activity is
obtained in nuclei and NE relative to the homogenate. The lack of
further purification of this enzyme in the NE fraction relative to

Table II
Enzyme Activities of Subcellular Fractions
Homogenate
Nuclei
NE
Mitochondria f
5'-Nucleotidase
(nmoles PO4 = hydrolyzed/
mg protein/hr)
49 (6)
ND
(2)
ND (2)
Succinate Dehydrogenase
(nmoles succinate oxidized/
mg protein/hr)
470 (2)
ND
(2)
ND (2)
Cytochrome c Oxidase
(nmoles cytochrome c oxidized/
mg protein/min)
0.7 (2)
ND
(2)
1.5 (2)
18.0 (2)
G1 ucose-6-Phosphatase
(pmoles PO4 = hydrolyzed/
mg protein/hr)
0.41 (2)
0.75
(2)
0.80 (2)
Each fraction was assayed as described in Materials and Methods, immediately following
isolation of NE fractions. ND — no detectable activity. The minimum detectable activity in succinate
dehydrogenase assays was 50 nmoles succinate oxidized/mg protein/hr and the minimum detectable activity
in 5' nucleotidase assays was 3 nmoles/mg protein/hr. The minimum detectable activity in cytochrome c
oxidase assay was 0.3 nmoles cytochrome c oxidized/mg protein/min. Numbers in parentheses are the number
of assays performed, t Digitonin treated mitochondria, isolated from bovine liver, were obtained from
Dr. T. W. O'Brien.

32
the nuclear fraction may be due to loss or denaturation of the
enzyme by the high salt conditions employed during the latter steps
of our isolation procedure (Kasper, 1974).
Chemical Composition
A preliminary analysis of the relative chemical composition
of the homogenate, nuclei and NE fractions are presented in Table III.
Among the points presented in Table III which should be stressed is
the fact that the "purified NE fraction" does contain small amounts
of residual DNA and RNA. Similar findings with regard to nucleic
acid composition of isolated NE have been reported in previously
published characterizations of NE from liver (Kasper, 1974) and may,
in fact, represent physiologically significant components of the NE.
It should also be noted that, not unexpectedly, if the nuclease
digestions (Figure 2) were omitted from the isolation procedure, sig¬
nificantly larger amounts of DNA and RNA were recovered in the
purified NE fraction.
To confirm the membranous nature of the isolated NE fraction,
phospholipid content was determined in the homogenate, nuclei, and
NE fraction contained 200pg phospholipid/mg protein indicating that
the fraction isolated is relatively protein rich (see pg. 31 of
Fleischer and Kervina, 1974) perhaps due to the proteinaceous lamina
associated with the inner nuclear membrane. In order to demonstrate
that this relatively low phospholipid/protein ratio was not due to
incomplete extraction of phospholipid, NE1s were prepared from cells
which had been maintained for 5 generations in medium containing
O.lpCi/ml [ H] choline. Ninety-seven percent of the [ H] choline

Table III
Chemical Content of Subcellular Fractions
pg DNA/mg pg RNA/mg pg Phospholipid/mg pg Cholesterol/mg
Fraction Protein Protein Protein Protein
Homogenate
90
220
91
—
Nuclei
200
90
30
—
NE
10
20
200
20
PM
117
Individual fractions were sampled during the isolation procedure and assayed for protein,
DNA, RNA, phospholipid or cholesterol as described in Materials and Methods. A plasma membrane
enriched fraction was prepared from CHO cells via the two-phase aqueous polymer technique of
Brunette and Till (1971).
CO
CO

34
present in the NE fraction was extracted by chloroform methanol and
accounted for in subsequent phosphorus determinations {data not
shown).
The cholesterol content of NE and plasma membrane were compared
in order to determine if there were any clear differences in choles¬
terol composition between these two fractions taken from CHO cells.
As can be seen in Table III, five-fold less cholesterol (per mg pro¬
tein) was found in the NE as compared to the PM. These data are in
agreement with previously published work concerning the cholesterol
content of bovine liver NE (Keenan et al., 1970).
Peptide and Glycopeptide Composition
When the purified NE fraction was examined by SDS gel electro¬
phoresis (Laemmli, 1970), a complex Coomasie blue staining profile
was obtained (Figure 6) which was distinguishable from the stained
profiles obtained from whole cell homogenate, purified nuclei and
plasma membrane derived from CHO cells (Figure 6).
The major Coomasie brilliant blue staining components of the
NE fraction ranged in molecular weight from 55,000 to 75,000 daltons.
The majority of the remaining NE peptides and glycopeptides were of
a MW higher than 75,000 daltons. It should be noted (compare nuclear
with NE fraction, Figure 6) that few, if any, peptides are found in
the NE fraction which co-migrate with the histones (arrows) found
in the nuclear fraction again arguing against significant contam¬
ination of the NE with DNA.

35
Figure 6. Coomasie Blue Stained Profiles of Subcellular
Fractions
Fractions were isolated and electrophoresed as
described in the Materials and Methods section.
Seventy-five micrograms of protein were applied
to each slot. From left to right: lane 1,
standards - (phosphoryase A, 100,000 MW; bovine
serum albumin, 69,000 MW; ovalbumin, 43,000 MW;
DNase I, 31,500 MW; soybean trypsin inhibitor,
23,000 MW; and cytochrome c, 13,500 MW); lane 2,
DNase I and RNase A; lane 3, NE; lane 4, nuclei;
lane 5, plasma membrane and lane 6, whole cell
homogenate. Arrows ( ) indicate the region in
which histones migrate in these gels.

CHAPTER III
REFORMATION OF THE NUCLEAR ENVELOPE DURING MITOSIS
Materials and Methods
Materials
All tissue culture materials were purchased from Grand Island
Biological Company (Grand Island, New York). Tissue culture plastic-
ware was purchased from Corning Glass Works (Corning, New York).
3 3
[4,5- H] L-leucine, [methyl- H] thymidine, and ultrapure sucrose
were purchased from Schwarz Mann, Division of Becton, Dickinson and
3
Company (Orangeburg, New York). [Methyl- H] choline chloride and
32
[ P] orthophosphate were purchased from Amersham Searle Corporation
(Arlington Heights, Illinois). All reagents for electrophoresis
were purchased from Bio-Rad Laboratories (Rockville Centre, New
York). All other reagents were obtained from Scientific Products
(Ocala, Florida).
Cell Culture and Synchrony
Chinese hamster ovary cells were grown in suspension culture
as described in Chapter II. Cells were synchronized via a modifica¬
tion of the isoleucine deprivation technique first introduced by
Tobey and Crissman (1972). In our procedure suspension cultures
(usually 6 liters) were allowed to grow to stationary phase
(6-8 x 10 cells/ml) in complete media. The cells were left for 12
to 24 hr at stationary phase and then harvested and resuspended in
36

37
medium containing 10% calf serum, 5% fetal calf serum and ImM
hydroxyurea (HU). Ten hours after the addition of the medium
supplemented with HU (when the majority of the cells were arrested
at the G^/S boundary), the cultures were re-harvested and resuspended
in fresh, complete medium. Immediately upon resuspension in fresh
medium the cells began to traverse the cell cycle. Attempts to
synchronize other CHO subclones using this technique were not
sucessful.
In order to make comparisons between experiments, I have plotted
synchronization curves as fraction of cells divided (N-N0/N0) versus
time. N0 is the number of cells before division and N is the number
of cells at any given time during division.
Labeling of Cell Cultures
Labeling of cell cultures in preparation for isotope dilution
experiments was performed by maintaining cells for 5 generations
prior to synchronization in a particular radioactive precursor (at
the specific activity indicated) and then synchronizing the cultures
3 3
in the presence of either 0.2pCi/ml [ H] leucine, O.lpCi/ml [ H]
32
choline, or 0.4uCi/ml [ P] orthophosphate. In order to determine
the dilution of radiolabel which occurred during the mitotic phase
of the cell cycle, the labeled cell cultures were harvested
immediately prior to entrance into M (i.e. in very late G,,) and
washed twice in PBS, pH 7.2. One aliquot of cells was used to
prepare NE while the remaining cells were returned to culture in
fresh media containing no radioactive precursor. After the cells had
completed M, this aliquot was used to prepare the G^ NE's.

38
3
Pulse label experiments with [ H] leucine were performed by
growing and synchronizing cultures in the absence of radiolabel.
Following release of the cells from HU, the cultures were resuspended
in media containing 3.3pg/liter leucine (25% of the leucine normally
present in F-10 medium). One hour before each sequential NE iso¬
lation, an aliquot of the culture was removed from the synchronized
stock culture and [3H] leucine was added to a final concentration
of 0.2pCi/ml [3H] leucine.
32
Pulse labeling of cultures with [ P] orthophosphate was per-
3
formed as in [ H] leucine pulses except that media added after
release of the cells from the HU block was complete F-10 with no
32
dilution of any component. [ P] Orthophosphate was added to a final
concentration of 0.8pCi/ml.
Isolation of Nuclei and NE
Preparation of nuclei and NE was described in detail in
Chapter II.
Determination of Specific Activities
3
The specific activity of [ H] leucine labeled cell components
was determined by liquid scintillation counting (LSC) and protein
assay according to Lowry et al. (1951) after either solubilization
of the cell component in 10% sodium dodecyl sulfate (SDS), lOmM
Tris (pH 8.5), and 1% g-mercaptoethanol ($ME) and dialysis for 36 hr
against 2% SDS, 62.5mM Tris (pH 6.8), 10% glycerol, 0.1% BME and
O.lmM phenylmethylsulfonyl fluoride (PMSF) or following precipitation
of the isolated component with 10% trichloroacetic acid (TCA). The
specific activity of [ P] or [ H] choline labeled phospholipid was

39
determined from phospholipid phosphorus assays (Rouser et al., 1966)
of chloroform-methanol extracts (Rouser and Fleischer, 1967) of the
specific cell component.
Determination of Precursor Pool Equilibration Rates
Cell cultures were grown for 5 generations in either 0.2pCi/ml
3 32
[ H] leucine or 0.4pCi/ml [ P] orthophosphate. The cells were then
3
synchronized in the presence of 0.2pCi/ml [ H] leucine or 0.4pCi/ml
32
[ P] orthophosphate. Five to six hours after release from HU, an
aliquot of cells was taken from the culture and the initial precursor
pool specific activity was determined as described below. Immediately
after removal of this initial aliquot, the remaining cells were
washed twice with PBS, sampled again, and returned to unlabeled
medium. Each hour for the next 4 hr after being returned to unlabeled
medium, a sample of cells was removed, and the precursor pool specific
activity was determined.
3
To determine the size of the [ H]leucine pool, cells were
collected on a glass fiber filter, washed quickly with 10 volumes of
ice cold PBS, and solubilized in 0.2N NaOH for 5 minutes at 70°C.
The solubilized material was chilled, precipitated with 10% trichloro¬
acetic (TCA) overnight at 4°C and the supernatant collected by cen-
3
trifugation. [ H] Leucine in the supernatant (i.e. the acid soluble
pool) was determined by LSC and total cell protein was assayed
according to Lowry et al. (1951).
Phospholipid precursor pool size was based on labeled phospha-
32
tidic acid. [ P] Phosphatidic acid specific activity was determined
by harvesting cells via centrifugation and immediately extracting

40
the harvested cells with chloroform-methanol (Rouser and Fleischer,
1967). After the Folch back washes of the chloroform-methanol
extract, phosphatidic acid was found in the lower organic phase.
Phosphatidic acid was purified by two sequential separations on
silica gel thin layer chromatography using solvent system I developed
by Skipski and Barclay (1969) for acidic phospholipids. The purified
phosphatidic acid was assayed for phosphorous (Rouser et al., 1966)
32
and the [ P] content determined by LSC.
Measurement of Nuclear Surface Areas
Nuclear surface areas were measured by point and intersection
planimetry on photographs of thick sections of embedded whole cells.
Samples of cells were taken from a synchronized culture 5h and 8h
after release from HU. Samples were fixed in 2% glutaraldehyde, 2%
formaldehyde and 50mM PO^ = (pH 7.4) for 30 minutes at 22°C. Post¬
fixation was performed in 1% OsO^, lOOmM PO^ = (pH 7.4) for 30
minutes at 22°C. Samples were then dehydrated and embedded as
described in Chapter II. Thick sections were cut on a Sorvall micro¬
tome and stained with 0.1% toludine blue 0 in 1% sodium borate for
15 seconds at approximately 200°C. Sections were photographed using
a Wild Mil microscope with camera attachment. The resulting photo¬
graphs were subsequently subjected to planimetry according to
Wei bel (1969).
Polyacrylamide Gel Electrophoresis
All electrophoresis was performed as described in Chapter II.
3
Fluorography of [ H] labeled NE gels was performed according to
Bonner and Laskey (1974).

