1 IDENTIFICATION AND CHARACTERIZATION OF NUCLEAR ENVELOPE PROTEINS By DAE IN KIM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCT OR OF PHILOSOPHY UNIVERSITY OF FLORIDA 201 2
2 201 2 Dae In Kim
3 To Jiyoung, June and Jayden
4 ACKNOWLEDGMENTS I would like to thank to my mentor, Dr.Brian Burke, for welcoming me into his lab and supporting me throughout my graduate studies. His suggestions always and amazingly worked and showed right direction for me I will never forget the care Brian took of me Whenever I needed, Brian welcomed to travel from Singapore. I would like to thank to Dr Kyle Roux He taught me how to present d ata and speak in scientific language I was lucky to be educated by Brian and Kyle. I would like to thank to my committee members, Drs. John Aris, Brian Harfe, and Lucia Notterpek for their insightful comments, suggestions and for generously sharing their expertise and individual point of view. I am indebted to my family. W ithout t heir unconditional love and support I could not finish this dissertation
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ................... 10 C HAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Overview: Organization of the Nuclear Envelope ................................ ................... 12 The N uclear E nvelope ................................ ................................ ............................ 13 The N uclear P ore C omplexes (NPCs) ................................ ................................ .... 17 The N uclear L amina ................................ ................................ ............................... 19 I nner N uclear M embrane P roteins ................................ ................................ .......... 2 3 The LINC complex ................................ ................................ ................................ .. 26 Nuclear P roteins and H uman D iseases ................................ ................................ .. 32 2 D ALEK6: A NOVEL MAMMALIAN MEIOSIS SPECIFIC KASH DOMAIN PROTEIN REQUIRED FOR GAMETOGENESIS ................................ ................... 38 Introduction ................................ ................................ ................................ ............. 38 Results ................................ ................................ ................................ .................... 41 Discussion ................................ ................................ ................................ .............. 48 Materials and Methods ................................ ................................ ............................ 56 Ge neration of Dalek6 null M ice ................................ ................................ ........ 56 Plasmids ................................ ................................ ................................ ........... 56 Cell Lines ................................ ................................ ................................ .......... 56 Antibo dies ................................ ................................ ................................ ......... 57 Immunofluorescence ................................ ................................ ........................ 57 I mmunoprecipitations ................................ ................................ ....................... 57 Isolation of M ouse S permatocytes ................................ ................................ ... 57 TEL FISH ................................ ................................ ................................ ......... 58 TUNEL Assay ................................ ................................ ................................ ... 58 3 IDENTIFICATION OF NUCLEAR PROTEINS BY A NO VEL METHOD USING A MUTATED BIOTIN LIGASE ................................ ................................ .................... 74 Introduction ................................ ................................ ................................ ............. 74 R esults ................................ ................................ ................................ .................... 75 D iscussion ................................ ................................ ................................ .............. 78 Materials and Methods ................................ ................................ ............................ 85
6 Plasmids ................................ ................................ ................................ ........... 85 Cell C ulture and G eneration of S table C ell L ines ................................ ............. 86 Immunofluorescence ................................ ................................ ........................ 86 Western B lotting ................................ ................................ ............................... 86 Affinity C apture of B iotinylated P roteins ................................ ........................... 86 Protein I dentification by M ass S pectrometry ................................ .................... 87 4 CONCLUSION ................................ ................................ ................................ ........ 94 Dalek6 Is an ONM Protein Required for Early Meiotic Progression in Mice ............ 94 Overview of Find ings ................................ ................................ ........................ 94 Significance ................................ ................................ ................................ ...... 95 Future Directions ................................ ................................ .............................. 96 BioID Is a Novel Me thod to Identify Protein Protein Interactions ............................ 99 Overview of Findings ................................ ................................ ........................ 99 Significance ................................ ................................ ................................ .... 100 Future Directions ................................ ................................ ............................ 102 LIST OF REFERENCES ................................ ................................ ............................. 104 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 122
