Title: Cell contact mediated differentiation in Dictyostelium / by Ning Yueh Yu
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Title: Cell contact mediated differentiation in Dictyostelium / by Ning Yueh Yu
Physical Description: vi, 49 leaves. : illus. ; 28 cm.
Language: English
Creator: Yu, Ning Yueh, 1947-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Dictyostelium discoideum   ( lcsh )
Zoology thesis Ph. D
Dissertations, Academic -- Zoology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis -- University of Florida.
Bibliography: Bibliography: leaves 45-48.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098323
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000580802
oclc - 14083240
notis - ADA8907

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CELL CONTACT MEDIATED DIFFERENTIATION
IN DICTYOSTELIUM
















By

NING YUEH YU












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

1975












ACKNOWLEDGEMENTS


I would like to express my gratitude to Dr. J. H. Gregg, the

chairman of the supervisory committee; Drs. J. W. Brookbank, F. C.

Davis and H. C. Aldrich for their participation on the committee

and advice on the work.

Special appreciation is made to Dr. Gregg for his kindly guid-

ance, technical instructions, as well as manuscript preparations.

Thanks also extend for the equipment provided and financial arrange-

ment to make all this work possible.

I would also like to thank Dr. Aldrich for his professional

supervision on electron microscopy and freeze-fracturing during the

course of the work.

The work of Mrs. Donna Gillis who typed the manuscript is also

appreciated.

Finally, I want to thank my parents, Mr. and Mrs. C. A. Yueh,

and parents-in-law, Mr. and Mrs. C. C. Yu, for their constant help

and encouragement during the entire process. The interest of my husband,

Hsi-Ling, who shared the experience throughout the work is also deeply

appreciated.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . . . . . . . . . .

ABSTRACT . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . .

METHODS AND MATERIALS . . . . . . . .

Cellular Slime Molds . . . . . . . .
Harvesting of D. discoideum Cells . . . .
Thin-section Electron Microscopy . . .....
Freeze-fracturing . . . . .. . .
Purification of Concanavalin A . . . . .

RESULTS . . . . . . . . . . . .

Vegetative Myxamoebae . . . . . .
a. Inhibition of morphogenesis and different
by Con A . . . . . . .
b. Cyclic AMP effects on prespore cell
differentiation . . . . . . .
Aggregating Streams . . . . . . .
a. Cyclic AMP effects on prespore cell
differentiation . . . . . . .
b. Cyclic AMP effects on plasma membrane
structure . . . . . . . .
c. Con A effects on prespore cell different
d. Con A effects on plasma membrane structure


Page

. . ii

v

. . . 1

. . . 14

. . . 14
. . . 14
. . . 14
. . . 15
. . . 15
. . . 16

. . . 17

. . . 17
iation
. . . 17

. . . 17
. . . 19

. . . 19

. . . 19
ation. . 19
e. . .. 21


e. Effect of mechanical disruption on prespore cell
differentiation . . . . . . . . .
f. Effect of mechanical disruption on plasma membrane
structure . . . . . . . . . .
Early Culminates . . . . . . . . . . .
a. Effect of Con A, distilled water and CAB
on culmination . . . . . . . .
b. Effect of Con A on mature spore differentiation .

DISCUSSION . . . . . . . . . . . . . .

Cyclic AMP in Prespore Differentiation . . . . .
Con A in Prespore Differentiation . . . . . .









Con A in Culmination and Mature Spore
Differentiation .... ........... . . 41
Role of Cell Contacts and Interaction in
Differentiation . . . . . . . . . . 42

BIBLIOGRAPHY . . . . . . . . . . . . . . 45

BIOGRAPHICAL SKETCH. . . . . . . . . .. ... . 49











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


CELL CONTACT MEDIATED DIFFERENTIATION
IN DICTYOSTELIUM

By

Ning Yueh Yu

March, 1975

Chairman: Dr. James H. Gregg
Major Department: Zoology

Upon spore germination in the cellular slime mold Dictyosteliwn

discoidewn amoeboid cells are liberated which proceed to engulf bacteria

as foodstuff and undergo binary fission. After large numbers of the

myxamoebae are produced aggregation of the cells in response to a

chemotactic substance, cyclic AMP, results in the formation of multi-

cellular pseudoplasmodia. Differentiation of two cell types, the pre-

stalks and the prespores, occurs as a result of cell association.