41
Results
Cell Synchrony
The relative synchrony obtained by the technique outlined in
3
the Materials and Methods section is presented in Figure 7. [ H]
Thymidine incorporation into DNA indicated that following release
from HU, DNA synthesis was initiated immediately and that the syn¬
thetic phase (S pahse of the cell cycle) lasted approximately 5 hr.
A 5 hr S phase is in good agreement with the value previously obtained
by Tobey and Crissman (1972) using these same cells. Concurrent with
cessation of DNA synthesis (i.e. 5 hr after release from HU),
mitotic cells, defined by metaphase figures appeared (Figure 7). The
mitotic phase of the cell cycle was complete in approximately 4-5 hr
(i.e. occurred 5-10 hr after release from HU). During this time
frame the cell population increased by approximately 80% (Figure 7).
It must be pointed out that from Figure 7 it is apparent that the G,,
phase of the CHO cell cycle synchronized by the technique outlined
in Materials and Methods is very short. The fact that mitotic
figures begin appearing as soon as DNA synthesis is terminated
(Figure 7) does not allow accurate measurement of the G^ phase of the
cell cycle. It should also be noted that the maximum mitotic index
at any time after release from HU is 25-30% of the total cell popula¬
tion. Thus, during the 4-5 hr mitotic phase of these synchronized
cells a proportion of the cells are either in the preceding G,, or
have progressed into the G^ phase and are therefore coexisting with
cells in mitosis. Since I well recognize that I do not have 100% of
the cells in mitosis at any one time, I refer to the 4-5 hr time
period during which the cell population divides as the G^-M-G^

42
Figure 7. Cell Cycle Parameters in Synchronized CHO Cells
A suspension culture was synchronized as
described in Materials and Methods. DNA syn¬
thesis was determined by pulsing duplicate 2 ml
aliquots of cells for 10 min at 37°C with
2.6 yCi/ml [3I1] thymidine. Labeled cells were
then washed three times with ice cold PBS, pre¬
cipitated and washed three times with ice cold
10% TCA, and radioactivity in the precipitate
was determined by LSC. Cell number was deter¬
mined from 4 replicate counts using a Levy-
Hausser counting chamber. Mitotic index is
expressed as percent of cells in mitosis
observed in phase microscopy. [^H] Thymidine
incorporation into TCA insoluble material 0-0
cell density A-A ; mitotic index â–¡-â–¡.

43
transition. It is during this transitional period of the ceil cycle
that the NE breaks down and reforms.
Peptide Composition of the NE during Different Phases of the Cell
Cycle
Maul et al. (1972) have reported that changes in the number of
nuclear pores per unit nuclear surface area occur as HeLa cells pro¬
gress through the cell cycle. Riley and Keller (1978) have reported
major morphological changes and minor compositional changes in non-
membranous nuclear ghosts isolated from HeLa cells at various stages
of the cell cycle. Hodge et al. (1977) have also examined polypep¬
tides of the HeLa cell nuclear matrix isolated from synchronized
populations of cells at various stages of the cell cycle. These
authors reported, in agreement with Riley and Keller, that minor
changes in the composition of nuclear matrix could be noted when
nuclear matrixes, isolated from cells in various phases of the cell
cycle, were compared by electrophoresis. Sieber-Blum and Burger
(1977), using CHO cells, have compared NE, isolated from synchronized
cell cultures, by electrophoresis and found no noticeable differences
in the stained gel profiles of cell cycle specific NE fractions.
In an effort to determine whether any changes occur in CHO NE
during the cell cycle which could be detected as changes in the
coomasie blue staining profile of NE peptides and glycopeptides
separated on SDS-PAGE, I have isolated NE from cells at various stages
of the cell cycle. Specifically NE were isolated from a) cells which
were in the logarithmic phase of growth (i.e. primarily G-| cells);
b) cells which had been grown to stationary phase, diluted, allowed
to reinitiate the cell cycle in fresh HU containing media and then

44
collected 10 hr after dilution (i.e. cells at G-j/S boundary, Figure 7);
c) cells 5 hr after release from HU (i.e. cells at the G^/M boundary);
and d) cells 10 hr after release from HU (i.e. cells in early G-j).
The NE's from these four distinct cell populations were then solu¬
bilized in SDS as described in Materials and Methods and equal
amounts of protein applied to individual wells of 7.5 - 12.5% Laemmli
discontinuous SDS-PAGE. As can be seen in Figure 8, coomasie blue
staining of the various isolates showed some differences in the
overall coomasie blue stained profile of NE's isolated from the
different stages of the cell cycle. However the cell cycle related
differences shown in Figure 8 were not consistently seen from exper¬
iment to experiment suggesting that these differences are of
questionable physiologic significance. It is my own bias, based on
observations drawn from a number of experiments identical to that
outlined in Figure 8, that there are no reproducible differences in
the coomasie blue stained profile of NE derived from the four phases
of the cell cycle discussed in this section of the dissertation.
3
Dilution of f Hi Leucine Labeled NE
Using synchronized and uniformly labeled CHO cells I have
examined the dilution of labeled NE in order to follow synthesis of
NE peptides during the G^-M-G^ transition. Such label dilution
experiments were directed at determining whether peptides present in
NE, isolated from early G^ cells (10-12 hr after release from HU),
were synthesized de novo during the G2-M-G1 transition or whether
the peptides present in the early G-j NE pre-existed in the cell
prior to mitosis.

45
~ B a “
-
12 3 4
Figure 8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells
Nuclear envelopes were isolated from cells in
logarithmic growth and from cells at various
stages of the cell cycle. Nuclear envelope
fractions were solubilized in SDS and electro¬
phoresis was performed as described in
Materials and Methods. Lane 1, log phase;
Lane 2, G2/M; Lane 3, M/G-j; Lane 4, G-|/S.

46
Specifically CHO cells were grown for 5 generations in 0.2pCi/ml
3 3
[ H] leucine and then synchronized in the presence of 0.2pCi/ml [ H]
leucine as described in Materials and Methods. In late (5 hr
after HU removal, Figure 7) the cell culture was harvested, washed
twice with PBS and then the 6 liters of cells divided into two equal
aliquots. From one aliquot NE were immediately isolated (described
in Table IV as the G^/M NE population). The remaining cells were
returned to fresh medium free of [ H] leucine but containing 13.2 mg/
liter cold leucine. Four hours later (after 80% of the cells had
completed M, Figure 7) these cells were harvested and NE immediately
isolated (described as the M/G^ NE population in Table IV). The
specific activity of these two NE fractions was then determined and
compared. As can be seen in Table IV, the average specific activity
of the M/G1 NE is approximately 25% less than the specific activity
of the NE's isolated from the G£/M cell population. These data
suggest that although some de novo synthesis of NE protein has
occurred over the 4 hr time span between the isolation of the G^/M
and M/G-| NE, the majority of the peptides present in the M/G^ NE's
pre-existed in the G^/M cells. The data presented in Table IV do
not, of course, take the precursor pool into account. Resolution
of this problem will be discussed below.
The reduction in specific activity broadly described in
Table IV was further investigated by isolating NE at a time point
(6.5 hr after release from HU) at which less than 5% of the cells
have completed M and every hour thereafter until greater than 80%
of the cells have completed M (Figure 9). Cells were grown and

Table IV
Dilution of [JH] Leucine Labeled NE Protein
Specific Activities of NE Protein (cpm/mg protein)
G,/M M/G, Ratio (M/G,:G,/M)
5.3 x 105 3.9
9.3 x 105 6.7
6.3 x 105 4.2
2.7 x 105 2.1
1.4 x 105 1.1
105
0.74
105
0.72
105
0.67
105
0.78
105
0.79
x = 0.74
SD = t 0.05
3
Cells were grown and synchronized in media containing 0.2uCi/ml [ H] leucine and NE
isolated from half of the cell population immediately before synchronous division (G2/M NE).
Four hours later (when n,80% of the cells had completed M) NE were isolated from the other
half of the cell population which had proceeded through M in the absence of [^H] leucine (M/G-| NE).
Specific activities were determined as described in Materials and Methods.

48
3
synchronized in the presence of [ H] leucine as described for the
experiments presented in Table IV. Six and one-half hours after
release from HU (Figure 9), cells were harvested and washed twice
in PBS. One aliquot of cells was used immediately for NE isolation
(G^/M NE), while the remaining cells were returned to culture in
complete medium lacking [ H] leucine. Each hour thereafter, for the
next 6 hr, an aliquot of cells was harvested, NE isolated and the
specific activity of NE protein was determined. The specific
activity of the NE's at each time point is displayed in Figure 9.
Clearly, NE's which are in the late phase of the cycle (8.5 hr
after release from HU) have lost 10% of the label associated with
the envelopes suggesting that during very late G^ labeled components
of the NE are rapidly diluted with unlabeled components. As the
NE's enter M there appears to be a sharp decrease in the rate of
dilution and then as NE's enter G-j a resumption of label dilution
occurs (Figure 9). Dilution of labeled NE peptides is greatly
reduced during that period of time in which the cell population is
dividing when compared to the dilution observed in NE’s isolated from
cells in the G^ and G^ phases of the cell cycle.
In order to determine whether a particular peptide or group of
NE peptides were preferentially lost during the G^-M-G^ transition,
3
[ H] leucine-labeled NE, isolated during the experiments described in
Table IV were solubilized in SDS, labeled peptides and glycopeptides
separated on a discontinuous SDS-PAGE, and the profile examined by
fluorography. Figure 10 contains both the coomasie blue stained
profile of the SDS-PAGE and the fluorogram obtained from the labeled

3
Figure 9. Dilution of [ H] Leucine Labeled NE Protein
A suspension culture was grown to stationary
phase in the presence of 0.2pCi/ml [3h] leucine
and synchronized as described in Materials and
Methods in 0.2pCi/ml [3h] leucine. Six and one-
half hours after release from HU NE were prepared
from an aliquot of cells. The remaining cells
were washed twice with PBS and returned to
culture in complete medium lacking [^H] leucine.
Nuclear envelopes were isolated each hour after
removal of [3h] leucine and specific activities
of each fraction were determined as described
in Materials and Methods. Fraction of cells divided
was determined from 4 replicate countings in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0-0; fraction divided

time after release
FROM HU (HRS.)
3H CPM X1Ó5/ mg PROTEIN
{j)-\ ' i i | ' n i | i | i | i i i
FRACTION DIVIDED
I 1 I
OD (D
cn
o

Figure 10. SOS Gel Electrophoresis of NE Peptides Isolated
After Removal of pH] Leucine at the G2/M
Boundary
A cell culture was grown for 5 generations in
0.2uCi/ml [3H] leucine and synchronized in
0.2yCi/ml pH] leucine as described in Materials
and Methods. Nuclear envelopes were isolated
before label removal (G2/M interface, 7 hr after
removal of HU) and after division in the absence
of label (M/Gi interface, 11 hr after removal of
HU). The isolated NE were solubilized in SDS,
electrophoresed and fluorographed as described
in Materials and Methods. (A) Fluroograph of
NE peptides isolated at G2/M (lane 1) and M/G-j
(lane 2) interfaces. Equal numbers of counts
(31,000 cpm) were applied to the gel. (B)
Coomasie blue stained profiles corresponding
to the fluorogram in A. The G2/M (lane 1)
fraction contained 75yg of protein while the
M/G-] (lane 2) fraction contained 50yg protein.