7 LIST OF FIGURES Figure page 2 1 Identification of Dalek6. ................................ ................................ ...................... 59 2 2 Dalek6 is a genuine KASH family member. ................................ ........................ 60 2 3 Dalek6 recruits cytoplasmic dynein to the NE ................................ .................... 61 2 4 Dalek6 antibody (anti Dalek6) specifically recognizes murine Dalek6 (MmDalek6) i n immunofluorescence and Western blot ................................ ...... 62 2 5 Dalek6 is predominantly expressed in the adult testis ................................ ........ 63 2 6 Dalek6 is localized at the meiotic attachment plates in primary spermatocytes 64 2 7 Dalek6 colocalizes with cytoplasmic dynactin in primary spermatocytes ............ 65 2 8 Dalek6 appears to be the only KASH domain protein expressed i n primary spermatocyte s ................................ ................................ ................................ .... 66 2 9 Validation of Dalek6 null mice ................................ ................................ ............ 67 2 10 Spermatogenesis is largely disrupted in the seminiferous tubule of Dalek6 null mice ................................ ................................ ................................ ............. 68 2 11 Cell death was occurred in Dalek6 null mice ................................ ...................... 69 2 12 Oogenesis is disrupted in Dalek6 null mice ................................ ........................ 70 2 13 Sun1 remains associated with the NE in sper matocytes of Dalek6 null mice ..... 71 2 14 Loss of Dalek6 does not prevent entry into meiotic prophase. ........................... 72 2 1 5 Model of Mammalian LINC complex fun ction during meiotic prophase I ............ 73 3 1 BirA* promiscuously biotinylates endogenous proteins in mammalian cells.. ..... 89 3 2 Proximity dependent promiscuous biotinylation by BioID LaA. .......................... 90 3 3 Temporal regulation of access to excess biotin controls biotinylation by BioID .. 91 3 4 SLAP75 is a novel NE consti tuent ide ntified with BioID LaA .............................. 92 3 5 SLA P75 is a constituent of the INM ................................ ................................ .... 93
8 LIST OF ABBREVIATION S ABD Actin binding domain AD EDMD Autsomal dominant EDMD BAF Barrie r to autointegration factor BioID Biotin based identification C. elegans Caenorhabditis elegans Dalek Dynein associated LIN C engaged KASH D. melanogaster Drosophila melanogaster DCM CD1 Dilated cardiomyopathy with conduction defects DHC Dynein heavy chai n DIC Dynein intermediate chain DNA Deoxyribonucleic acid ER E ndoplasmic reticulum EDMD Emery Dreifuss Muscular Dystrophy FPLD2 Dunnigan type familial partial lipodystrophy GFP Green fluorescence protein HGPS Hutchinson Gilford progeria syndrome HRP Hor se radish peroxidase IF Immunofluorescence INM Inner nuclear membrane KASH Klarsicht, ANC 1, Syne homology kD a Kilodalton LaA Lamin A LAP Lamin A polypeptide LBR Lamin B receptor
9 LEM LAP, emerin, MAN1 LGMD1B L imb girdle muscular dystrophy type 1B LINC Li nker of nucleoskeleton and cytoskeleton MAD Mandibuloacral dysplasia MDa Megadalton MSC Messenchymal stem cell (MSC). MTOC Microtubule organizing center NE Nuclear envelope Nesprin Nuclear envelope spectrin repeat NLS Nuclear localization signal NMJ Neur omuscular junction NPC Nuclear pore complex Nup Nucleoporin ONM Outer nuclear membrane PNS Perinuclear space RD Restrictive dermopathy RNA Ribonucleic acid SREBP1 S terol response element binding protein 1 SUN Sad1p Unc 84 Syne Synaptic nuclear envelope TX 100 Triton X 100 WS Werner's syndrome WT Wild type X EDMD X linked EDMD Zmpste24 Zinc Metalloproteinase STE24
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirement s for the Degree of Doctor of Philosophy IDENTIFICATION AND CHARACTERIZATION OF NUCLEAR ENVELOPE PROTEINS By Dae In Kim M ay 20 1 2 Chair: Brian Burke Major: Medical Sciences Molecular Cell Biology The nuclear envelope (NE) is composed of inner an d outer nuclear membranes, a nuclear lamina, and nuclear pore complexes. M utations in NE proteins particularly the A type lamins, which are major constituents of the nuclear lamina, are linked to a variety of human diseases. These range from striated musc le disease, including muscular dystrophy and cardiomyopathy to lipodystrophy and premature aging syndromes. Given that the A type lamins and many other components of the NE are expressed in the majority of cell types, it remains a puzzle how defects in th ese proteins can give rise to such an array of tissue specific disorders At present none of these so called laminopathies is truly understood at the molecular level. One potential explanation for the tissue specific phenotypes associated with lamin mutati ons is that these defects perturb interactions with other proteins which are themselves expressed in a tissue specific fashion. Clearly an important goal must be to identify new NE associated proteins as well as to define interaction networks at the nuclea r periphery. However, the insolubility of many NE components, the lamins in particular, has represented a significant barrier to such analyses. I have applied two very different, but complementary approaches to overcome this barrier. The first approach inv olves the
11 identification and characterization of novel nuclear membrane components based on homology to other known NE constituents. Such analyses yielded an unknown protein which we have named Dalek 6 T his protein is a member of the KASH domain family and is a resident of the outer nuclear membrane. It is found predominantly in germ cells during meiotic prophase I It is specifically localized to meiotic attachment plates sites at which telomeres are associated with the NE Dalek 6 functions as a binding p artner for cytoplasmic dynein/dynactin which it will recruit to the outer nuclear membrane Dalek6 knockout mice lack post meiotic germ cells illustrating the requirement of this protein for events that occur during early meiosis This finding expands our understanding of the role of the NE in mammalian biology and reproduction The second approach that I have taken to define protein interactions at the NE involved the development of an entirely new method that takes advantage of proximity dependent biotiny lation in vivo Called BioID, this method capitalizes on a promiscuous biotin protein ligase that when fused to a targeting protein results in labeling of neighboring proteins, permitting their isolation and identification. Conjugati on of this bi otin ligase to lamin A (LaA), resulted in the specific biotinylation of known LaA associated proteins, thus validating the overall strategy. Furthermore, the BioID approach led to the identification of an entirely new NE constituent that we have called SLA P75. We propose that this method will prove valuable, not just for the characterization of protein interaction within the NE, but that it will provide a general approach for the analysis of protein interactions and proximity in a wide variety of cell types and biological systems.