Eventually each aggregate forms a mature sorocarp composed of a slender

stalk bearing a mass of spores. Major biochemical and ultrastructural

changes occur at the cell surfaces following cell association. Cyclic

AMP which effected the formation of certain plasma membrane particles

during aggregation and Con A which binds to specific membrane sites

were used to clarify the nature of the cellular interactions. Cyclic

AMP induced major increases in the sizes of the plasma membrane

particles among isolated aggregating cells but prespore vacuoles

indicative of prespore differentiation did not appear. Aggregating









streams exposed to Con A failed to synthesize large plasma membrane

particles and prespore differentiation was inhibited. A few cells

bearing large particles were found among the mechanically disrupted

aggregates. Differentiation did not occur among these cells suggest-

ing that a continuous period of cell association and interaction is

essential. Prespore cells exposed to Con A just prior to differen-

tiation into mature spores formed aberrant spore walls and irregularly

shaped spores. The inhibitory effect of Con A on prespore and mature

spore differentiation is discussed. The large plasma membrane

particles were considered to be the components through which cell

interactions are mediated resulting in differentiation.













INTRODUCTION


The existence of cellular interactions as a factor in cyto-

differentiation has long been recognized. The classical experiment

of Spemann and Mangold (1924) demonstrated that the induction of

neural structures in amphibian embryos depended upon an interaction

between the ectoderm and chorda mesoderm. The concept of inductive

interactions was exploited and a similar phenomenon was shown by

Spemann (1901) to occur in the induction of a lens by the eyecup in

Rana fusca. Subsequently numerous other examples of a similar nature

were shown to exist among developing organisms.

Eventually the question arose as to the nature of the inductive

processes and the degree of contact necessary between two tissue types

to effect cytodifferentiation. This led in one instance to the insertion

of a semi-permeable barrier between the inducing optic vesicle of a

chick and the overlying ectoderm (McKeehan, 1951). The failure of lens

induction under these circumstances emphasized the necessity for some

form of macromolecular communication during the inductive period.

A series of experiments were conducted to determine the degree

of association required in inductive interactions. By the use of

millipore filters separating mesenchymal components of various types

of rudiments from appropriate epithelium it was demonstrated that

induction of the epithelium was still possible (Grobstein, 1961, 1963;

Golosow and Grobstein, 1962). The inductions were attributed primarily

to the transmission of large molecular weight substances through the









20 thick filters. However, the average pore diameter of the filter

was 5,000 A which could not exclude certain cytoplasmic processes.

A unique type of cellular interaction is recognized in the

differentiation of skeletal muscle cells. The primordial muscle cells

or myoblasts following a period of proliferation fuse to form multi-

nucleated myotubes. The myotubes subsequently differentiate exhibiting

cross-striations characteristic of skeletal muscle in the myofibrils

(Konigsberg, 1963).

Among the systems employed in studies of cellular interactions

the cellular slime mold Dictyostelium discoidewu offers certain ad-

vantages. The germination of mature spores provides population of

vegetative myxamoebae which feed upon appropriate bacteria and undergo

binary fission (Figures la, lb). The myxamoebae of the vegetative

stage exist independently until they aggregate in response to the

chemotactic agent acrasin (cyclic AMP) which is initially secreted by

individual or small groups of cells (Bonner, 1947; Konijn et al.,

1967)(Figures 1c, Id). Ultrastructurally the myxamoebae appear to be

identical. However, by late aggregation two cell types may be dis-

tinguished in the pseudoplasmodium (Figure le). The cells which

become prespore cells contain prominent organelles named prespore vacuoles

(PV) while the other cell type, the prestalk, is devoid of these 6,000 A

structures (Hohl and Hamamoto, 1969) (Figure 2). The posterior two

thirds of the migrating pseudoplasmodium is composed of prespore cells

while the anterior prestalk cells occupy the remainder of the cell

mass (Bonner, 1952). By further morphogenetic movements each pseudo-

plasmodium forms a mature fruiting body consisting of a slender stalk

bearing a mass of mature spores at the apex (Figure la).



















Figure 1. Life cycle of the cellular slime mold Dictyostelium
discoideum-lH.

a. mature sorocarp (inset: group of spores)
b. vegetative myxamoebae
c. aggregating myxamoebae
d. aggregating center with streams
e. late aggregate
f. migrating pseudoplasmodium
g. preculminate
h. early culmination
i. culmination stage just prior to mature spore
differentiation
j. culmination stage following mature spore
formation















S,0 mm.
e f
^iI f






























Figure 2. Prespore cell in an early culminating stage, X19,566.

AV autophagic vacuole
M mitochondrion
N nucleus
PV prespore vacuole












The discrete groups of prestalk and prespore cells in the

migrating pseudoplasmodium may be isolated to determine their capacity

for cell redifferentiation and the regulation of proportions. Raper

(1941) noted that both isolated prestalk and prespore cell groups

could redifferentiate the missing cell types and form normal fruiting

bodies. Gregg and Badman (1970) observed that prestalk and prespore

cell group isolates were capable of synthesizing or losing PV respectively,

in the course of redifferentiating. If aggregating myxamoebae or pre-

stalk and prespore cells were isolated not in groups but as single

cells, neither differentiation nor redifferentiation occurred (Gregg,

1971). Evidence is also available which suggests that cell association

regulates the synthesis of certain enzymes in Dictyastdium. Disaggregated

cells maintained in isolation were observed to cease production of the

enzymes (Loomis, 1970; Newell et al., 1971).