52

53
components. From the fluorogram presented in Figure 10 it is apparent
that, within the detection limit of the fluorographic process and
one dimensional gel electrophoresis, no individual NE peptide or
glycopeptide is preferentially lost during the G^-M-G^ transition.
3
Incorporation of the T Hi Leucine into NE Protein
One likely, if not the most likely, explanation for the reduced
specific activity found in M/G^ NE's relative to G^/M NE's (Table IV)
is dilution of the pre-existing (i.e. G^/M) label with unlabeled NE
components synthesized and inserted into the NE during either G^, M,
3
or G^ phase of the cell cycle after removal of [ H] leucine from the
culture medium. The data presented in Figure 9 suggests that some
dilution of label occurs in G,, immediately after removal of [ H]
leucine from the culture medium as well as in G^. In order to
determine whether this dilution results from phase-specific biosyn¬
thesis of NE peptides, cells were synchronized as described above and
then 5.25 hr after release from HU an aliquot of cells was taken and
pulsed for 1 hr in medium containing 0.2pCi/ml [ H] leucine but only
25l of the standard F-10 leucine concentration. After 1 hr in labeled
precursor an aliquot was taken from the homogenate and the NE was
isolated. Cells were pulsed with 0.2yCi/ml [ H] leucine and NE iso¬
lated every hour from 5.25 - 11.5 hr after release of cells from an
HU blockade (Figure 11).
The specific activity of each NE and homogenate fraction was
determined and the specific activities plotted as a function of time
after release from HU block. As can be seen, the specific activity
of the NE fractions remain relatively constant (± 5%) from 6 - 9 hr

3
Figure 11. Incorporation of [ H] Leucine into NE Protein
A suspension culture was grown to stationary
phase and synchronized as described in Materials
and Methods. The culture was released from HU
blockade into media containing 25% of the normal
F-10 leucine concentration. One hour before
synchronous division (i.e. 5.25 hr after removal
from HU), an aliquot of cells was removed, pulsed
with 0.2yCi/ml [^H] leucine for one hour and
harvested for NE isolation. Each hour thereafter,
an additional aliquot of cells was pulsed for 1 hr
with 0.2yCi/ml [^H] leucine, NE isolated, and the
specific activity determined on each NE and
homogenate fraction. Fraction of cells divided
was determined from 4 replicate countings in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0-0; homogenate specific
activity A-A ; fraction dividedn-Q.

TIME AFTER RELEASE
FROM HU (HRS.)
3H CPM x105/ mg PROTEIN
ru gj

56
after release from HU (by which time approximately 40% of the cells
in the population have divided). However between 9 and 10 hr after
release from HU when the majority of the cell population is in the
early portion of the G-| phase of the cell cycle (Figure 11), incor-
3
poration of [ H] leucine into the NE's increased 1.25 times that
3
seen in M and by 11 hr after release, incorporation of [ H] leucine
into the NE is 1.56 that seen in M. Taken together, the data in
Figure 9 and Figure 11 suggest that the majority of NE synthesis (and
consequently dilution of pre-existing label) occurs in late G,, and
early to mid G^, with NE specific synthesis in M being low relative
to either of these time periods.
Determination of acid soluble [ H] leucine counts (cpm/mg protein)
in the whole cell homogenate at each time point clearly demonstrated
that the increase in specific activity of NE and homogenate protein
seen in G^ was not due to increased transport of labeled precursor
but rather was due to enhanced de novo biosynthesis.
To determine whether specific peptides of the NE might be
preferentially synthesized during the G,,-M-G.| segment of the cell cycle
NE fractions, obtained from the time points taken in Figure 11, were
solubilized in SDS and the peptide and glycopeptide composition dis¬
played by coomasie blue staining and fluorography on a 7.5 - 12.5%
discontinuous SDS-PAGE. The fluorogram presented in Figure 12A
indicates that, at the limits of detection by one dimensional SDS gel
electrophoresis and fluorography, all NE peptides are synthesized at
some point in the G^-M-G-j transition. Comparison of the coomasie
blue stained profile (Figure 12B) with the intensities of the individ¬
ual bands in the exposed fluorogram (Figure 12A) suggests that the

3
Figure 12. SDS Gel Electrophoresis of [ H] Leucine Pulse Labeled
NE Peptides
Nuclear envelopes isolated during the experiment
described in Figure 11 were solubilized and subjected
to electrophoresis and fluorography as described in
Materials and Methods. (A) Fluorogram depicting
[3H] leucine labeled NE isolated at the six time
points indicated in Figure 11. Lane 1, 5.75 hr
after HU removal; Lane 2, 6.75 hr after HU removal;
Lane 3, 7.75 hr after HU removal; Lane 4, 8.75 hr
after HU removal; Lane 5, 9.75 after HU removal;
Lane 6, 11 hr after HU removal. Equal numbers of
counts (11,000 cpm) were applied to each lane.
(B) Coomasie blue stained profile corresponding
to the fluorogram in (A). Lane 1, 72pg protein;
Lane 2, 74pg protein; Lane 3, 76yg protein; Lane 4,
71yg protein; Lane 5, 56yg protein; Lane 6, 42yg
protein.

58

59
NE peptides were labeled approximately in proportion to their relative
staining with coomasie blue.
Dilution of r^H~| Choline Labeled NE
Using [ H] choline as a precursor of phosphatidyl choline, I
have examined the relative reutilization of NE phospholipids during
3
the G^-M-G^ transition. As in the work with [ H] leucine labeled NE,
I have initially followed the dilution of label at two time points
within the cell cycle, the so-called stage and the M/G-| stage of
the cell cycle (Table V).
Chinese hamster ovary cell cultures were grown for 5 generations
3
in medium supplemented with O.lyCi/ml [ H] choline and then syn-
3
chronized in complete medium containing O.lpCi/ml [ H] choline. Five
hours after release from HU (i.e. at the G^/H transition) the labeled
culture was harvested, washed twice with PBS, and divided into equal
aliquots. G^/M NE were prepared from one aliquot while the other
aliquot was returned to culture in complete F-10 medium without
3
[ H] choline but containing 0.7 mg/liter cold choline. Following
division (i.e. 9 hr after release from HU, see Figure 7), M/G^ NE
were prepared and chloroform-methanol extracts (Rouser and Fleischer,
1967) were prepared from both preparations. The specific radio¬
activity of the G^/M NE and the M/G^ NE phospholipid (cpm/pg lipid
phosphorus) was determined and the specific activities compared
(Table V).
The simplest interpretation of the data in Table V would suggest
that approximately 65% of the M/G. NE phospholipid was present in the
cell prior to mitosis while 35% of the M/G^ phospholipid was

Table V
Dilution of Labeled NE Phospholipids
NE Phospholipid Specific Activity (cpm/yg lipid phosphorus)
Radiolabel
[%] choline
[32pj orthophosphatet
G2/M
2.0 x lCf
0.8 x 104
M/G
1.4 x 10^
0.5 x 104
Ratio (M/G-j :G2/M)
0.70
0.63
x = 0.67
Cells were grown and synchronized in media containing 0.1yCi/ml [^H] choline or 0.4pCi/ml [ P]
orthophosphate and NE isolated from half of the cell population immediately before synchronous
division (G2/M NE). Four hours later (when^805i of the cells had completed M) NE were isolated
from the other half of the cell population which had proceeded through M in the absence of
label (M/G-| NE). Specific activities were determined as described in Materials and Methods,
t [32p] data were calculated from experiment shown in Figure 13.
CD
O

61
synthesized during the G^-M^ transition. Reutilization of label
is not considered in these data but will be discussed below.
32
Dilution of [ P] Orthophosphate Labeled NE Phospholipid
Since choline labels a specific phospholipid which could con¬
ceivably behave differently than the majority of the NE phospholipid,
I felt it important to determine the dilution of total NE phospho¬
lipid. To do this I chose to label cells to constant specific
32
activity with [ P] orthophosphate. In these experiments CHO cell
cultures were maintained and synchronized in complete F-10 medium
32
supplemented with 0.4pCi/ml [ P] orthophosphate. As in the previous
radiolabel dilution experiments (Tables IV and V, Figure 9), the
culture, labeled to constant specific activity with [ P], was
harvested approximately 6.5 hr after release from HU (at the
transition) and washed twice with PBS. One aliquot of the washed cells
was immediately used for preparation of NE while the remaining cells
32
were returned to complete medium lacking [ P] and allowed to proceed
through M. Each hour after the cells were returned to unlabeled media,
a percentage of the cells was harvested, homogenate prepared, and NE
isolated. After the last isolation was completed (11.5 hr after
release from HU, Figure 13), phospholipid was extracted into chloro¬
form methanol, phosphorous anlaysis was performed on each fraction,
and the NE phospholipid specific activity (cpm/ug lipid phosphorus)
determined. Figure 13 describes the time course of the change in
phospholipid specific activity in cell homogenate and the NE fraction.
Clearly the specific activity of both the NE and homogenate remains
relatively constant while the majority of the cells are in late G,,

Figure 13. Dilution of [^P] Labeled NE Phospholipid
A suspension culture was grown to stationary phase
in the presence of 0.4pCi/ml [32p] orthophosphate
and synchronized as described in Materials and
Methods in 0.4pCi/ml [32p] orthophosphate. Six
and one-half hours after release from HU NE were
prepared from an aliquot of cells. The remain¬
ing cells were washed twice with PBS and returned
to culture in complete medium lacking [32p].
Nuclear envelope and homogenate were isolated each
hour after removal of [32p] and phospholipid
specific activities were determined on each frac¬
tion as described in Materials and Methods.
Fraction of cells divided was determined from 4
replicate countings in a Levy-Hausser counting
chamber. Nuclear envelope specific activity
0-0; homogenate specific activity A-A; fraction
divided D-D .

P CPM x 1Ó In g LIPID PHOSPHORUS
TIME AFTER RELEASE
FROM HU (HRS.)