12 CHAPTER 1 INTRODUCTION Overview: Organization of the Nuclear Envelope The eukaryotic cell partitions its genetic material into a separate compartment called the nucleus. Originally considered a simple container for DNA it has b ecome increasingly clear that the interphase nucleus is in fact a highly compartmentalized organelle (Schermelleh et al., 2008) C hromosomes are now known to occupy discrete territories (Zhang and Pugh, 2011) and various regulatory proteins are present in a plethora of nuclear bodies that are distributed throughout the nucleoplasm (Kumaran and Spector, 2008) Some of these structures, such as nucleoli are stable for exte nded periods. Others, such as replication factories are more transient. The nucleus has several unique architectural components, the most peripheral of which is the nuclear envelope (NE). The NE is comprised of two lipid bilayers, the outer nuclear membra ne (ONM) and inner nuclear membrane (INM). The nuclear pore complexes (NPC) that regulate n ucleo cytoplasmic transport of macromolecules form at site s where the ONM and INM are connected (Gerace and Burke, 1988) Under lying the INM is the nuclear lamina, a protein meshwork serving at least in part as a structural scaffold. In addition to the nuclear lamina at least 60 integral membrane proteins are enriched in the INM These may c ontribute to organization of the NE as well as imparting specialized functions ONM specific proteins are also known. Certain of these define a recent identified structur al component of the NE known as the LINC complex (LInker of Nucleoskeleton and Cytoskeleton) that mechanically couples the nucleus and the cytoskeleton (Crisp et al., 2006; Padmakumar et al., 2004; Starr and Han, 2002;
13 Zhang et al., 2001; Zhen et al., 2002) This is discussed more extensively in later sections. It has become ever more evident that the NE is fundamental to the maintenance of nuclear organization, nuclear positioning and overall cytoarchitecture. The sig nificance of the NE is best illustrated by the growing list of mu tations in NE constituents particularly the A type lamins, which are major co mpon ents of the nuclear lamina and which are linked to a myriad of human diseases (Burke and Stewart, 2006) P roteomic analys e s of NE s, isolated from a variety of sources have identified at least 60 nuclear membrane proteins the functions of which are larg ely unknown (Schirmer et al., 2003a) Many of these are enriched in some cell types but not in others (Korfali et al., 2010; Schirmer et al., 2003a; Wilkie et al., 2011; Wilki e and Schirmer, 2008) The implication is that NE may have functions that are themselves tissue specific In this chapter, I will introduce our current understanding of the structure and function of the NE including the nuclear pore complexes, nuclear la mina, nuclear membrane proteins and the LINC complex. Finally, I will describe human diseases linked to mutations in genes encoding NE specific proteins. A complete understanding of the etiology of these diseases or novel therapeutic strategies requires that we grasp how individual NE constituents function together as a whole. At the same time, these diseases, and corresponding animal models, are providing new insight into the role that the NE plays as a determinant of both nuclear and cytoplasmic architecture. The N uclear E nvelope The NE is a selective barrier that separat es the nucleus from the cytoplasm and in many respects defines the biochemical identities of both compartments Small molecules and ions have free passage across the NE while larger molecules ( for
14 instance globular proteins >40 kDa) require active transport mechanisms (Stewart, 2007; Terry et al., 2007) While the ONM and INM are separated by a fairly uniform 30 50 nm wide lumen, known as the perinuclear space (PNS), they are periodically connected at annular junctions that form aqueous channels between the cytoplasm and the nucleoplasm and which are occupied by large (50 120MDa) multiprotein assemblies calle d the nuclear pore complexes (Watson, 1955) It is the NPCs that mediate the movement of molecules, both large and small, across the NE. In addition to its junctions with the INM at NP Cs, the ONM also displays periodic connections to the peripheral endoplasmic reticulum (ER). In this way, the ER, ONM and INM represent discrete domains within a single continuous membrane system with the PNS forming a perinuclear extension of the ER lumen Despite their continuities, each of these membrane domains is biochemically distinct. The ONM like the ER is studded with ribosomes. However, the ONM features a number of integral membrane proteins that are absent from the ER. The INM lacks bound ribosom es but is intimately associated with the underlying nuclear lamina and possesses and array of resident integral membrane proteins that found in neither the ER nor the ONM (Padmakumar et al., 2004; Zhen et al., 2002) In metazoa, underlying the INM is a network of intermedi ate filament proteins that form the nuclear lamina. This protein meshwork is primarily composed of A and B type lamins that, in part, functions to structurally support the NE. Like all intermediat e filament proteins A and B type lamins feature a central alpha helical rod domain flanked by non helical head and tail domains. Although B type lamins are present in all nucleated cell types the A type lamins are not found in early embryonic cells. The y are
15 similarly absent from many stem cell populations. In mice, A type lamin expression commences only after embryonic day 7 8 initially in visceral endoderm (Lenz Bhme et al., 1997; Liu et al., 2000; Sullivan et al., 1999b) Mutations in A type lamins are associated with a wide range of human diseases including muscular dystrophy, lipodystrophy, neuropathy and progeria (Burke and Stewart, 2006) T o date, over 2 5 0 disease linked mutations have been documented in the human LMNA gene that encodes A type lamins (Worman et al., 2010) M ore recently characterized components of the NE are the LINC complex es (for LI nker of N ucleoskleton and C ytoskeleton). These consist of members of the KASH domain family of ONM proteins that physically interact across the PNS with SUN domain proteins in the INM. SUN domain proteins are known to interact with lamins and other nuclear components while KASH domain proteins typically interact with elements of the cytoskeleton. Together, KASH and SUN domain proteins represent a pair links in a molecular chain that mechanically couples nuclear and cytoplasmic structures (Crisp et al., 2006) Both KASH and SUN domains are highly conserved during evolution. The KASH do main, which can be recognized in yeast, consists of 50 60 C terminal amino acids. It features a single membrane spanning domain of roughly 20 22 amino acid residues followed by a short luminal domain of 30 40 residues. KASH domains typically feature a C te rminal cluster of 2 3 proline residues terminating with a single aliphatic residue. KASH domain proteins lack an N terminal signal sequence. They are always oriented with the N terminus exposed to the cytoplasm and the C terminus residing within the PNS. B ecause KASH domain family members are tail anchored, they must be inserted in to the ER/ONM post translationally. Fusion of KASH domains to reporter