The necessity of cell contact in the differentiation of D. discoidewn

myxamoebae suggests that the plasma membranes must be intimately involved.

Ample evidence exists that the plasma membranes undergo changes during

the transition of the myxamoebae from unicellular state to a multi-

cellular existence. Shaffer (1958) first noted that the myxamoebae

upon aggregation became adhesive in response to the effect of acrasin.

Simultaneously new surface antigens appear (Gregg, 1956) which may be

involved in the maintenance of contacts between apposing cells (Beug

et al., 1970). Freeze-fracture studies revealed that particulate

structures in the plasma membranes increased 1.7X in average diameter

from the vegetative stage to the prespore cells in migrating pseudo-

plasmodia (Figures 3,4) (Aldrich and Gregg, 1973). Plasma membranes


































Figure 3. Freeze-fractured vegetative myxamoebae plasma
membrane exhibiting particles averaging 60 A
in diameter, X300,000.




9










rr


a 6








*I
,Y


9 Jtd)






























Figure 4. Freeze-fractured late aggregate prespore cell
plasma membrane exhibiting particles averaging
106 A in diameter, X300,000.





~,
-.- -- rl
:~ ~~L~P




12



of many species are characterized by the presence of particulate

structures. In red blood cells the membrane particles are considered

to be composed of glycoproteins which interact with intramembranous

proteins or lipids. The glycoproteins which extend through the membrane

expose carbohydrate moieties at the cell surface (Marchesi et at.,

1972). Indeed carbohydrate is bound to all the major surface proteins

(Steck, 1974). The large plasma membrane particles in Dictyostelium

are considered to project through the membrane and be exposed at the

cell surface (Aldrich and Gregg, 1973). Singer and Nicolson (1972)

suggest that contacts between the particulate structures of apposing

cells may result in the transmission of stimuli into the cytoplasm

thereby initiating cell differentiation.

Dictyostelium cells may be quickly agglutinated by Concanavalin

A (Con A) (Weeks, 1973). Con A, a phytagglutinin isolated from the

jack bean, CanavaZia ensiformis, is known to bind reversibly to

oligosaccharides containing terminal a-D-glucopyranosyl, a-D-

mannopyranosyl and sterically related sugar residues. Thus, Con A

can bind to glycoproteins and possibly glycolipids on the cell surfaces

of a variety of cell types (Edelman, 1973).

It is clear that differentiation in Dictyostelium normally depends

upon an association between the cells beginning at the aggregation phase.

The ultrastructural changes which appear in the plasma membranes follow-

ing aggregation may provide the morphological and functional basis upon

which cellular interactions depend. Cyclic AMP and Con A, both of which

bind to specific cell sites on the plasma membranes,were utilized in the

course of this study. The effects of these substances on development




13



may yield information on the origin and nature of the interactions

which affect the transformation of the cells.













METHODS AND MATERIALS


Cellular Slime Molds

Wild-type Dictyostelium discoideum-lH was used throughout this

investigation. The myxamoebae were cultured at 22-23C on nutrient

agar plates using Escherichia coZi as the bacterial associate (Bonner,

1947).

Harvesting of D. discoideu cells

a. Vegetative myxamoebae harvested from 17-22-hour cultures were

washed free of bacteria 3X in 0.013 M phosphate buffer (PB) at

pH 7 by low speed centrifugation in a clinical centrifuge. The

cells to be used in subsequent experiments were concentrated at

1 X 106/ml as determined by counts made with a hemocytometer.

b. Aggregating stages were prepared from 24-hour cultures of vege-

tative myxamoebae. The washed cells were distributed on 2%

non-nutrient (NN) agar buffered at pH 7. The myxamoebae formed

aggregating streams within 11 hours.

c. Early culminating stage pseudoplasmodia were collected as

individuals by removing 5 mm cylinders of agar each bearing a

slime mold. This procedure utilized a sharp metal tube connected

to a rubber tube mouth aspirator to apply the small vacuum

necessary in retaining the agar plug as it was transferred

to another culture dish containing NN agar.









Thin-section Electron Microscopy

Vegetative and aggregation stage myxamoebae were fixed in 2%

glutaraldehyde (Ladd Research Industries) containing standard salt

solution (SS) (Bonner, 1947) at 40C for 15-20 minutes. The fixative

was adjusted to pH 7 just prior to use by stirring with barium carbonate

and filtration through Whatman #50 paper. The cells were post-fixed

in 1% osmium tetroxide containing SS for 20 minutes and stained with

0.5% uranyl acetate at pH 3.9 for 20 minutes (Terzakis, 1968). De-

hydration was effected in ethanol and acetone followed by embedding

in Epon 812 (Ladd Research Industries). Thin-sections were post-

stained in 2% aqueous uranyl acetate for 15 minutes and Reynolds (1963)

lead citrate for 4 minutes. The sections were examined and photographed

by an Hitachi HU 11-E electron microscope at 75 KV.