64
and M and then begins to drop as the cells enter . After 80% of
the cells have completed division (Figure 13, 11.5 hr after release
from HU), the specific activity of the NE is approximately 65% of
the NE phospholipid specific activity before M (Figure 13 and Table V).
The simplest explanation for these data is that 65% of the phospho¬
lipid present in NE 12 hr after release from HU (i.e. early , see
Figure 13) was present in the cell prior to M.
Interestingly the specific activity of the NE phospholipid
begins to decrease after 20-30% of the cells have entered the G^ phase
of the cell cycle whereas the specific activity of the whole cell
phospholipid drops only after ^80% of the cells have completed M
(i.e. the majority of the cells are in early to mid G^. The data
in Figure 13 clearly suggest that synthesis of phospholipid to be
incorporated into NE and other cellular phospholipid increases as the
cel 1s enter G^.
32
Incorporation of [ P] Orthophosphate into NE Phospholipid
In order to confirm that the dilution of NE phospholipid specific
activity, seen in Table V and Figure 13, is due to synthesis of new
phospholipid, which is, in turn incorporated into NE, cell cultures
were grown to stationary phase and then synchronized as described
previously in complete medium containing no label. Seven hours after
release from HU (i.e. at the G^/M transition, Figure 14), an aliquot
of cells was removed from the stock culture and pulsed for 1 hr with
32 32
0.8pCi/ml [ P] orthophosphate. After one hour in [ P], the labeled
culture was harvested and both NE and homogenate prepared. Similarly
treated aliquots of cells were pulsed for 1 hr with [ P] over the

65
next 5 hr. The specific activity of phospholipid was then deter¬
mined by chloroform-methanol extraction and phosphorus analysis on
32
each homogenate and NE fraction. The time course of [ P] incorpora¬
tion into NE and homogenate phospholipid can be seen in Figure 14.
As would have been predicted from Figure 13, after 30-40% of the
cells have completed division, the homogenate and NE phospholipid
specific activity begins to rise. This increase in specific activity
of the two fractions remains linear over the remainder of the time
course studied. These data (Figure 14) clearly suggest that a major
fraction of the phospholipids which are destined to become part of the
NE are synthesized and incorporated into the organelle during early .
Rate of Precursor Pool Equilibration
To more rigorously interpret the label dilution data described
throughout the previous experiments (Tables IV and V and Figures 9
and 13), I examined the kinetics of precursor pool equilibration in
synchronized cultures which were manipulated in a fashion identical
to that already reported in Tables IV and V and Figures 9 and 13.
Such data is, of course, essential if I am to make more precise
estimates of the amount of NE peptide and phospholipid synthesis (and
subsequently dilution of pre-existing label) which occurs during the
G^-M-Gi transition in our synchronized cultures.
In the experiments presented in Figure 15, CHO cells were
grown for 5 generations in medium supplemented with 0.2pCi/ml [ H]
leucine (i.e. until the peptides in the cell had reached a constant
specific activity) and then synchronized as described in Materials
and Methods in the presence of 0.2pCi/ml [ H] leucine. Five hours

32
Figure 14. Incorporation of [ P] into NE Phospholipid
A suspension culture was grown to stationary
phase and synchronized as described in Materials
and Methods. Seven hours after removal of HU an
aliquot of cells was removed, pulsed with 0.8pCi/ml
[32P] orthophosphate for one hour and NE isolated.
Each hour thereafter (for 6 hr) another aliquot of
cells was pulsed with 0.8pCi/ml [32p] for 1 hr and
NE isolated. Phospholipid specific activity was
determined on NE and homogenate isolated after
each pulse. Fraction of cells divided was deter¬
mined from 4 replicate countings in a Levy-Hausser
counting chamber. Nuclear envelope specific
activity 0-0; homogenate specific activity A-A ;
fraction divided

TIME AFTER RELEASE
FROM HU (HRS.)
FRACTION DIVIDED

68
after release from HU an aliquot of cells was removed from the culture,
collected on a glass fiber filter, quickly washed with 10 volumes of
ice cold PBS and solubilized in 0.2 N NaOH. The remaining cells were
harvested, washed twice with PBS (as was done in all my previously
presented dilution data). Immediately after the PBS washes (6 hr
after HU removal) another aliquot of cells was collected on a glass
fiber filter, washed and solubilized. The remaining cells were
returned to complete media without [ H] leucine. Each hour after
returning the cells to unlabeled media an aliquot of cells was
collected, washed, and placed in 0.2 N NaOH. After all the samples
had been collected (10 hr after removal from HU, 90% of the cells
having divided, Figure 15), the filters were heated at 70°C for 5 min,
cooled on ice, and precipitated overnight in 10% TCA (4°C). The
specific activity of the TCA soluble [ H] leucine counts was then
determined and plotted as a function of time (Figure 15). As can
be seen in Figure 15, approximately 55% of the [ H] leucine, which
was in the cytoplasmic precursor pool before the cells were harvested,
was removed during the initial two PBS washes. Subsequent culturing
3
of the washed cells in [ H] leucine-free media reduced the soluble
3
[ H] leucine pool to a specific activity with was 30% of the initial
specific activity of the initial time point (Figure 13).
3
By first setting the initial, pre-PBS wash, soluble [ H] leucine
pool at 100% (initial time point, Figure 15) and then averaging the
fractional value of this initial pool which was measured during the
G^-M-G^ transition in the absence of label, I have calculated a mean
soluble [ H] leucine pool specific activity during the G2-M-G-|. This

3
Figure 15. [ H] Leucine Pool Dilution
A suspension culture was grown for 5 generations
in 0.2pCi/ml [3h] leucine and synchronized in
0.2yCi/ml [3h] leucine as described in Materials
and Methods. Five and one-half hours after HU
removal a sample was taken and acid soluble [^H]
leucine determined as described in Materials and
Methods. The remaining cells were washed 2 times
with PBS and another sample taken for determina¬
tion of TCA soluble [3h] leucine counts. The
washed cells were then returned to unlabeled
media and samples taken each hour for determination
of TCA soluble [3H] leucine counts. Fraction of
cells divided was determined from 4 replicate
countings in a Levy-Hausser counting chamber.
Acid soluble [^H] cpm/mg total protein 0-0;
fraction dividedO-O .

TIME AFTER RELEASE
FROM HU (HRS)
ACID SOLUBLE 3H CPM X104 / MG. TOTAL PROTEIN

71
calculation results in a mean value equal to 36% (average of two
3
experiments) of the [ H] leucine found in the soluble pool of cells
prior to removal of label. This mean value represents the percentage
of [ H] leucine in the soluble pool of cells dividing in the absence
of label relative to those cells (before PBS washes) whose cytoplasm
had reached equilibrium with [ H] leucine in the medium. Since the
leucine pool size has been shown to remain constant in HeLa cells
during this phase of the cell cycle (Robbins and Scharff, 1966), I
have concluded that the protein synthesized following removal of
[ H] leucine from the medium, is synthesized with a specific activity
that is, on the average, 36% of the specific activity of proteins
3
synthesized when [ H] leucine was maintained in the culture.
Using the formula below (which is derived in the appendix), I
have estimated the amount of early G-j NE protein synthesized during
the G^-M-G^ transition. I have based these calculations on the ratio
of specific activities of G2/M NE to M/G^ NE presented in Table I.
P =
SAG1 “ SAG2
SA' - SA
G2
where: p = proportion of G^ NE synthesized during G^-M-G^,
SAgi = specific activity of H/G^-NE
SAg2 = specific activity of G^/H-NE
SA' = relative specific activity of pool after label removal
Setting SAG2 equal to unity gives SAG1 a value of 0.74 (Table IV).
Substituting these values into the equation we have:
= (0-74) - 1.0
p (0.36) - 1.0
= 0.41

72
Thus when one takes into account the fact that the soluble pool
3 3
is not completely depleted of [ H] leucine after removal of [ H]
leucine from the media, one calculates that''-40% of the M/G-j-NE
protein was synthesized during the G2-M-G1 transition. By simple sub¬
traction then ^60% of the M/G1 NE protein must have pre-existed M.
I have used the same experimental design and logic to examine
32
the dilution of the [ P] labeled phosphatidic acid pool in order to
more precisely estimate NE phospholipid biosynthesis during the
G^-M-G-j transition. Phosphatidic acid was chosen since it is a
precursor of phospholipids and is not shunted into other pathways
(Howard and Howard, 1974; Spector, 1972).
In these experiments (Figure 16) CHO cells were grown for 5
32
generations in 0.4 pCi/ml [ P] and then synchronized as described
32
in Materials and Methods in 0.4 pCi/ml [ P] orthophosphate. Six
hours after release from HU (Figure 16) an aliquot of cells was
harvested and immediately extracted with chloroform-methanol to give
me a base value for phosphatidic acid specific activity. The
remaining cells were also harvested, washed twice in PBS and another
aliquot of cell population extracted with chloroform-methanol
(Figure 16, 6.5 hr after HU removal). The remaining cells were then
returned to media containing 60 mg/liter cold phosphate but free of
32
[ P]. As shown in Figure 16, aliquots were harvested from the cell
population each hour through the G^-M-G^ transition and extracted
with chloroform-methanol. After all samples had been extracted, each
was Folch backwashed (Rouser and Fleischer, 1967), filtered, and
concentrated for application to thin layer chromatography plates.

Figure 16. [^P] Phosphatidic Acid Pool Dilution
A suspension culture was grown for 5 generations
in 0.4pCi/ml [32p] and synchronized in 0.4pCi/ml
[32P] as described in Materials and Methods.
Five hours after HU removal an aliquot of cells
was harvested by centrifugation and phosphatidic
acid specific activity was determined as
described in Materials and Methods. The remain¬
ing cells were washed twice with PBS, another
aliquot removed for phosphatidic acid specific
activity determination and the bulk of the
cells returned to unlabeled media. Aliquots
were removed for phosphatidic acid specific
activity determination each hour thereafter
until 90% of the cells had completed division.
Fraction of cells divided was determined from
4 replicate countings in a Levy-Hausser counting
chamber. Phosphatidic acid (PA) specific
activity 0-0; fraction divided .

10
(/)
=5
QC
O
I
CL

? 15-
<
£L
O)
=1
10-
co
•o
X
2
Q.
O
Q.
5-
ni| mj iii| 1111 â– â–  ij in
5 6 7 8 9 10 11
TIME AFTER RELEASE
FROM HU (HRS.)

75
Thin layer chromatography was run in solvent system I of Skipski and
Barclay (1969) for acidic phospholipids. After development, the
phosphatidic acid spot was visualized by spraying with distilled
water, scraped from the plate, eluted from the silica gel with
chloroform-methanol, and rechromatographed in the same developing
solvents. The phosphatidic acid spot was visualized in an iodine
chamber, scraped, and eluted as before. The specific activities of
phosphatidic acid at each time were determined on the eluted material
as described in Materials and Methods.
32
The rate of decrease in the specific activity of [ P] labeled
phosphatidic acid is presented in Figure 16. The arithmetic mean
intracellular specific activity during the G2“M'G1 transition was
calculated to be 38% (average of two experiments) of that found in
32
cells prior to removal of [ P] from the media. Calculating from
32
the observed dilution of [ P] labeled NE phospholipid (Table V) I
find the proportion of M/G^-NE phospholipid synthesized during the
G^-M-G^ transition to be 0.53.
0.67 - 1.0
0.38 - 1.0
0.53
Again by subtraction, I estimate that 50% of the M/G^NE pre-existed
M.
Changes in Nuclear Surface Area
Since a change in total nuclear surface area might reflect
synthesis of NE and at the same time affect the interpretation of
my data, I have determined mean nuclear surface areas in a population
of cells before and after the G^-M-G^ transition (i.e. at 5 hr and

76
8 hr in Figure 7). Cells were synchronized as described in Materials
and Methods and 5 hr after release from HU, an aliquot of cells was
removed, and prepared for sectioning as described in Materials and
Methods. After 75% of the cells had completed division (8 hr after
HU removal) another aliquot of cells was removed and prepared for
sectioning. Thick sections were taken from both cell samples,
stained, and photographed as described in Materials and Methods.
Measurements of the surface area of individual nuclei were made by
planimetry (Weibel, 1969). The nuclei in both cell populations were
2
found to have a mean nuclear surface area of 'â– vlOOp . If we assume
that the mean nuclear surface area after the cells have completed
mitosis represents the mean surface area of "daughter" nuclei only,
then the magnitude of the G-j (8 hr after release from HU) nuclear
surface area relative to G,, nuclear surface area will reflect what
increase, if any, has occurred during the G^-M-G. transition. If no
increase in NE occurred during the G^-M-G^ transition, this would be
reflected by a mean nuclear surface area in G^ which is exactly one-
half of the G^ mean nuclear surface area. My measurements suggest
that the total nuclear surface area has doubled during the G2“M-G^
transition (G^ and G., mean nuclear surface areas are approximately
equal). If such a doubling in nuclear surface area was due entirely
to NE synthesis (i.e. no stretching or expansion of available NE),
I would expect to see a 50% dilution of NE protein during the G^-M-G^
transition. My label dilution experiments have indicated that the G-j
NE peptides have a specific activity which is 60% of the G^ NE while
the G.| NE phospholipids have a specific activity which is 50% that
of the G,, NE. In my opinion both my labeling data and my surface
area data are, within the limits of resolution, complementary.