16 molecules such as GFP, reveal that the KASH domain alone that is sufficient to confer stable localization t o the ONM (Crisp et al., 2006; Roux et al., 2009) The SUN domain consists of a sequence of approximately 200 amino acid residues. It is typically located at the C terminus, although there is a single exception to this rule. Like the KASH domain, with which it interacts, the globular SUN domain extends in to the PNS usually at the end of a coiled terminus of SUN domain proteins, which lack an N terminal signal sequence, always faces the nucleopl asm (or cytoplasm). Thus SUN domain proteins have the typical topology of type II membrane proteins. In mammalian somatic cells there are two known SUN domain proteins, Sun1 and Sun2. Both of these localize exclusively to the INM T argeting of KASH domain p roteins to the ONM is critically dependent on the interaction with SUN domain proteins in the PNS. SUN domain protein localization is KASH independent. Th e interaction between SUN and KASH domain proteins contribute s to the maintenance of the NE structure Perturbation of the LINC complex either by RNA interference mediated down regulation of Sun1 and 2 or expression of a dominant negative form of S un 1 led to periodic expansion of the PNS and increased separation of the INM and ONM (Crisp et al., 2006) SUN domain interactions appear to be quite promiscuous. The result is that there is a high degree of redundancy between Sun1 and Sun2. This is evident from studies in mice, where animals deficient in either Sun1 or S un2 are viable. In contrast, deficiency of both Sun1 and Sun2 is peri natal lethal (Lei et al., 2009; Zhang et al., 2009) The large N terminal domains of KASH proteins physically connect the nucleus to cytoskeletal components. At the time that I began my thesis work, four members of this
17 family were known, Nesprins 1, 2, 3 and 4 (Nesp1 4). The largest isoforms of Nesp1 and 2 (~1,000 and 800kDa respectively) both feature pairs of N terminal calponin homology domains (CHDs) which function in actin binding. In addition, at least Nesp2 is also a binding partner for both conventional k inesin 1 and cytoplasmic dynein, providing links to the microtubule system. Nesp3 and 4 are significantly smaller (~100 and 42kDa respectiv ely). Nesp3 is a binding partner for plectin, a versatile cytolinker that provides a connection to the intermediate filament system. Nesp4 on the other hand, which is found primarily in epithelial cells, functions as a NE adaptor for k inesin 1 (Burke and Roux, 2009; Schneider et al., 2011; Zhang et al., 2009) As the only known mechanism t o link the cytoskeleton to the nucleus, the LINC complex has important roles in nuclear positioning, organization of nuclear conte nts (a conclusion of this thesis) nuclear anchoring, nuclear structure and the establishment and maintenance of the cytoarchitecture. The N uclear P ore C omplexes (NPCs) The NPC s are extremely large roughly cylindrical structure s with 8 fold radial symmetr y. Each NPC is anchored in the pore membrane domain of the nuclear envelope, where the inner and outer nuclear membranes are contiguous Anchoring is mediated by at least two NPC specific integral membrane proteins. Th e NPC is composed of ~30 distinct prot eins, collectively called nucl e oporins (Nups) Each Nup is present in multiples of eight copies per NPC and are organized within discrete subcomplex e s. The basic structure of the NPC is organized around a massive central framework which resides at the leve l of the nuclear membranes. This core structure embraces a central channel of 30 40nm and displays 2 fold symmetry about an axis parallel to the plane of the nuclear membranes. Thus the central framework lacks any
18 polarity and could not itself, impose dire ctionality on the nucleocytoplasmic transport process (Akey and Radermacher, 1993; Hinshaw et al., 1 992) The overall asymmetry of the NPC is determined by peripheral structures. The cytoplasmic face of the NPC features eight short (50 100nm) flexible filaments that contain Nup358 (also known as Ran binding protein 2, RanBP2) (Dworetzky et al., 1988; Feldherr et al., 1984) The nucleoplasmic face of the NPC features a basket like structure consisting of eight stiff filame nts of 50 100nm that are joined at their tips to a 50nm distal ring. Major components of this basket structure, which resides at the level of the nuclear lamina, are Tpr, Nup153 and Nup50. (Melchior et al., 1995; Rou t et al., 2000; Stoffler et al., 1999) The major function of NPCs is the regulation of bidirectional trafficking of macromolecules between the nucleus and cytoplasm. Although molecules smaller than 40kDa can passively diffuse across the NPC, passage o f larger molecules is unfavorable (Terry et al., 2007) In other words, there are facilitated transport mechanisms to regulate transit of these larger macromolecules Several models have been proposed that describe the mechanism for movement through NPCs. All invoke facilitated diffusion controlled by association and disassociation of transport receptors, belonging to the karyophe rin family (also known as importin s, exportin s and transportins ) (King et al., 2006; Saksena et al., 2006) that coordinate with a s ubset of Phe Gly (FG) repeat containing Nups (Mattaj and Englmeier, 1998; Talcott and Moore, 1999) In addition, the directionality of Kap mediated transport is exquisitely controlled by the small GTPase Ran, whose nucleotide bound state dictates cargo binding and release in a compartmentalized manner (Fried and Kutay, 2003; Pemberton and Kay,
19 2005) However, different Kaps may require different FG rich Nups, which allow the existence of multiple, nonequivalent transport pathways within each NPC (Rout and Aitchison, 2001) In metazoa, the distribution of NPCs within the NE is tightly controlled. Studies in mammalian cells revealed that NP Cs are stably anchored within the NE, forming immobile arrays with little independent movement (Daigle et al., 2001; Rabut et al., 2004) Evidence that NPC movement might be restricted in part by interactions with the nuclear lamina came from early cell fractionation studies. Blobel and colleagues extracted purified rat liver nuclear envelopes in nonionic detergent leaving behind a porecomplex lamina fraction in which morphologically identifiable NPCs could be seen associated with the fibrous nuclear lamina (Aaronson and Blobel, 1975; Dwyer and Blobel, 1976) The N uclear L amina Lining the nuclear face of the INM is a network of intermediate filament prot eins that constitutes the nuclear lamina. B y binding both INM proteins and chrom atin components, t his protein lattice serves as a structural scaffold for the NE while at the same time providing an anchoring site for chromosome domains at the nuclear periph ery (Stewart et al., 2007) Disruption of the nuclear lamina leads to defects in both nuclear and NE morphology (Aebi et al., 1986; Burke and Stewart, 2006; Gerace and Burke, 1988a; Liu et al., 2000; Mounkes et al., 2003; Ris, 1997; Schirmer and Gerace, 2004; Stuurman et al., 1998) The major co nstitu ent s of the nuclear lamina are the A and B type lamins, type V intermediate filament proteins (Moir et al., 2000; Stuurman et al., 1998) Like all intermediate filaments, lamins have a central alpha helical rod domain ( of about 40 kD a