Migrating pseudoplasmodia, culminating stages and mature sorocarps

were fixed in 2% glutaraldehyde containing 0.1 M cacodylate buffer at

pH 7 at 4C for 1 hour. The cells were post-fixed in cacodylate-buffered

1% osmium tetroxide at 4C for 2 hours. The remainder of the preparation

was identical to that described for the vegetative and aggregation stage

myxamoebae.


Freeze-fracturing

Myxamoebae composing aggregating streams and late aggregation

stages were fixed for 30 minutes at 4C in 2% glutaraldehyde in SS

which had been adjusted to pH 7. The cells were washed 3X in SS for

10 minutes/wash. Following exposure to 20% glycerol for 3-24 hours the

cells were mounted on gold-nickel discs, frozen in Freon 22 and stored

in liquid nitrogen. The freeze-fracturing technique and production of









carbon-platinum replicas were essentially identical to those used

by Moor and MUhlethaler (1963). A Balzers BA 360 M apparatus was

used to produce 13 replicas which were subsequently photographed at

150,00X. The plasma membrane particle sizes were derived by

analyzing 27 photographic negatives in the manner described by

Goodenough and Staehelin (1971) and Gregg and Nesom (1973).


Purification of Concanavalin A

Con A (Calbiochem) was dissolved in 0.001 M phosphate buffer

with the addition of 0.086 M NaC1, 0.0027 M KC1, 0.0001 M CaC12 and

0.0001 M MnCl2. Centrifugation at low speed in a clinical centrifuge

for 5 minutes was necessary to remove any large aggregates. The

supernatant was passed through a 2.2 X 40 cm G-75 Sephadex column

which had been equilibrated with the Con A buffer (CAB). Bonding of the

Con A to the Sephadex enabled the column to be eluted with CAB to

eliminate contaminating proteins. The Con A was then recovered by

eluting the column with CAB containing 0.1 M a-D-glucose or a-D-mannose.

Two-milliliter fractions were collected for spectrophotometric deter-

minations at 280 nm. The fractions containing Con A were dialyzed

against CAB to remove excess sugar. Following dialysis the Con A was

lyophilized and subsequently diluted to the concentrations required

for a particular experiment.













RESULTS


Vegetative Myxamoebae

a. Inhibition of morphogenesis and differentiation by Con A

NN agar plates were prepared containing Con A ranging in

concentration from 50-800 ig/ml agar. Control plates consisted

of NN agar containing only CAB. Using a micropipette 200 pl of

22-hour vegetative myxamoebae cultures at a concentration of

1 X 106 cells/ml were delivered to the plates. The cells were

uniformly distributed over the agar surface with a glass rod.

Observations over a period of 75 hours disclosed that Con A

causes a delay in the appearance of aggregates. The amount of

delay depends upon the dosage which also affects the number of

fruiting bodies which ultimately appear (Table 1).

b: Cyclic AMP effects on prespore cell differentiation

Vegetative myxamoebae from 17-hour cultures following the SS

wash to free them from bacteria were further washed for approximately

5 minutes in 0.001 M cyclic AMP in SS. The cells were then isolated

as individuals on NN agar containing 0.001 M cyclic AMP for 3 hours.

At the end of this period the cells were fixed and prepared for

thin-sectioning. Subsequent examination with the electron microscope

revealed that the cells had not synthesized PV typically found in

prespore cells.










TABLE 1

Inhibition of morphogenesis in vegetative myxamoebae
exposed to agar containing Con A*


Hours at which




Aggregates


various degrees of development
were observed
First
appearance Maximum no.
of mature of mature
sorocarps sorocarps


0 9 26 31 (control)

50 20 26 50 (100)

100 20 44 75 (100)

200 20 44 75 (100)

300 26 44 75 (90)

400 26 44 75 (80)

500 26 44 75 (70)

800 26 44 75 (20)

*The cultures were examined to determine the stage of development
following 9, 20, 26, 31, 44, 50, and 75 hours of exposure to
Con A.
**Percentage of mature sorocarps compared to control.


Con A
solutions
in
ug/ml agar









Aggregating Streams

a. Cyclic AMP effects on prespore cell differentiation

The aggregating streams were washed once in SS containing

0.001 M cyclic AMP and dispersed as individuals on NN agar con-

taining 0.001 M cyclic AMP for a period of 6 hours. The control

streams washed only in SS were dispensed as small dense groups of

myxamoebae on NN agar for a similar period.

The dispersed cyclic AMP treated cells examined in thin-section

did not exhibit PV formation. However, cyclic AMP promotes adhesive-

ness between the cells which resulted in a number of small aggregates.