CHAPTER IV
DISCUSSION
In Chapter II of this dissertation I have described a procedure
for the isolation of highly enriched nuclear and NE fractions from
CHO cells. The nuclei and NE fractions obtained by use of this
procedure have been characterized by light and electron microscopy;
chemical and enzymatic assay; as well as SDS gel electrophoresis.
These characterizations have demonstrated that the nuclei and NE
fractions isolated are distinct from each other and that both frac¬
tions are distinct from the plasma membrane and whole cell homogen¬
ate.
Although electron microscopy and marker enzyme assays indicate
that contamination of the nuclei and NE fraction with other organ¬
elles is very low, the true enrichment of NE in the final pellet as
compared to the homogenate is impossible to determine since no
dependable enzymatic marker has been unequivocally localized to this
organelle in CHO cells. However, it must be again stressed that
the final NE fraction obtained by the procedures outlined in this
dissertation is morphologically similar to the intact NE found in
the cell in that the isolated NE's contain a double membrane bilayer
as well as the pore-lamina complex, thus strongly suggesting that
the isolated procedure described produces a highly enriched NE
fraction.
77

78
The NE fraction isolated from CHO cells is composed primarily
of protein and phospholipid having 200pg phospholipid/mg protein.
This phospholipid/protein ratio suggests that, in comparison to other
isolated cellular membranes (Fleischer and Kervina, 1974), the isolated
NE fraction is relatively protein rich. This finding may derive
from the fact that the isolation procedure described, isolates both
the nuclear membrane and the so-called proteinaceous lamina. The
inclusion of the proteinaceous lamina in the NE fraction would be
expected to increase the protein to lipid ratio.
Examination of the peptide composition of the NE fraction by
SDS gel electrophoresis demonstrates that the major coomasie blue
staining peptides of the NE fraction chromatograph with molecular
weights between 55,000 and 75,000 daltons. These peptides are
undoubtedly related to the pore-lamina and nuclear matrix proteins
identified in rat liver (Dwyer and Blobel, 1976) and HeLa cell
(Hodge et al., 1977) NE's. It is of interest to note that the
majority of the remaining NE peptides chromatograph with a molecular
weight greater than 75,000 daltons. Relatively few coomasie blue
staining bands are found to run at molecular weights below 50,000
daltons and no peptides appear to co-migrate with the histones seen
in the nuclear fraction.
It can not be emphasized too much that although the isolation
technique described required careful handling of material during the
preparation of the nuclei and NE, it does produce material in
sufficient quantity and of sufficient purity to permit biochemical
studies directed at elucidating the molecular basis of both NE
structure and biosynthesis.

79
In Chapter III of this dissertation the NE isolation procedure
(described in Chapter II) has been employed to examine the question
of whether or not cellular proteins and phospholipids which pre-existed
the mitotic phase of the CHO cell cycle are used by the CHO cell in
the reformation of the NE which occurs during telophase.
Sodium dodecyl sulfate gel electrophoresis of NE isolated at
various stages of the CHO cell cycle showed no consistent differences
in peptide or glycopeptide composition which could be assigned to
any phase of the cell cycle. It is of interest to note that I did not
see any cell cycle dependent changes in the peptides migrating between
55,000 and 75,000 MW, as have been reported by Hodge et al. (1977) to
occur in the HeLa cell nuclear matrix. In this regard, the data in
this dissertation agree with those of Sieber-Blum and Burger (1977)
who also did not see cycle specific changes in CHO cell NE's.
In Chapter III of this dissertation,evidence, obtained primarily
from what I have dubbed "label dilution" studies, has been presented
which strongly suggest that at least 60% of the early NE protein
and at least 50% of the early G-j NE phospholipid existed in the cell
prior to mitotic breakdown and reformation (i.e. in late G^). These
data further suggest that the remaining 40% of early G.-NE protein
and the remaining 50% of early G.-NE phospholipid come from de novo
synthesis of NE components. Pulse label experiments suggest that
relatively little NE protein or phospholipid synthesis occurs in M
but rather that the majority of this NE synthesis occurs in late G2
and early G-j. Unfortunately the degree of synchrony obtainable with
such a large number of cells does not allow me to determine whether

80
some NE components are specifically synthesized in the G,,, M, or G-|
phases of the cell cycle. However, my data do indicate that at some
point in the G^-M-G^ transition all of the NE peptides separable on
3
a one-dimensional SDS-PAGE are labeled with [ H] leucine suggesting
that no specific component(s) is carried through the transition while
another component(s) is degraded and totally resynthesized during
the G2-M-G-| transition.
Measurement of nuclear surface area in synchronized populations
of cells in G^ and G^ indicated that there was no significant differ¬
ence in the average surface area of the two nuclear populations.
These data suggest that the total nuclear surface has increased
n-2 fold between G,, and early G^. If one assumes that increases in
surface area are directly correlated to NE biosynthesis, these data
predict that "label dilution" experiments would detect an^SOJ!
dilution of NE protein during the time period examined. This is in
good agreement with our calculated results of 40% dilution and 60%
reutilization of pre-existing peptides.
The 60% of the early G^-NE protein which the data suggest
pre-existed M, when taken together with the data suggesting a burst
of early G^ NE synthesis does strongly imply that a majority of
G.| NE proteins come from pre-existing cellular components. Thus,
in my opinion, these data unequivocably rule out "complete" de novo
synthesis of NE protein as being responsible for mitotic reassembly.
Unfortunately I cannot state unequivocally that the early G1 NE
protein which pre-existed M resided solely in the G,, NE. The
possibility clearly exists that G, NE components are made prior to

81
M and then stored in some cellular compartment for use in restruc¬
turing the NE after division. The most likely organelle for such a
"storage function" would be the ER. Testing such a possibility must
await the availability of NE specific probes which can be applied
to the cells at various phases of the cell cycle to determine whether
"NE specific" components exist in other cellular organelles prior
to or during M.
The same problems and interpretation just discussed with regard
to NE peptides apply to those experiments which indicate that 50% of
the early G-j NE phospholipid pre-existed M. This apparently higher
rate of NE phospholipid biosynthesis relative to NE protein bio¬
synthesis over the same G^-M-G^ transition may reflect a high turn¬
over has been reported in other tissue culture cells (Cunningham,
1972).
I feel that my data can be interpreted to rule out complete
de novo synthesis of the NE during mitotic breakdown and reformation.
I also feel that the majority of the very early G1 NE components
pre-exist mitosis. A question which should now be asked is what is
the exact fate of NE peptides during mitosis. This can be ascer¬
tained only by applying probes (e.g. antibodies) specific for NE
peptides to small populations of mitotic cells. Such probes should
allow identification of the precise location of NE peptides during
M and thus determine the "storage" and reutilization of such com¬
ponents.

APPENDIX
DERIVATION OF THE EQUATION FOR CALCULATING THE PROPORTION
OF M/Gi NE SYNTHESIZED DE NOVO DURING THE G2-M-Gi TRANSITION
p = proportion of M/G^ NE synthesized during the G2-M-G.|
transition
SAgi = specific activity of M/G^ NE
SAG2 = specific activity of G^/H NE
SA1 = mean intracellular leucine pool specific activity during
the G2-M-G1 transition assuming an initial pool specific
activity of 1.0
The NE specific activity at M/G-j (SAQ^) is equal to some pro¬
portion (p) which was synthesized at a diluted pool specific activity
(SA1) plus some proportion (1-p) synthesized before label removal at
the G^/M NE specific activity (SAG2). This can be expressed in the
following equation:
SAG1
= P(SA1
) + (1-P)
SAG2
(1)
tion
1 gives
SAG1
= P(SA1
) + saG2
- p(saG2)
(2)
SAG1
- SAG2
= P(SA')
- p(sag2)
(3)
sagi
SAG2
= p(SA' -
sag2)
(4)
sagi
SA' -
SAG2
SAG2
P
(5)
82

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BIOGRAPHICAL SKETCH
Gregory E. Conner was born on December 21, 1950 in Jacksonville,
Florida, where he received his primary and secondary education in
parochial schools. In June of 1972 he received a Bachelor of Arts
degree from Vanderbilt University in Nashville, Tennessee. In
September of 1973 the author began graduate studies in the Department
of Biochemistry and Molecular Biology at the University of Florida.
Upon completion of these studies he will assume a post-doctoral
position at the Rockefeller University, New York, New York.
The author is married to the former Daphne Ann Wales of
Jacksonville, Florida.
88

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
tljn fr.
‘IKenneth D. Npom
ponan, Chairman
‘Assistant Professor of
Biochemistry and Molecular Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
<%á i?u2gA.
Charles M. A1len, Jr.
Associate Professor of
Biochemistry and Molecular Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
c
Carl M. Feldherr
Associate Professor of
Anatomy

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Thomas W. O'Brien
Associate Professor of
Biochemistry and Molecular Biology
This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
August 1978

CTi3r
ISili
3 1262 08554 oZU/



40
the harvested cells with chloroform-methanol (Rouser and Fleischer,
1967). After the Folch back washes of the chloroform-methanol
extract, phosphatidic acid was found in the lower organic phase.
Phosphatidic acid was purified by two sequential separations on
silica gel thin layer chromatography using solvent system I developed
by Skipski and Barclay (1969) for acidic phospholipids. The purified
phosphatidic acid was assayed for phosphorous (Rouser et al., 1966)
32
and the [ P] content determined by LSC.
Measurement of Nuclear Surface Areas
Nuclear surface areas were measured by point and intersection
planimetry on photographs of thick sections of embedded whole cells.
Samples of cells were taken from a synchronized culture 5h and 8h
after release from HU. Samples were fixed in 2% glutaraldehyde, 2%
formaldehyde and 50mM PO^ = (pH 7.4) for 30 minutes at 22C. Post
fixation was performed in 1% OsO^, lOOmM PO^ = (pH 7.4) for 30
minutes at 22C. Samples were then dehydrated and embedded as
described in Chapter II. Thick sections were cut on a Sorvall micro
tome and stained with 0.1% toludine blue 0 in 1% sodium borate for
15 seconds at approximately 200C. Sections were photographed using
a Wild Mil microscope with camera attachment. The resulting photo
graphs were subsequently subjected to planimetry according to
Wei bel (1969).
Polyacrylamide Gel Electrophoresis
All electrophoresis was performed as described in Chapter II.
3
Fluorography of [ H] labeled NE gels was performed according to
Bonner and Laskey (1974).