20 in the case of the lamins) flanked by a small globular head domain (N terminal) and a much larger tail (C terminal) domain (Fisher et al., 1986; McKeon et al., 1986) The core of the lamin tail consists of a n Ig like fold (Dhe Paganon et al., 2002; Krimm et al., 2002) The central rod domain enables lamin monomer s to intertwine with one another as coiled coil homodimers in a parallel, unstaggered fashion. These dimers can then assemble into head to tail linear polymers that are thought to associate laterally to form higher order filamentous structures that make up the lamina meshwork. Features that distinguish lamins from other intermediate filament proteins include a basic domain nuclear localization signal (NLS) and highly conserved phosphoacceptor sites (P sites) that flank the central coiled coil (Haas and Jost, 1993; Loewinger and McKeon, 1988) These P sites act as substrates for a protein kinase which phosphorylates lamins during mitotic prophase, promoting nuclear lamina disassembly (Dessev et al., 1990; Gerace and Blobel, 1980) L amins are generally classified as A type or B type and are products of three separate genes: LMNA LMNB1 and LMNB2 (human designation ) Alternate splicing of a single LMNA transcript yields at le ast four variants: lamin A, lamin C, lamin A 10 and lamin C2 (Goldman et al., 2002b; Lin and Worman, 1993; Mounkes et al., 2001) The two major B type lamins (B1 and B2) are encoded by LMNB1 and LMNB2 respectively (Goldman et al., 2002b; Mounkes et al., 2003) Lamin B3 (a splice variant of LMNB2 ), is expressed in mammalian male germ cells and is thought to contribute to the characteristic morphology of sperm nuclei (Furukawa and Hot ta, 1993) A and B type lamins are distinguished by two main features; expression pattern and post translational modifications. A type lamins are absent from early embryonic
21 cells and are only expressed upon differentiation in a tissue specific and async hronous manner (Mattout Drubezki and Gruenbaum, 2003; Rober et al., 1989; Zastrow et al., 2004) In contrast to B type lamins, A type lamins are have been detected in the nuclear interior frequently associated wit h intranuclear foci (Hozk et al., 1995; Vlcek et al., 2001) B type lamins are expressed in all nucleated cell types, both embryonic and adult (Burke et al., 2001; Gerace an d Burke, 1988; Mounkes et al., 2003; Nigg, 1989) However, this should not impl y that B type lamins are essential proteins, at least at the cellular level. While mice deficient in lamin B1 are unusually small during embryonic development, they do develop to term. Post natal mortality is associated with lung and bone defects (Yang et al., 2011) Lamin B2 deficient mice also develop to term but display abnormal organization of the cerebral cortex and cerebellum (Coffinier et al., 2011; Yang et al., 2011) They invariably die within an hour of birth. Mice have also been derived that display keratinocyte specific deletion of both lamins B1 and B2. Remarkably, these mice display no defects associated with skin or hair development, and display apparently normal keratinocyte proliferation (Coffinier et al., 2011; Yang et al., 2011) Taken together, these various studies show that B type lami ns are dispensable in at least some cell types Given the limited expression of A type lamins during embryonic development and their absence from certain stem cell populations it is obvious that at the cellular level these proteins are dispensable. This conclusion is reinforced by findings that RNAi mediated down regulation of A type lamin expression in cultured cells has few adverse consequences (Harborth et al., 2001; Sullivan et al., 1999b) However, A type lami ns are not entirely dispensable for a normal lifespan, as postnatal growth is severely
22 compromised in Lmna / mice that lack both lamins A and C. These animals invariably develop muscular dystrophy and die within eight weeks of birth (Raharjo et al., 2001; Sullivan et al., 1999b) In humans, LMNA haplo insufficiency is associated with autosomal dominant Emery Dreyfuss muscular dystrophy while complete loss of LMNA expression is peri natal lethal. Recent research sugg ests significant functional redunda ncy of lamins A and C, since lamin C only mice ( Lmna LCO/LCO ) are indistinguishable from their wild type counterparts. (Fong et al., 2006) Whether the same holds true in humans is unknown. The two major A type lamins A and C are identical for the first 566 amino acids (Zastrow et al., 2004) While lamin C has a unique six residue C terminal tail, lamin A has a 98 amino acid extension that includes a C terminal CaaX motif (where C=cystine, a=ali phatic amino acid, X=any amino acid; methionine in vertebrate lamins). The CaaX motif is common to all lamins except lamin C and lamin C2 and is subject to a series of post translational modifications to generate the mature proteins (Zastrow et al., 2004) Shortly after synthesis, all CaaX motif containing lamins (prelamin A or B 1/2 ) are farnesylated on the CaaX c ys teine residue by a farnesyl transferase. The farnesyl cysteine linkage is formed by a thioether bond. Following farnesylation, the carboxy terminus is proteoly tically cleaved to remove the triplet aaX sequence This is catalyzed by ZmpS te24 (also called FACE 1) in the case of lamin A, and the Ras converting enzyme proteinase (RCE1) in the case of the B type lamins. The exposed C terminal farnesyl cysteine residu e is then carboxymethylated (Kitten and Nigg, 1991 ) Farnesylated lamins are imported into the nucle us, via NPCs by virtue of their nuclear localization signal s (NLS). Farnesylation appears to be required for efficient