The cells composing the aggregates were found to have synthesized PV

and were considered to have differentiated into prespore cells. The

control streams within the 6-hour period had formed late aggregates

and migrating pseudoplasmodia (Figures le, If) which emphasized that

sufficient developmental time had elapsed for prestalk and prespore

cells to differentiate.

b. Cyclic AMP effects on plasma membrane structure

The aggregating streams exposed to cyclic AMP as described in

"a" above were subjected to freeze-fracturing.

Freeze-fracturing of the cyclic AMP treated cells revealed

that plasma membrane particles had been synthesized which averaged

97 A (Table 2). The control preparation had formed late aggregates

and migrating pseudoplasmodia. The prespore cells composing these

stages exhibited plasma membrane particles averaging 101 A (Table 2).

c. Con A effects on prespore cell differentiation

The streams from several aggregates were collected carefully

with a hair loop and placed upon small rectangles of PB NN agar.











TABLE 2

Analysis of plasma membrane particles appearing
in the cells under various conditions


Average size
Negative area Numbers of of particles
analyzed particles in A and their
in cm2 measured S.D.

Immediately
fixed control 291.6 511 62 + 14**
aggregates*

Mechanically 217.1 849 61 + 11
disturbed 288.0 519 90 22
aggregates

Control late 345.6 994 101 + 27
aggregates

20 vg/2 il
Con A added 342.6 1,323 70 + 16
to aggregates

1 X 10-3 M cyclic AMP
on dispersed 327.7 1,130 97 + 22
aggregates


*Aldrich and Gregg (1973).

**The difference In particle sizes between the immediately fixed control
aggregates (62 A) agd the smaller particles among the mechanically
disturbed pair (61 A) was not considered to be statistically signif-
icant (p < 0.2). The differences between the immediately fixed control
aggregates and the four remaining aggregate preparations were all con-
sidered to be statistically significant (p < 0.001 in each instance).









Approximately 2 u1 of Con A solution (10 mg/ml) was added to

each aggregating stream which was sufficient to cover the entire

surface area. The cells were exposed to the Con A by a gentle

mixing process using a fine-tipped glass rod. Control preparations

were identical in every respect with the exception that they were

treated only with CAB.

Control aggregating streams required 4 hours to form late

aggregates. The volume and concentration of Con A was carefully

selected to effect a 4-5-hour morphogenetic delay in the aggregates.

Consequently during the experimental period further development of

the Con A treated streams was inhibited. At the end of 4-5-hour

period each preparation treated with Con A or CAB was transferred

with a hair loop into a tiny depression in the agar block to prevent

its loss during the fixation and embedding process (Gregg, 1971).

The control preparations upon thin-sectioning and examination with

the electron microscope were shown to have synthesized PV typical

of prespore cells. No evidence of prespore cell differentiation

could be detected in the delayed aggregating streams. The Con A

inhibited aggregates following the period of delay gradually

recovered and formed mature sorocarps.

d. Con A effects on plasma membrane structure

Aggregating streams delayed for 4 hours with Con A and

control aggregates exposed only to CAB were freeze-fractured.

The control preparations which had formed late aggregates within

4 hours exhibited plasma membrane particles averaging 101 A in

diameter. The membranes of the Con A inhibited cells contained

particles averaging only 70 A (Table 2).









e. Effect of mechanical disruption on prespore cell differentiation

The aggregating streams were disrupted with a tiny glass rod

every 20 minutes for 5 hours to prevent migrating pseudoplasmodium

formation. Although this procedure did not disperse the cells many

cell contacts may have been broken momentarily. This experiment

was conducted to determine if cell differentiation will occur in

spite of the periodically disrupted cell contacts which prevent

the attainment of normal polarized late aggregates. Aggregating

streams which were left undisturbed for a 5-hour period served as

controls.

The disrupted aggregates examined by thin-section electron

microscopy were devoid of PV although control late aggregates

had differentiated prespore cells.

f. Effect of mechanical disruption on plasma membrane structure

The aggregating streams were prepared for freeze-fracture

studies as described above under "e."

The freeze-fractured disrupted aggregates were found to have

plasma membranes in two states of development. Approximately 80-

90% of the cells exhibited membrane particles having an average

size of 61 A in diameter which is typical of the aggregation stage.

The other 10-20% of the cells had much larger membrane particles

averaging 90 A which approaches the size of those found in prespore

cells (Table 2). The undisturbed controls formed late aggregates

within 4 or 5 hours which exhibited large particles averaging 101 A

in diameter.