Employing this isolation technique I have examined the breakdown
and reformation of the NE during a limited stage (late G^, M, and early
G.|) of the replicative cycle in synchronized populations of CHO cells.
3 3
Using [ H] leucine as a precursor for protein and either [ H] choline
32
or [ P] orthophosphate as precursors for phospholipid, I have shown
that a minimum of 60'/ of the early G1, NE protein and a minimum of 50%
of the early NE phospholipid were present during the preceeding G,,
phase of the cell cycle and were reutilized in the reformation of the
NE which occurs during late M and early G-j. Pulse label studies
employing [ H] leucine or [ P] orthophosphate indicate that a burst
of NE biosynthesis occurs in early G^. Autoradiographic examination
of NE peptides isolated during these pulse label and label dilution
studies shows neither preferential loss nor preferential biosynthesis
of specific NE peptides during the G^ to M or M to G^ transition. My
evidence suggests that all the peptides of the NE, which I can resolve
in one dimensional gel electrophoresis, are synthesized during this
portion of the cell cycle. Examination of NE peptides by one dimension
al gel electrophoresis does not highlight any reproducible changes in
NE peptide composition which can be correlated with specific phase of
the cell cycle.
IX


75
Thin layer chromatography was run in solvent system I of Skipski and
Barclay (1969) for acidic phospholipids. After development, the
phosphatidic acid spot was visualized by spraying with distilled
water, scraped from the plate, eluted from the silica gel with
chloroform-methanol, and rechromatographed in the same developing
solvents. The phosphatidic acid spot was visualized in an iodine
chamber, scraped, and eluted as before. The specific activities of
phosphatidic acid at each time were determined on the eluted material
as described in Materials and Methods.
32
The rate of decrease in the specific activity of [ P] labeled
phosphatidic acid is presented in Figure 16. The arithmetic mean
intracellular specific activity during the G2M'G1 transition was
calculated to be 38% (average of two experiments) of that found in
32
cells prior to removal of [ P] from the media. Calculating from
32
the observed dilution of [ P] labeled NE phospholipid (Table V) I
find the proportion of M/G^-NE phospholipid synthesized during the
G^-M-G^ transition to be 0.53.
0.67 1.0
0.38 1.0
0.53
Again by subtraction, I estimate that 50% of the M/G^NE pre-existed
M.
Changes in Nuclear Surface Area
Since a change in total nuclear surface area might reflect
synthesis of NE and at the same time affect the interpretation of
my data, I have determined mean nuclear surface areas in a population
of cells before and after the G^-M-G^ transition (i.e. at 5 hr and


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS
By
Gregory E. Conner
August 1978
Chairman: Kenneth D. Noonan
Major Department: Biochemistry
A technique for isolating nuclei and nuclear envelopes (NE) from
Chinese hamster ovary (CHO) cells has been developed. This technique
does not depend on the use of detergents to solubilize contaminating
chromatin. In this procedure NE are prepared from purified nuclei by
nuclease digestion and subsequent high salt-sucrose gradient centrifu
gation. The nuclei and NE fractions are free of significant contamina
tion by other subcellular organelles as judged by electron microscopy
and enzymatic analysis. Electron microscopic observation of the puri
fied NE clearly demonstrates that the isolated material retains both a
double bilayer and the pore complexes observed in intact nuclei,
strongly suggesting that the isolation procedure described in this
dissertation permits the recovery of "intact" NE. Biochemical analysis
of the isolated nuclei and NE shows the NE fraction to be composed
primarily of protein and phospholipid, while containing only small
amounts of DNA and RNA. Examination of the peptide composition of the
NE fraction by SDS polyacrylamide gel electrophoresis reveals a very
complex coomasie blue staining profile with prominent bands in the
55,000 to 75,000 dalton molecular weight range.


PS
Figure 1. Diagrammatic representation of NE structure.
ONM outer nuclear membrane; INM inner
nuclear membrane; PS pore structure; PL -
proteinaceous lamina; HC heterochromatin.


Figure 10. SOS Gel Electrophoresis of NE Peptides Isolated
After Removal of pH] Leucine at the G2/M
Boundary
A cell culture was grown for 5 generations in
0.2uCi/ml [3H] leucine and synchronized in
0.2yCi/ml pH] leucine as described in Materials
and Methods. Nuclear envelopes were isolated
before label removal (G2/M interface, 7 hr after
removal of HU) and after division in the absence
of label (M/Gi interface, 11 hr after removal of
HU). The isolated NE were solubilized in SDS,
electrophoresed and fluorographed as described
in Materials and Methods. (A) Fluroograph of
NE peptides isolated at G2/M (lane 1) and M/G-j
(lane 2) interfaces. Equal numbers of counts
(31,000 cpm) were applied to the gel. (B)
Coomasie blue stained profiles corresponding
to the fluorogram in A. The G2/M (lane 1)
fraction contained 75yg of protein while the
M/G-] (lane 2) fraction contained 50yg protein.


TIME AFTER RELEASE
FROM HU (HRS.)
3H CPM x105/ mg PROTEIN
ru gj


32
Figure 14. Incorporation of [ P] into NE Phospholipid
A suspension culture was grown to stationary
phase and synchronized as described in Materials
and Methods. Seven hours after removal of HU an
aliquot of cells was removed, pulsed with 0.8pCi/ml
[32P] orthophosphate for one hour and NE isolated.
Each hour thereafter (for 6 hr) another aliquot of
cells was pulsed with 0.8pCi/ml [32p] for 1 hr and
NE isolated. Phospholipid specific activity was
determined on NE and homogenate isolated after
each pulse. Fraction of cells divided was deter
mined from 4 replicate countings in a Levy-Hausser
counting chamber. Nuclear envelope specific
activity 0-0; homogenate specific activity A-A ;
fraction divided


35
Figure 6. Coomasie Blue Stained Profiles of Subcellular
Fractions
Fractions were isolated and electrophoresed as
described in the Materials and Methods section.
Seventy-five micrograms of protein were applied
to each slot. From left to right: lane 1,
standards (phosphoryase A, 100,000 MW; bovine
serum albumin, 69,000 MW; ovalbumin, 43,000 MW;
DNase I, 31,500 MW; soybean trypsin inhibitor,
23,000 MW; and cytochrome c, 13,500 MW); lane 2,
DNase I and RNase A; lane 3, NE; lane 4, nuclei;
lane 5, plasma membrane and lane 6, whole cell
homogenate. Arrows ( ) indicate the region in
which histones migrate in these gels.


79
In Chapter III of this dissertation the NE isolation procedure
(described in Chapter II) has been employed to examine the question
of whether or not cellular proteins and phospholipids which pre-existed
the mitotic phase of the CHO cell cycle are used by the CHO cell in
the reformation of the NE which occurs during telophase.
Sodium dodecyl sulfate gel electrophoresis of NE isolated at
various stages of the CHO cell cycle showed no consistent differences
in peptide or glycopeptide composition which could be assigned to
any phase of the cell cycle. It is of interest to note that I did not
see any cell cycle dependent changes in the peptides migrating between
55,000 and 75,000 MW, as have been reported by Hodge et al. (1977) to
occur in the HeLa cell nuclear matrix. In this regard, the data in
this dissertation agree with those of Sieber-Blum and Burger (1977)
who also did not see cycle specific changes in CHO cell NE's.
In Chapter III of this dissertation,evidence, obtained primarily
from what I have dubbed "label dilution" studies, has been presented
which strongly suggest that at least 60% of the early NE protein
and at least 50% of the early G-j NE phospholipid existed in the cell
prior to mitotic breakdown and reformation (i.e. in late G^). These
data further suggest that the remaining 40% of early G.-NE protein
and the remaining 50% of early G.-NE phospholipid come from de novo
synthesis of NE components. Pulse label experiments suggest that
relatively little NE protein or phospholipid synthesis occurs in M
but rather that the majority of this NE synthesis occurs in late G2
and early G-j. Unfortunately the degree of synchrony obtainable with
such a large number of cells does not allow me to determine whether


TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS USED vii
ABSTRACT viii
CHAPTER I-INTRODUCTION 1
NE Structure 1
Membranes of the NE 3
Pore Structures 3
Proteinaceous Lamina 4
Association of Chromatin with NE 5
Isolation and Composition of NE 5
Isolation Procedures 5
Composition 7
Morphological Changes in the NE
During Cell Cycle 9
Objectives 12
CHAPTER II-ISOLATION AND CHARACTERIZATION 13
Materials and Methods 13
Materials 13
Cell Culture 13
Isolation of Nuclei and NE 14
Isolation of Plasma Membrane 15
Chemical Assays 16
Enzyme Assays 16
Electron Microscopy 17
SDS Polyacrylamide Disc Gel
Electrophoresis (PADGE) 17
Results 18
Isolation of Nuclei 18
Morphology of the Purified Nuclear
Fraction 22
Isolation of NE 26
Morphology of the Purified NE Fraction 26
Recovery of Isolated Fractions 28
Characterization and Purity of Nuclear
and NE Fractions 28
Enzyme Activity 30
Chemical Composition 32
Peptide and Glycopeptide Composition 34
CHAPTER 11¡-REFORMATION OF THE NUCLEAR ENVELOPE
DURING MITOSIS 36
Materials and Methods 36
Materials 36
Cell Culture and Synchrony 36


43
transition. It is during this transitional period of the ceil cycle
that the NE breaks down and reforms.
Peptide Composition of the NE during Different Phases of the Cell
Cycle
Maul et al. (1972) have reported that changes in the number of
nuclear pores per unit nuclear surface area occur as HeLa cells pro
gress through the cell cycle. Riley and Keller (1978) have reported
major morphological changes and minor compositional changes in non-
membranous nuclear ghosts isolated from HeLa cells at various stages
of the cell cycle. Hodge et al. (1977) have also examined polypep
tides of the HeLa cell nuclear matrix isolated from synchronized
populations of cells at various stages of the cell cycle. These
authors reported, in agreement with Riley and Keller, that minor
changes in the composition of nuclear matrix could be noted when
nuclear matrixes, isolated from cells in various phases of the cell
cycle, were compared by electrophoresis. Sieber-Blum and Burger
(1977), using CHO cells, have compared NE, isolated from synchronized
cell cultures, by electrophoresis and found no noticeable differences
in the stained gel profiles of cell cycle specific NE fractions.
In an effort to determine whether any changes occur in CHO NE
during the cell cycle which could be detected as changes in the
coomasie blue staining profile of NE peptides and glycopeptides
separated on SDS-PAGE, I have isolated NE from cells at various stages
of the cell cycle. Specifically NE were isolated from a) cells which
were in the logarithmic phase of growth (i.e. primarily G-| cells);
b) cells which had been grown to stationary phase, diluted, allowed
to reinitiate the cell cycle in fresh HU containing media and then


23
Figure 3. Phase Contrast Micrograph of the Purified
Nuclear Fraction. (xl340)


Figure 4. Electron Micrograph of the Purified Nuclear Fraction
Purified nuclei were fixed, embedded, and stained as
described in Materials and Methods.
A. Representative section through "purified nuclear
fraction (x3775).
B. High magnification EM of two randomly chosen nuclei
with the attention being directed to the nuclear
membranes (x24,350) (]]') outer bilayer; (T) inner
bilayer; CfTT) Pres-