23 incorporation into the nuclear lamina After assembly its assembly at the nuclear per iphery prelamin A undergoes a n additional cleavage event at Y646 catalyzed by ZmpSte24 releasing the modified 15 C terminal residues and resulting in the loss of its (Bergo et al., 2002) B type lamins do not undergo th is second cleavage and c onsequently remain farnesylated The retention of this modification is thought to mediate interaction s with the INM (Navarro et al., 2006) Inner N uclear M embrane P roteins To date, only a bout a dozen INM resident integral proteins have been extensively characterized. Most of these INM proteins interact with lamins either directly or indirectly However, recent proteomic analys e s o f purified NE s ha ve revealed at least 60 additional integral membrane proteins that are enriched to a greater or lesser extent within the NE (Schirmer et al., 2003a; Schirmer et al., 2003b) Many of the se are likely to represent novel INM proteins. Several well characterize d INM proteins belong to the LEM domain family. Prototype members of this family are lamin associated polypeptide 2 (LAP2), emerin and MAN1 (Cai et al., 2001; Laguri et al., 2001; Wolff et al., 2001) The LEM domai n is thought t o directly bind to barrier autointegration factor (BAF) (Lee and Wilson, 2004; Lin et al., 2000) an essential soluble DNA and lamin binding protein. TMPO the gene encoding L AP 2 proteins, is transcri ) that contain transmembrane domains (Berger et al., 1996; Harris et al., 1995) a transmembrane domain and is detected throughout the nucleoplasm (Foisner and Gerace, 1993 ) Nuclear envelope localization of at least one LEM domain family member, emerin, is dependent in part upon interactions with A type lamins. Loss of A type lamin expression in a variety of cell types is associated with mislocalization of
24 emerin to the pe ripheral ER. This is not, however, unique to LEM domain proteins. Retention of both LAP1 and lamin B receptor (LBR) in the INM is facilitated by interaction s with both lamins a chromatin binding proteins (Foisner an d Gerace, 1993; Gruenbaum et al., 2005) Similarly, INM localization of Unc84, a C. elegans SUN domain protein is dependent upon the single B type lamin. M ost INM proteins interact with lamins and/or chromatin. Such interaction s are generally may contribu te to the structural stability of the NE. In addition to this structural function, certain INM proteins are known to regulate gene expression. One of the better described examples of this is the attenuation of TGF and bone morphogenic protein (BMP) signa ling pathways by the sequestration of R Smad s 2/3 at the NE by MAN1 (also known as LEMD3). MAN1 contains two membrane spanning helices. Its topology is such that both its N and C termini are exposed to the nucleoplasm with only a small loop extending in t o the PNS. The N terminal region contains the LEM domain as well as binding sites for both A and B type lamins. The R SMAD binding site is contained within the C terminal domain and in this way confers MAN1 with the function of a negative regulator of TGF /BMP activated gene expression (Holaska et al., 2003; Lin et al., 2005) One model proposed to explain the mechanism of transit of integral membrane proteins to the INM involves a process of selective retention that depends on the interconnected nature of the ER, ONM and INM membranes (Gerace and Burke, 1988; Newport and Forbes, 1987) Newly synthesized integral proteins move into the ONM by lateral diffusion from the ER a nd gain access to the INM via membrane continuities of the nuclear pore complexes. Only those proteins that are capable of binding to stable
25 nuclear components such as lamins or chromatin are subsequently concentrated within the INM. Clearly appropriate lo calization of proteins such as emerin MAN1, LAP2 and LBR should be contingent upon their ability to bind lamins and/or chromatin and independent of any nuclear localization signal (Furukawa et al., 1995; Ohba et al ., 2004; Ostlund et al., 1999; Smith and Blobel, 1993; Soullam and Worman, 1995; Wu et al., 2002) Indeed, incorporation of a nuclear localization signal ( NLS ) into the truncated cytoplasmic domain of a type II membrane protein, chicken hepatic lectin (CH L), failed to provide access to the INM (Soullam and Worman, 1995) This selective retention hypothesis predicts a significant reduction in mobility of INM proteins in the NE relative to the ER Quantitative fluorescence recovery after photobleaching (FRAP) analyses indicate that GFP LBR and GFP emerin diffused with unrestricted mobility in the ER while the fraction localized to the INM was virtually immobil e (Ellenberg and Lippincott Schwartz, 1999; Ostlund et al., 1999) Furthermore, emerin and MAN1 experienced increase d mobility at the NE of LMNA / cells, indicating at least a partial dependence on lamin A for retention at the INM (Ostlund et al., 2006) The size restriction imposed by the NPC for the INM proteins appears to be ~60 kD a of nucleoplasmic domains (Burke, 1990; Holmer and Worman, 2001; Ostlund et al., 1999; Powell and Burke, 1990; Wu et al., 2002) Soullam and Worman demonstrated that LBR mutants containing an extended nucleoplasmic domain approximately of 70 kD a were excluded from the INM (Soulla m and Worman, 1995) In addition to the selective retention hypothesis, there is evidence for active import of INM proteins. In one model, ATP driven changes in nucleoporin interactions might allow membrane proteins to travel across the NPC (Ohba et al., 2004) In an alterna tive
26 model, integral INM proteins might directly, or through an associated factor promote their movement past the NPC. An important component of the latter model might be the specific recognition of discrete NLS on integral membrane cargo molecules by ded icated transport mechanism (Soullam and Worman, 1995) Other studies suggest a role for the classical nuclear import receptor, karyopherin/importin in INM protein targeting (King et al., 2006; Saksena et al., 2006) The interaction between the INM proteins and karyopherin is formed in NLS dependent manner. Either mutat ion or deleti on the putative NLS of Heh2, a yeast INM protein that is related to MAN1 /LEMD3 caused the loss of a ssociati on of Heh2 and karyopherin, resulting in dispersal of the mutant proteins throughout the ER (King et al., 2006 ) This clearly suggest s a role for NLS in karyopherin dependent active targeting of the INM proteins. This is at odds with the findings of Soullam and Worman (1995) on LBR and CHL targeting. The most reasonable solution to this paradox is that both selective retention and NLS dependent active import may be employed as mechanisms to localize integral membrane prot eins to the INM. The LINC complex Nuclear positioning is crucial for many cellular events such as cell migration, differentiation, polarization and mitosis. There is a considerable body of evidence, some stretching back several decades, for an associatio n between the NE and elements of the cytoskeleton. However, until relatively recently the mediators of this association remained obscure. A mechanical link between the nucleus the cytoskeleton and cell surface molecules was experimentally demonstrated wit h RGD peptide coated beads bound to integrin s on the plasma membrane (Maniotis et al., 1997) Movement of the