Early Culminates

The small cylinders of agar bearing the beginning culminates were

positioned such that the long axis of the slime mold was horizontal

(Figure 5). This position prevented the drops of Con A solution

(20 mg/ml) from touching the basal disc and the agar base which would

result in a loss of the solution. The volume of Con A applied was

approximately that of the volume of the prespore mass. By restricting

the volume of Con A, which was applied with a fine-tipped glass pipette

to the prespore area, the majority of the prestalk cells did not appear

to be affected. The controls consisted of early culminates which had

distilled water or CAB applied to the prespore area.

a. Effect of Con A, distilled water and CAB on culmination

Beginning culminates in which the prespore area only was

exposed to any one of the three agents formed fruiting bodies of

the type illustrated in Figure 5. The prespore cells remained at

the original level they had attained at the time the agent was

applied. A group of cells composed primarily of prestalk cells

but possibly including a small number of prespore cells continued

to culminate. This resulted in a mature stalk bearing a small

mass of normal mature spores at the apex.

b. Effect of Con A on mature spore differentiation

The cells on the peripheral layers of the prespore mass having

the greatest exposure to Con A were devoid of PV and contained large

autophagic vacuoles. Although no cell walls had developed, a layer

of amorphous electron-dense material surrounded the cells which were

abnormally irregular in shape. The mitochondria retained the form

























Figure 5. Application of Con A or control solutions to the
prespore area of early culminates.

a. early culminate on an agar base.
b. early culminate tipped to 900 angle for
exposure to small volumes of reagents in
the prespore area
c. prespore area exposed to reagents
d. type of mature sorocarp produced following
exposure to reagents. (Apex composed of small
mass of normal mature spores in both Con A
and control preparations.)













- prestalks


-- prespores




26




normally observed in prespore cells (Figures 6, 7). The mitochondria

in the remaining cells of the prespore mass were essentially typical

of those found in normal mature spores. However, the PV were absent

and cell walls failed to appear (Figure 8).

All of the cells composing the prespore mass upon exposure

to distilled water or CAB differentiated into normal spores,

lacking PV and exhibiting a cell wall (Figures 9, 10). Occasionally

a few spores exhibited empty spaces in the cytoplasm which may have

resulted from a loss of glycogen during the preparation for electron

microscopy.





























Figure 6. Cell from the peripheral region of the prespore
mass after exposure to Con A, X19,566. Note the
defective cell wall composed of an amorphous layer
of electron-dense substance surrounding the
cell surface.

AV autophagic vacuole
M mitochondrion, typical form found in
prespore cells
N nucleus

























.i.

/.-
3j"


y j


























Figure 7. Cell from the mid-prespore region after exposure to
Con A, X26,088. Note the layer of electron-dense
substance composing the defective cell wall and the
irregular cytoplasmic spaces which may have contained
deposits of glycogen.

M mitochondrion, typical form found in prespore
cells














^ ~~ ANA1/



























Figure 8. Cell from the central region of the prespore mass
after exposure to Con A, X26,088. Note that the cell
wall is atypical in thickness and associated with the
cell surface by a layer of electron-dense substance.
Arrows denote mitochondria essentially similar to those
observed in controls.





s;,,2
\h~%
































Figure 9. Cell from the prespore region after exposure to
distilled water exhibiting normal cell wall,
X26,088. Arrows denote mitochondria typical
of normal mature spores.

N nucleus





34


















Z
/-* 11. N


-."Yti"i~;i~,~-;g)





























Figure 10. Cell from the prespore region after exposure to
CAB exhibiting normal cell wall and the irregular
cytoplasmic spaces which may have contained deposits
of glycogen, X34,784. Arrows denote mitochondria
typical of normal mature spores.







































































































--













DISCUSSION


Cyclic AMP in Prespore Differentiation

Cell differentiation in Dictyosteiwnm depends upon cell association.

Aggregation of the cells in response to the chemotactic stimuli from

cyclic AMP results in the necessary contacts (Konijn et al., 1967).

The close association between the cells provides the opportunity for

a potential accumulation or exchange of critical metabolites as well

as interaction between components of the plasma membranes. The identity

of metabolites possibly involved in effecting cell differentiation are

obscure, with the exception of the report by Bonner (1970) that cyclic

AMP induces mature stalk cell formation. A major increase in the average

size of the plasma membrane particles has been correlated with the

differentiation of the prespore cells (Aldrich and Gregg, 1973). The

particles are the only ultrastructural components in the membrane known

to change during this period although their precise role in development

has not been established.

Gregg and Nesom (1973) determined that the synthesis or assembly

of the large plasma membrane particles could be induced in vegetative

myxamoebae within 2 hours by exposure to cyclic AMP. Myxamoebae from

aggregating streams dispersed as single cells for 6 hours upon agar

containing cyclic AMP synthesized particles averaging 97 A in size.

This represented a 1.6X increase above the immediately fixed control

preparations at 62 A (Table 2). Neither vegetative myxamoebae nor









aggregating streams were found to contain PV (Gregg and Badman, 1970).

Upon thin-sectioning and examining dispersed vegetative and aggregating

stage cells exposed to cyclic AMP it was determined that PV had not

formed indicating that the cells had failed to differentiate into

prespores. Thus, the synthesis of large particles may occur independently

of PV formation and prior to cell differentiation. Occasionally a few

small aggregates formed on the cyclic AMP agar. These cells had

synthesized large particles and also PV. This emphasizes the necessity

of cell contact in promoting cell differentiation. Preparations of

aggregating streams left undisturbed during the 6-hour period as

controls had formed late aggregates containing PV and particles

averaging 101 A.