11
movement begins. Rows of elongated membranous cisternae appear at
the polar aspect of the spindle. As the chromatids begin to fuse into
a chromatin mass, the row of cisternae becomes juxtaposed and extends
further toward the equatorial constriction (cytokinesis and telophase).
The NE is completely reconstructed before the finish of cytokinesis
which marks the end of the mitotic cycle.
Unfortunately very little is known concerning the fate of the
NE during mitotic breakdown nor is there good evidence relating to the
origin of the components which reform the NE in late M and early .
Similarly the mechanism(s) whereby the NE is disassembled at the mitosis
and reformed in late mitosis have not been elucidated.
It has been suggested by a number of workers (Porter and Machado,
1960; Moses, 1964; Robbins and Sonatas, 1964; Murray et a!., 1965;
Brinkley et a!., 1967) that following NE breakdown, fragments of NE
mingle with and become indistinguishable from the enaoplasmic reticulum
(ER). Furthermore, it has been argued that components of the ER are
utilized in reformation of tne G-j-NE (Porter and Machado, 1960; Moses,
1964). Other authors have suggested that the NE, or components of the
NE, persist through mitosis as distinct entities, biochemically differ
ent from the ER, and that these components are specifically reutilized
to reform NE at the completion of M (Erlandson and De Harven, 1971;
Maruta and Goldstein, 1975; Maul, 1977). Finally some workers in the
field have suggested that the NE which reappears at the end of M is a
product of de novo synthesis of all the NE components (e.g. Jones,
1960).
The multiplicity and diversity of hypotheses which have been pre
sented to explain NE breakdown and reformation during M are probably


APPENDIX
DERIVATION OF THE EQUATION FOR CALCULATING THE PROPORTION
OF M/Gi NE SYNTHESIZED DE NOVO DURING THE G2-M-Gi TRANSITION
p = proportion of M/G^ NE synthesized during the G2-M-G.|
transition
SAgi = specific activity of M/G^ NE
SAG2 = specific activity of G^/H NE
SA1 = mean intracellular leucine pool specific activity during
the G2-M-G1 transition assuming an initial pool specific
activity of 1.0
The NE specific activity at M/G-j (SAQ^) is equal to some pro
portion (p) which was synthesized at a diluted pool specific activity
(SA1) plus some proportion (1-p) synthesized before label removal at
the G^/M NE specific activity (SAG2). This can be expressed in the
following equation:
SAG1
= P(SA1
) + (1-P)
SAG2
(1)
tion
1 gives
SAG1
= P(SA1
) + saG2
- p(saG2)
(2)
SAG1
- SAG2
= P(SA')
- p(sag2)
(3)
sagi
SAG2
= p(SA' -
sag2)
(4)
sagi
SA' -
SAG2
SAG2
P
(5)
82


59
NE peptides were labeled approximately in proportion to their relative
staining with coomasie blue.
Dilution of r^H~| Choline Labeled NE
Using [ H] choline as a precursor of phosphatidyl choline, I
have examined the relative reutilization of NE phospholipids during
3
the G^-M-G^ transition. As in the work with [ H] leucine labeled NE,
I have initially followed the dilution of label at two time points
within the cell cycle, the so-called stage and the M/G-| stage of
the cell cycle (Table V).
Chinese hamster ovary cell cultures were grown for 5 generations
3
in medium supplemented with O.lyCi/ml [ H] choline and then syn-
3
chronized in complete medium containing O.lpCi/ml [ H] choline. Five
hours after release from HU (i.e. at the G^/H transition) the labeled
culture was harvested, washed twice with PBS, and divided into equal
aliquots. G^/M NE were prepared from one aliquot while the other
aliquot was returned to culture in complete F-10 medium without
3
[ H] choline but containing 0.7 mg/liter cold choline. Following
division (i.e. 9 hr after release from HU, see Figure 7), M/G^ NE
were prepared and chloroform-methanol extracts (Rouser and Fleischer,
1967) were prepared from both preparations. The specific radio
activity of the G^/M NE and the M/G^ NE phospholipid (cpm/pg lipid
phosphorus) was determined and the specific activities compared
(Table V).
The simplest interpretation of the data in Table V would suggest
that approximately 65% of the M/G. NE phospholipid was present in the
cell prior to mitosis while 35% of the M/G^ phospholipid was


53
components. From the fluorogram presented in Figure 10 it is apparent
that, within the detection limit of the fluorographic process and
one dimensional gel electrophoresis, no individual NE peptide or
glycopeptide is preferentially lost during the G^-M-G^ transition.
3
Incorporation of the T Hi Leucine into NE Protein
One likely, if not the most likely, explanation for the reduced
specific activity found in M/G^ NE's relative to G^/M NE's (Table IV)
is dilution of the pre-existing (i.e. G^/M) label with unlabeled NE
components synthesized and inserted into the NE during either G^, M,
3
or G^ phase of the cell cycle after removal of [ H] leucine from the
culture medium. The data presented in Figure 9 suggests that some
dilution of label occurs in G,, immediately after removal of [ H]
leucine from the culture medium as well as in G^. In order to
determine whether this dilution results from phase-specific biosyn
thesis of NE peptides, cells were synchronized as described above and
then 5.25 hr after release from HU an aliquot of cells was taken and
pulsed for 1 hr in medium containing 0.2pCi/ml [ H] leucine but only
25l of the standard F-10 leucine concentration. After 1 hr in labeled
precursor an aliquot was taken from the homogenate and the NE was
isolated. Cells were pulsed with 0.2yCi/ml [ H] leucine and NE iso
lated every hour from 5.25 11.5 hr after release of cells from an
HU blockade (Figure 11).
The specific activity of each NE and homogenate fraction was
determined and the specific activities plotted as a function of time
after release from HU block. As can be seen, the specific activity
of the NE fractions remain relatively constant ( 5%) from 6 9 hr


12
due to the variety of animal and plant species in which the predom
inantly microscopic work was performed and to the fact that the evidence
available concerning NE breakdown and reformation consists almost
entirely of morphological studies which suffer from the difficulty of
positively identifying cytoplasmic components which might be NE frag
ments released during mitosis.
Objectives
The main objective of the studies presented in this dissertation
is to investigate the mechanism whereby the NE disassembles and reforms
during mitosis. To examine biochemical changes and the biosynthesis
of NE during the cell cycle it is necessary to develop a procedure to
use with cultured cells which would permit the isolation of "clean"
NE containing both lipid bilayers of the nuclear membranes as well as
the proteinaceous lamina.
In Chapter II of this dissertation I will report a preparation
technique which permits the isolation of nuclei from Chinese hamster
ovary (CHO) cells without the use of reagents (such as detergents) which
might disrupt membranes. The isolated nuclear fraction has been used
to purify sufficient quantities of NE to allow both biochemical and
EM studies of this organelle.
In Chapter III of this dissertation I will present a biochemical
investigation of the breakdown and reformation of the NE during mitosis
in CHO cells. Using radioactive precursors of protein and phospho
lipid I have studied the reutilization and biosynthesis of NE in the
late G^-M-early portion of the cell cycle.


13
CHAPTER II
ISOLATION AND CHARACTERIZATION
Materials and Methods
Materials
All tissue culture materials were purchased from Grand Island
Biological Company (Grand Island, New York). Tissue culture plastic-
ware was obtained from Corning Glass Works (Corning, N.Y.). Ultra-
3 3
pure sucrose, [methyl- H] thymidine, and [5- H] uridine were obtained
from Schwarz-Mann Division, Becton, Dickinson and Company (Orangeburg,
New York). Deoxyribonuclease I (DNase I) and ribonuclease A (RNase A)
were purchased from Worthington Biochemical Corporation (Freehold, New
Jersey) and were shown to be protease-free via the assay of Tomarelli
et al. (1949). [Methyl- H] choline chloride was obtained from Amer-
sham Searle Corporation (Arlington Heights, Illinois). All reagents
for electrophoresis were purchased from Bio-Rad Laboratories (Rockville
Centre, New York). All other reagents were obtained from Scientific
Products (Ocala, Florida).
Cell Culture
Chinese hamster ovary cells (originally obtained from Dr. Kenneth
Ley, Sandia Laboratories, Albuquerque, New Mexico) were maintained at
37C in suspension culture in Ham's F-10 nutrient medium, supplemented
with 10% (v/v) calf serum and 5% (v/v) fetal calf serum. Cell density
5 S
was monitored daily and maintained between 1.2 x 10 4 x 10 cells/ml.


34
present in the NE fraction was extracted by chloroform methanol and
accounted for in subsequent phosphorus determinations {data not
shown).
The cholesterol content of NE and plasma membrane were compared
in order to determine if there were any clear differences in choles
terol composition between these two fractions taken from CHO cells.
As can be seen in Table III, five-fold less cholesterol (per mg pro
tein) was found in the NE as compared to the PM. These data are in
agreement with previously published work concerning the cholesterol
content of bovine liver NE (Keenan et al., 1970).
Peptide and Glycopeptide Composition
When the purified NE fraction was examined by SDS gel electro
phoresis (Laemmli, 1970), a complex Coomasie blue staining profile
was obtained (Figure 6) which was distinguishable from the stained
profiles obtained from whole cell homogenate, purified nuclei and
plasma membrane derived from CHO cells (Figure 6).
The major Coomasie brilliant blue staining components of the
NE fraction ranged in molecular weight from 55,000 to 75,000 daltons.
The majority of the remaining NE peptides and glycopeptides were of
a MW higher than 75,000 daltons. It should be noted (compare nuclear
with NE fraction, Figure 6) that few, if any, peptides are found in
the NE fraction which co-migrate with the histones (arrows) found
in the nuclear fraction again arguing against significant contam
ination of the NE with DNA.


REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS
By
GREGORY E. CONNER
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


membranes seem to be fused (Figure 1). When visualized tangentially,
the pore structures appear as complexes of 8 or 9 granules arranged as
an annulus. The central zone of the pore structure is less electron
dense than the surrounding annular material and may coincide with the
gaps seen in transverse sections. The annular pore complexes have
o
an overall diameter of 1000 2000 A suggesting that the pore structure
extends beyond the gaps visualized in transverse sections possibly
overlapping part of the nuclear membranes. Electron dense granules
can occasionally be seen in the interior of the pores. Some data have
been obtained which suggest that these granules are material in transit
between nucleus and cytoplasm. Many authors have suggested that the
pore complexes function in the control of nucleo-cytoplasmic communi
cation (for review see Feldherr, 1972).
Proteinaceous Lamina
In some cell types the inner aspect of the inner nuclear membrane
appears to be associated with an electron dense material. This layer,
variously called the dense lamella (Kalifat et al., 1967), zonula
nucleum limitans (Patrizi and Poger, 1967), and the fibrous lamina
O O
(Fawcett, 1966), ranges in thickness from 2800 A in amoeba to 150 200 A
in vertebrate cell types. This layer, apparently proteinaceous in com
position (Stelly et al., 1970), appears to be tightly associated with
the interphase NE, in that it may copurify with NE during subcellular
isolation procedures. The exact function of this proteinaceous lamina
has not been determined, however Stelly et al. (1970) have suggested
that it is responsible for the shape and rigidity of the nucleus.