27 beads led to corresponding displacement of nucleus and nuclear contents (Maniotis et al., 1997) strongly suggest ing that there are physical connections between the plasma membrane and the NE presumably mediate d by the cytoskeleton. These observations raised the question of how this cytoskeletal force is transferred to the nucleus The resolution to this problem involves, at least in part, the LINC complexes described in an earlier section (above). Initial evid ence for the existence of these structures came from analysis of C. elegans Anc 1 mutants that displayed aberrant nuclear positioning in hypodermal cells. Anc 1 is very large (956kDa) actin binding protein and prototype member of the KASH domain family. It appears to be localized exclusively to the ONM where it functions as an attachment site for actin f i laments. Localization of Anc 1 was found to be dependent upon Unc 84, an inner nuclear membrane protein and a founder member of the SUN domain protein fami ly. Localization of Unc 84 itself was also found to be dependent up the single C. elegans lamin. A suggestion at the time was that perhaps Unc 84 functioned as a trans luminal tether for Anc 1. This hypothesis was later tested and confirmed in mammalian sy stems. Localization of the giant (800kDa) isoform of Nesp2 was found to be dependent upon the expression of the two somatic SUN domain proteins, Sun1 and Sun2. Indeed a dominant negative Sun1 mutant, consisting of a soluble form of the Sun1 lumenal domain (containing the coiled coil region and SUN domain) equipped with sequence elements that would target it to the ER lumen and PNS, will efficiently evict KASH domain proteins, including Nesp2 from the ONM (Crisp et al. 2006) Subsequent studies have revealed that Sun1 and Sun2 form homotypic oligomers mediated by their luminal coiled coil domains and have the capacity to interact with components of the
28 nuclear lamina via their nucleoplasmic domains. However, while the nature of the lamin interactions are still not entirely clear Sun1 has a preferences for both pre lamin A (Crisp et al., 2006) and lamin B2 (Roux, Burke, and Stewart,. unpublished yeast two hybrid data). Mammals ha ve four other SUN domain proteins in addition to Sun1 and Sun2 S unc 1 (S un 3), Spag4 (S un 4), Spag4L (Spag4 like, S un 5) and Osteopotentia. Sun3 5 appear to be restricted largely to the testis where their functions have yet to be resolved. (Crisp et al., 2006) Osteopotentia is an unusual member of the family in that it has an internal (as opposed to C terminal) SUN domain. Whether Osteopotentia has the capacity to bind KASH domain proteins remains unclear. However, it does not appear to be an ONM protein, and instead is found throughout the ER. Although its function is still not understood, mice deficient in Osteopotentia display impaired bone formation (Sohaskey et al., 2010) KASH domain protein s display a wide variety of cytoskeletal binding activities whic h provide the LINC complexes with their functional heterogeneity in diverse organisms from yeast to hu man. In Drosophila, Klarsich t (a prototype KASH domain protein) was shown to be required for normal eye development. Klarsicht mutants display aberrant ph otoreceptor nuclear migration resulting in retinal disorganization (Fischer Vize and Mosley, 1994) Kalrsicht is an ONM protein that functions as an adaptor for cytoplasmic dynein, a microtubule minus ( )end directed motor protein. Klarsicht is tethered in the ONM by virtue of its interaction with Klaroid, an INM SUN domain protein which itself associates with lamin Dm 0 Together, Klarsicht and Klar oid represent a Drosophila photoreceptor LINC complex that mediates microtubule and dynein dependent nuclear migration.
29 In mammals, the first KASH domain protein to be described was Syne1 (now known as N esprin 1 ). It was identified on the basis of specifi c enrichment in the nuclear envelopes of the minor population of muscle cell nuclei that are clustered beneath the post synaptic membrane. Syne2/Nesp2 was by homology with Syne1/Nesp1. The two proteins display overlapping but nonetheless distinct expressio n patterns. The suggestion at the time was that Syne1/Nesp1 might be responsible for the post synaptic nuclear clustering. This notion has been born out in mouse models. In mice harboring a mutation in the Nesp1 gene in which exons encoding the KASH domain were deleted, nuclear clustering at the NMJ was lost. These mice were viable and did not display any serious muscle pathology. Similarly, overexpression of the Nesp1 KASH domain which will displace endogenous Nesp1 from the ONM also resulted in a decline in the number of postsynaptic nuclei. (Grady et al., 2005; Zhang et al., 2007a; Zhang et al., 2007b) Deletion of exons encoding the Nesp2 KASH domain had no obvious effect on muscle organization. Like the Nesp1 mut ant mice they were also found to be viable (Lke et al., 2008; Zhang et al., 2007a) However, mice homozygous for mutations at both loci died within minutes of birth, possibly due to respiratory failure (Zhang et al., 2007a) The conclusion of course is that Nesp 1 and Nesp2 are to a certain degree functionally redundant More recent studies have revealed that there is an important requirement for both Nesp1 and 2 in neurogenesis and neural migration In addition to the well established actin binding capabilities o f the giant isoforms of both Nesp1 and 2, both proteins also interact with dynein /dynactin (Zhang et al., 2009) Furthermore, Nesp2 has a membrane proximal binding site for conventional k inesin 1. In this way, Nesp 1 and 2 have the capacity to move the nucleus relative to the centrosome, an important feature
30 of both intrakinetic nuclear movement and nucleokinesis, processes that are critical to CNS development (Schneider et al. 2011; Zhang et al., 2009) The third mammalian KASH domain protein is Nesp 3 which employs plectin to anchor intermediate filament to the NE (Ketema et al., 2007; Wilhelmsen et al., 2005) The importance of Nesp3 is still a matter of some debate. Nesp3 deficient mice have no discernable phenotype. Similarly, while association of intermediate filaments with the NE was reduced in N esp 3 deficient zebrafish (Postel et al., 2011) there were no developmental or behavioral consequence. Nesp3 has been implicated in nuclear and centrosomal positioning in aortic vascular endothelial cells in vitro when subjected to shear stress (Morgan et al., 2011) However, whether this is the case in vivo is not yet clear. Nesp4 is the most recently identified mammalian KASH domain pr otein. It was found on the basis of C terminal homology to Nesp2. Expression Nesp4 appears to be restricted to epithelial cells, particular those in secretory tissues. It can also be detected in mouse ES cell lines. Nesp4 is a binding partner for k inesin 1 which it will recruit to the NE. When expressed inappropriately in non polarized cells, Nesp4 will cause the nucleus and centrosome to move apart, often quite dramatically so (Roux et al., 2009) The Nesp4 gene ha s been disrupted in mice. While no secretory epithelial phenotype apparent, these mice exhibit progressive hearing loss. This led to the finding that Nesp4 is highly expressed in outer hair cells (OHCs) of the cochlea. In the absence of Nesp4, OHCs show a rapid decline in numbers during the month after birth. This loss of OHCs is linked in some way to aberrant nuclear positioning (Burke and Stewart, unpublished data)