Cyclic AMP also induces adhesiveness between the cells (Konijn

et aZ., 1968) and simultaneously causes an efflux of Ca++ (Chi and

Francis, 1971) which may also be involved in the appearance of large

membrane particles (Gregg and Nesom, 1973). The plasma membrane in

D. discoideum is approximately 70 A in thickness. It is conceivable

that the larger particles and their associated carbohydrate components

project through the membrane and are exposed at the cell surface. This

suggests that cell adhesion may be effected by the interaction between

the projecting particles. Under these circumstances movement or re-

arrangement of the particles within a fluid plasma membrane might occur.

As a result of the shifting particles stimuli may be transmitted to

intracellular components which in turn initiate the process of differ-

entiation.

During normal development of Dictyostelimw it is probable that

following aggregation the concentration of cyclic AMP increases within









the cell mass and initiates large particle formation. A period of

cell interactions possibly mediated through the particles results

in prespore cell differentiation. The necessity of the cells to

maintain contact in order to differentiate was determined by mechani-

cally disrupting the aggregate every 20 minutes for 5 hours. The

greater proportion of the cells examined by freeze-fracturing had

not synthesized large particles suggesting that cyclic AMP secretion

may have been inhibited. A small proportion of the cells exhibited

particles averaging 90 A which approaches the average size of those

found in prespore cells. Neither group of cells had differentiated

into prespore cells as indicated by the absence of PV. The controls

had attained the late aggregate stage in 5 hours and exhibited both

large membrane particles and PV. Consequently it appears that a

certain period of continuous contact between the cells is essential

in cell differentiation.


Con A in Prespore Differentiation

Con A binds to vegetative myxamoebae cell surfaces and quickly

effects agglutination under appropriate conditions. If the cells are

exposed to Con A in agar the rate of aggregation is inhibited as well

as the number of mature sorocarps which ultimately appear (Table 1).

The ability of Con A to bind to cell surfaces provided a tool to

elucidate the nature and role of cell contacts in differentiation.

Groups of cells isolated from aggregating streams require approximately

4 or 5 hours to attain the late aggregate stage. If such preparations

are exposed to Con A the cells are held in contact to the degree









allowed by the Con A bonded to their plasma membranes. These aggregating

streams were delayed throughout the period required for the controls to

form late aggregates. Electron microscopy of the delayed preparations

revealed that membrane particles had formed which were only 1.1X larger

than the immediately fixed controls. However, the synthesis of particles

of this magnitude was not followed by PV formation. The controls forming

late aggregates contained PV and particles 1.6X greater than the

immediately fixed controls (Table 2).

The chemotactic response to cyclic AMP also depends upon a membrane

bound phosphodiesterase (PDE). PDE increases sharply in activity just

prior to aggregation but returns to lower levels shortly after this

morphogenetic event is initiated (Malchow et aZ., 1972). The failure of

the aggregates in differentiating prespore cells may be attributed to

the ability of Con A to cause the synthesis of excessive amounts of PDE

in vegetative myxamoebae prior to its customary appearance at aggregation

(Gillette and Filosa, 1973). Observations of other systems suggest that

alterations in the activity of PDE or adenyl cyclase may result from the

clustering of Con A glycoprotein receptors at the cell surface (Edelman

et aZ., 1972). Gudrin et aZ. (1974) have determined that Con A induced

agglutination of murine plasmocytoma cells was accompanied by the clumping

of intramembranous particles. The Con A binding sites are not considered

to be associated with the particles in either plasmocytoma or in E.

histoZytica membranes (Martfnez-Palomo et aZ., 1974).

The existence of cyclic AMP binding sites on the cell surface of

D. discoideum has been reported by Moens and Konijn (1974). The

possibility exists that Con A masks these sites and prevents cyclic









AMP from bonding to the membrane. It is conceivable that PDE activity

may also be increased by Con A among the cells in an aggregating stream

as well as in vegetative myxamoebae. Both PDE and masked cyclic AMP

binding sites could have the effect of reducing the concentration of

cyclic AMP available at the cell surfaces. Consequently normal

morphogenetic movements could be delayed as well as the assembly of

large membrane particles which depend upon the presence of cyclic

AMP.


Con A in Culmination and Mature Spore Differentiation

By the late aggregation stage large plasma membrane particles have

been synthesized and both prestalk and prespore cells have differentiated.

If a group of prespore cells are isolated from a migrating pseudoplasmodium

a certain proportion will redifferentiate into prestalk cells devoid of

PV (Gregg and Badman, 1970). If the prespores are isolated as single

cells neither redifferentiation into prestalk nor differentiation into

mature spores can occur (Gregg, 1971).