32
the nuclear fraction may be due to loss or denaturation of the
enzyme by the high salt conditions employed during the latter steps
of our isolation procedure (Kasper, 1974).
Chemical Composition
A preliminary analysis of the relative chemical composition
of the homogenate, nuclei and NE fractions are presented in Table III.
Among the points presented in Table III which should be stressed is
the fact that the "purified NE fraction" does contain small amounts
of residual DNA and RNA. Similar findings with regard to nucleic
acid composition of isolated NE have been reported in previously
published characterizations of NE from liver (Kasper, 1974) and may,
in fact, represent physiologically significant components of the NE.
It should also be noted that, not unexpectedly, if the nuclease
digestions (Figure 2) were omitted from the isolation procedure, sig
nificantly larger amounts of DNA and RNA were recovered in the
purified NE fraction.
To confirm the membranous nature of the isolated NE fraction,
phospholipid content was determined in the homogenate, nuclei, and
NE fraction contained 200pg phospholipid/mg protein indicating that
the fraction isolated is relatively protein rich (see pg. 31 of
Fleischer and Kervina, 1974) perhaps due to the proteinaceous lamina
associated with the inner nuclear membrane. In order to demonstrate
that this relatively low phospholipid/protein ratio was not due to
incomplete extraction of phospholipid, NE1s were prepared from cells
which had been maintained for 5 generations in medium containing
O.lpCi/ml [ H] choline. Ninety-seven percent of the [ H] choline


46
Specifically CHO cells were grown for 5 generations in 0.2pCi/ml
3 3
[ H] leucine and then synchronized in the presence of 0.2pCi/ml [ H]
leucine as described in Materials and Methods. In late (5 hr
after HU removal, Figure 7) the cell culture was harvested, washed
twice with PBS and then the 6 liters of cells divided into two equal
aliquots. From one aliquot NE were immediately isolated (described
in Table IV as the G^/M NE population). The remaining cells were
returned to fresh medium free of [ H] leucine but containing 13.2 mg/
liter cold leucine. Four hours later (after 80% of the cells had
completed M, Figure 7) these cells were harvested and NE immediately
isolated (described as the M/G^ NE population in Table IV). The
specific activity of these two NE fractions was then determined and
compared. As can be seen in Table IV, the average specific activity
of the M/G1 NE is approximately 25% less than the specific activity
of the NE's isolated from the G£/M cell population. These data
suggest that although some de novo synthesis of NE protein has
occurred over the 4 hr time span between the isolation of the G^/M
and M/G-| NE, the majority of the peptides present in the M/G^ NE's
pre-existed in the G^/M cells. The data presented in Table IV do
not, of course, take the precursor pool into account. Resolution
of this problem will be discussed below.
The reduction in specific activity broadly described in
Table IV was further investigated by isolating NE at a time point
(6.5 hr after release from HU) at which less than 5% of the cells
have completed M and every hour thereafter until greater than 80%
of the cells have completed M (Figure 9). Cells were grown and


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39
determined from phospholipid phosphorus assays (Rouser et al., 1966)
of chloroform-methanol extracts (Rouser and Fleischer, 1967) of the
specific cell component.
Determination of Precursor Pool Equilibration Rates
Cell cultures were grown for 5 generations in either 0.2pCi/ml
3 32
[ H] leucine or 0.4pCi/ml [ P] orthophosphate. The cells were then
3
synchronized in the presence of 0.2pCi/ml [ H] leucine or 0.4pCi/ml
32
[ P] orthophosphate. Five to six hours after release from HU, an
aliquot of cells was taken from the culture and the initial precursor
pool specific activity was determined as described below. Immediately
after removal of this initial aliquot, the remaining cells were
washed twice with PBS, sampled again, and returned to unlabeled
medium. Each hour for the next 4 hr after being returned to unlabeled
medium, a sample of cells was removed, and the precursor pool specific
activity was determined.
3
To determine the size of the [ H]leucine pool, cells were
collected on a glass fiber filter, washed quickly with 10 volumes of
ice cold PBS, and solubilized in 0.2N NaOH for 5 minutes at 70C.
The solubilized material was chilled, precipitated with 10% trichloro
acetic (TCA) overnight at 4C and the supernatant collected by cen-
3
trifugation. [ H] Leucine in the supernatant (i.e. the acid soluble
pool) was determined by LSC and total cell protein was assayed
according to Lowry et al. (1951).
Phospholipid precursor pool size was based on labeled phospha-
32
tidic acid. [ P] Phosphatidic acid specific activity was determined
by harvesting cells via centrifugation and immediately extracting


3
nuclear membrane is often studded with ribosomes in a manner similar to
rough endoplasmic reticulum (RER) and occasionally has been observed to
be continuous with RER (Watson, 1955). The inner aspect of the inner
nuclear membrane appears to be in close association with a proteinaceous
lamina (Fawcett, 1966) and to be intimately associated with hetero
chromatin (i.e. dense inactive chromatin).
Membranes of the NE
The membranes of the NE, when visualized by EM, are most fre-
O
quently found to be 60-S0 A in thickness. The two membranes are normally
O
separated by 100-700 A except at the pore structures where they appear
to be joined, thus forming a perinuclear cisternum. The frequent local
ization of ribosomes and the occasional continuity of the outer membrane
with RER indicates the strong morphological similarity between membranes
of the NE and RER. As will be discussed later, the enzymatic and bio
chemical composition of the NE serves to strengthen the resemblance of
these two membranous organelles. Currently no data are available which
conclusively demonstrate that components of the NE arise from RER or
that components of the RER arise from those of the NE.
Recent studies by Feldherr et al. (1977) and Virtanen (1977,1978)
and Virtanen and Wartiovaara(1976) suggest that the nuclear membranes
possess a property of sidedness similar to that of other cellular mem
branes. Specifically these authors have reported that the carbohydrate
containing portions of NE glycopeptides are located on only one side,
the cisternal side, of each nuclear membrane.
Pore Structures
When pore structures of the NE are visualized by EM in transverse
c o
sections, they appear as gaps (150 A 700 A wide) at which the nuclear


Figure 13. Dilution of [^P] Labeled NE Phospholipid
A suspension culture was grown to stationary phase
in the presence of 0.4pCi/ml [32p] orthophosphate
and synchronized as described in Materials and
Methods in 0.4pCi/ml [32p] orthophosphate. Six
and one-half hours after release from HU NE were
prepared from an aliquot of cells. The remain
ing cells were washed twice with PBS and returned
to culture in complete medium lacking [32p].
Nuclear envelope and homogenate were isolated each
hour after removal of [32p] and phospholipid
specific activities were determined on each frac
tion as described in Materials and Methods.
Fraction of cells divided was determined from 4
replicate countings in a Levy-Hausser counting
chamber. Nuclear envelope specific activity
0-0; homogenate specific activity A-A; fraction
divided D-D .


87
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TIME AFTER RELEASE
FROM HU (HRS.)
FRACTION DIVIDED


Table II
Enzyme Activities of Subcellular Fractions
Homogenate
Nuclei
NE
Mitochondria f
5'-Nucleotidase
(nmoles PO4 = hydrolyzed/
mg protein/hr)
49 (6)
ND
(2)
ND (2)
Succinate Dehydrogenase
(nmoles succinate oxidized/
mg protein/hr)
470 (2)
ND
(2)
ND (2)
Cytochrome c Oxidase
(nmoles cytochrome c oxidized/
mg protein/min)
0.7 (2)
ND
(2)
1.5 (2)
18.0 (2)
G1 ucose-6-Phosphatase
(pmoles PO4 = hydrolyzed/
mg protein/hr)
0.41 (2)
0.75
(2)
0.80 (2)
Each fraction was assayed as described in Materials and Methods, immediately following
isolation of NE fractions. ND no detectable activity. The minimum detectable activity in succinate
dehydrogenase assays was 50 nmoles succinate oxidized/mg protein/hr and the minimum detectable activity
in 5' nucleotidase assays was 3 nmoles/mg protein/hr. The minimum detectable activity in cytochrome c
oxidase assay was 0.3 nmoles cytochrome c oxidized/mg protein/min. Numbers in parentheses are the number
of assays performed, t Digitonin treated mitochondria, isolated from bovine liver, were obtained from
Dr. T. W. O'Brien.


LIST OF TABLES
I. Recovery of Protein, DNA, and
Phospholipid in Subcellular Fractions 29
II. Enzyme Activities of Subcellular
Fractions 31
III. Chemical Content of Subcellular Fractions 33
IV. Dilution of [^H] Leucine Labeled NE
Protein 47
V. Dilution of Labeled NE Phospholipids 60
v


68
after release from HU an aliquot of cells was removed from the culture,
collected on a glass fiber filter, quickly washed with 10 volumes of
ice cold PBS and solubilized in 0.2 N NaOH. The remaining cells were
harvested, washed twice with PBS (as was done in all my previously
presented dilution data). Immediately after the PBS washes (6 hr
after HU removal) another aliquot of cells was collected on a glass
fiber filter, washed and solubilized. The remaining cells were
returned to complete media without [ H] leucine. Each hour after
returning the cells to unlabeled media an aliquot of cells was
collected, washed, and placed in 0.2 N NaOH. After all the samples
had been collected (10 hr after removal from HU, 90% of the cells
having divided, Figure 15), the filters were heated at 70C for 5 min,
cooled on ice, and precipitated overnight in 10% TCA (4C). The
specific activity of the TCA soluble [ H] leucine counts was then
determined and plotted as a function of time (Figure 15). As can
be seen in Figure 15, approximately 55% of the [ H] leucine, which
was in the cytoplasmic precursor pool before the cells were harvested,
was removed during the initial two PBS washes. Subsequent culturing
3
of the washed cells in [ H] leucine-free media reduced the soluble
3
[ H] leucine pool to a specific activity with was 30% of the initial
specific activity of the initial time point (Figure 13).
3
By first setting the initial, pre-PBS wash, soluble [ H] leucine
pool at 100% (initial time point, Figure 15) and then averaging the
fractional value of this initial pool which was measured during the
G^-M-G^ transition in the absence of label, I have calculated a mean
soluble [ H] leucine pool specific activity during the G2-M-G-|. This


6
remaining elements of the nucleus (e.g. nucleoli, chromatin, and soluble
proteins). Because separation of NE from other nuclear components is
usually dependent on the membranous characteristic of this organelle,
the purified nuclei from which NE are to be isolated, must be prepared
in a fashion which retains both nuclear membranes and yet removes other
contaminating organelles of the cell. Such nuclear isolation procedures
have been described and are usually modifications of the technique of
Chauveau et al. (1956). The lack of a well characterized preparation
of NE from cultured cells is most probably due to difficulties in iso
lation of large quantities of "clean" nuclei which contain both nuclear
membranes. The use of detergents to remove cytoplasmic contamination
from nuclei most likely strips the nuclei of the lipid bilayers of the
nuclear membranes.
Disruption of nuclei for isolation of NE is usually accomplished
by mechanical and/or chemical treatments. The most frequently used
mechanical techniques are sonication (e.g. Franke, 1967; Kashnig and
Kasper, 1969) and hypotonic shock (e.g. Zbarsky et al., 1969; Kartenbeck
et al., 1971). High concentrations of MgC^ (Berezney et al., 1970;
Monneron et al., 1972), sodium citrate (Bornens, 1968; Kashnig and
Kasper, 1969), NaCl and KC1 (Franke et al., 1970; Matsuura and Ueda,
1972), and heparin (Bornens, 1973,1978) have been shown to disrupt
nuclei and aid in solubilization of chromatin during NE isolation.
Deoxyribonuclease I (DNase 1) has often been utilized to reduce the
viscosity of ruptured nuclei and to reduce the level of DNA contamina
tion in isolated NE fractions (e.g. Berezney et al., 1970; Kay et al.,
1972).