The differentiation of the prespore cells into mature spores begins

during the mid-point of the culmination process (Figure li). An early

culminate which has formed a basal disc and whose prespore cell mass

has barely cleared the agar substratum will not have differentiated

mature spores (Figures lh, 5a). Early culminates disorganized period-

ically for 6 hours with a glass needle cannot differentiate mature

spores (Gregg and Badman, 1970). Obviously cell contact is required

for either redifferentiation or differentiation at various points in

the development of DictyosteZiun.






L ________








During the normal transition of a prespore into a mature spore, PV

disappear from the cytoplasm and are considered to be incorporated into

the spore wall (Hohl and Hamamoto, 1969). Although small volumes of

distilled water, CAB and Con A affected the morphogenetic process of

culmination (Figure 5) only Con A prevented normal spore differentiation.

The major defect observed was in the formation of the spore wall. PV

also were not found among the defective spores induced by Con A. This

suggests that Con A is not bound to the sites involved in the cell inter-

actions which may effect the synthesis or loss of PV. However, the presence

of bound and free Con A at the surface of the prespores produced irregular

shaped spores surrounded by a layer of amorphous electron-dense material

in place of a cell wall (Figures 6, 7,8). This aberrant wall may have

resulted from Con A agglutinating and interfering with the PV and other

constituents which were liberated at the cell surface in the course of

spore differentiation. The defective spores retained mitochondria having

the typical form of those in prespore cells (Figures 6,7). This may be a

consequence of the failure of the spore to attain its normal morphogenetic

shape resulting in further effects upon the morphology of these cytoplasmic

organelles.

Role of Cell Contacts and Interaction in Differentiation

The induction of plasma membrane particles by cyclic AMP averaging

approximately 100 A in diameter is always associated with cell differ-

entiation in DictyosteZium. Although the particles may be synthesized

among isolated single vegetative or aggregating cells this event is not

simultaneously accompanied by prespore cell differentiation. If

aggregates are mechanically disrupted periodically for 5 hours a few

cells exhibit large particles but PV formation does not occur. Obviously









several hours of continuous contact between the cell surfaces is

necessary to effect prespore cell differentiation. Upon aggregation

in response to cyclic AMP the cells become adhesive (Konijn et at.,

1968). The contact sites active in the adhesive process may be

inhibited by specific univalent antiserum. Although random cell

movement continues migrating pseudoplasmodium formation is prevented

(Beug et al., 1970). Univalent Con A, however, does not interfere

with normal morphogenesis in Dictyostelizu (Weeks and Weeks, 1975).

The possibility that Dictyosteliwn adhesive sites are associated

with the membrane particles has not been established. The relative

effects of univalent Con A and antiserum suggest that the adhesive

sites could be associated with the particles considering that Con A

fails to bind to those membrane constituents (Martinez-Palomo et al.,

1974). Divalent Con A delays morphogenetic movements and prevents

cell differentiation probably by indirectly effecting a reduction in

the cyclic AMP necessary in particle formation. Thus, any cellular

interactions mediated through contacts among these membrane particles

could not occur.

During the process of culmination prespore cells bearing large

membrane particles differentiate into mature spores. The PV distributed

in the prespore cytoplasm break down and become part of the spore wall

(Hohl and Hamamoto, 1969). Exposure of the prespores to Con A just

prior to spore differentiation does not hinder the disappearance of

the PV. Since cell contact is essential in spore formation (Gregg and

Badman, 1970) the Con A evidently does not mask nor affect the sites

necessary in effecting PV loss. However, Con A does act at the cell




44



surface causing defective cell walls to appear as well as abnormally

formed spores containing atypical mitochondria.













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48




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BIOGRAPHICAL SKETCH


Ning Yueh Yu was born September 10, 1947 at Wu Ching, China and

raised in Taiwan. In June 1969, she received the Bachelor of Science

degree in Biology from the Tunghai University, Taiwan, and was employed

after graduation as a research assistant until March 1970 in the

Zoology Department Academic Sinica, Taipei. Enrolling in the Graduate

School of the University of Florida in March 1970, she completed her

Master of Science degree in Zoology in June 1971. Continuing in

Graduate School she pursued work toward the degree of Doctor of

Philosophy. She was appointed as a research assistant to Dr. J. H.

Gregg, Professor of Zoology, for the period September 1970, to the

present.

Ning Yueh Yu is married to Hsi-Ling Yu.









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.




James H. Gregg, Ct~irman
Professor of Zoology



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.




JohnNW. Brookbank
/Professor of Zoology



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 .
Frances C. Davis
Assistant Professor of Zoology



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.




Henry C. Aldrich
Associate Professor of Botany










This dissertation was submitted to the Graduate Faculty of the Department
of Zoology in the College of Arts and Sciences and to the Graduate Council,
and was accepted as partial fulfillment of the requirements for the degree
of Doctor of Philosophy.

March, 1975



Dean, Graduate School




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