31 Further roles for the LINC complex have emerged from studies in C. elegans as well as in both budding and fission yeast. In addition to Anc 1, C. elegans contains two more KASH domain proteins, Unc 83 and Zyg 12. Unc 83 has both kinesin and dynein binding activities and plays an essential role in modulating nuclear migration in embryonic hypo dermal cells (Fridolfsson et al., 2010; McGee et al., 2006; Starr et al., 2001) Zyg 12 is also a dynein binding protein and has an essential role in pronuclear migration and centrosome attachment to the male pronuc leus in fertilized eggs (Malone et al., 2003) Centrosome anchoring at the nuclear envelope is dependent homotypic interactions between full length Zyg 12 in the ONM and a soluble KASH less splice isoform that assoc iates with the centrosome (Malone et al., 2003) Zyg 12 also has an additional role in meiotic prophase I The C. elegans SUN domain protein Sun1 (also known as Matefin) is involved in anchoring telomeres at the nuc lear periphery during meiotic prophase I (Malone et al., 2003; Penkner et al., 2009; Sato et al., 2009) At the same time it functions as a tether for Zyg 12 in the ONM. Since Zyg 12 is an adaptor for dynein, the me iotic Sun1/Zyg 12 interaction is able to mediate microtubule dependent movement of telomeres (Sato et al., 2009) This is thought to facilitate faithful h omologue pairing as well as providing a physical check for non homologous pairing (Sato et al., 2009) Similar LINC dependent chromosome dynamics can be o bserved in both budding and fission yeast. In budding yeast, however, chromosome movement is actin mediated ( Koszul and Fischer, 2009; Koszul et al., 2008) In contrast, fission yeast employs a microtubule and dynein based system (Bhalla and Dernburg, 2008; Chikashige et al., 2007; Chikashige et al., 2006; Ding et al., 1998; Koszul and Fischer, 2009; Koszul et al., 2008; Sato et al., 2009)
32 In mammalian meiosis I there is also a clear role for LINC complex componen ts. Sun1 deficient mice are viable. However, males and females are infertile. Males are completely aspermic and display a meiotic arrest in primary spermatocytes (Ding et al., 2007; Schmitt et al., 2007) Sun1 funct ions in a manner analogous to its C. elegans counterpart in that it is essential for telomere attachment to the nuclear envelope at so called meiotic attachment plates. Prophase chromosomes movements are known to occur during mammalian meiosis. The implica tion of course is that there must be a germ cell KASH protein that can engage with Sun1 and which can mediate telomere movement and clustering (Chi et al., 2009; Ding et al., 2007; Schmitt et al., 2007) However, no ne of the known mammalian KASH proteins appear to be present at the meiotic attachment plates Furthermore there is no identifiable Zyg 12 homologue in mammals. This clearly raises the possibility that mammals must have an additional novel member of the KA SH domain family. As will be described in the next chapter we have identified and characterized just such a protein and have been able to demonstrate that it is the functional homologue of Zyg 12 Nuclear P roteins and H uman D iseases Over the last 15 years a range of human diseases has been linked to mutations in genes encoding NE components. The human A type lamin gene ( LMNA ) has proved to be a particular hotspot with more than 250 known mutations that can give rise to about a dozen distinct disorders. The se lamin linked diseases are frequently referred to as laminopathies (Burke and Stewart, 2006) The first laminopathy to be described was X link ed Emery Dreifuss muscular dystrophy (X EDMD) a disease characterized by progressive muscle atrophy and cardiac conduction defects with an onset before the age of 20 (Emery and Dreifuss,
33 1966) M utations in the gene encodin g the INM protein emerin are responsible for X EDMD (Bione et al., 1994b) Subsequently, mutations in LMNA were f ound to be associated with autosomal dominant EDMD (autosomal dominant EDMD, AD EDMD) (Bonne et al., 1999; Fairley et al., 1999; Sullivan et al., 1999b) Much rarer autosomal recessive EDMD has also been linked to L MNA Mutations that give rise to AD EDMD are found scattered throughout the lamin A molecule. Certain mutations also cause two related disorders. Limb girdle muscular dystrophy type 1B (LGMD1B) (Muchir et al., 2000) differs from EDMD in that it features different muscle group involvement. D ilated cardiomyopathy with conduction defects (DCM CD1) (Fatkin et al., 1999) is actually life threatening complication of both EDMD and LGMD1B. The overlap of phenotypes associated with these three laminopathies is highlighted by the fact that a single LMNA mutation has been shown to give rise to each of these three disorders in different members of the same family. Clearly some aspects of the disease phenotype must be influenced by genetic background or environment. Mutations in LMNA are not only associated with striated muscle disease. Dunnigan type familial partial lipodystrophy (FPLD2) (Cao and He gele, 2000; Shackleton et al., 2000; Speckman et al., 2000) is a disorder that features loss of subcutaneous fat from the limbs and lower trunk, with accumulation of fat in the face and neck. M andibuloacral dysplasia (MAD) (Novelli et al., 2002) features skeletal abnormalities in the face and shoulders. A utosomal recessive Charcot Marie Tooth type 2B1 disorder (CMT2B1) (De Sandre Giovannoli et al., 2002) is a peripheral axonal neuropathy. Hutchinson Gilford progeria syndrome (HGPS) (De Sandre Giovannoli et al., 2003; Eriksson et al., 2003) is a so called premature aging disease. It features multisystem pathologies with death from cardiovascular disease in
34 the mid teens. R estrictive dermopathy (RD) (Nava rro et al., 2004) is a perinatal lethal disorder that has progeroid features. A (Chen et al., 2003) is another, albeit less severe premature aging d isorder. A major unanswered question at the moment concerns how mutatio ns in a near ubiquitously expressed gene (at least in adult animals) can give rise to such a peculiar range of tissue specific disorders. Several models have been suggested, none of which can provide a universal answer. It is possible that structural chang es in the nuclear lamina may render nuclei more susceptible to mechanical damage. This could be most significant in muscle cells. It is also been proposed that changes in lamina organization could cause global changes in gene expression patterns. It is cer tainly the case that some LMNA mutations are associated with changes in the distribution and amount of heterochromatin. Furthermore, in progeric mouse models as well as in human progeria fibroblasts aberrant expression of Wnt responsive and perhaps also No tch responsive genes have been documented (Hernandez et al., 2010; Scaffidi and Misteli, 2008) It is also possible that lamin mutations may affect interactions with other proteins that are themselves expressed in a tissue specific manner. Indeed it was my aim in Chapter 3 of this thesis to develop a generally applicable method to identify lamin interacting proteins, tissue specific and otherwise. The LINC complex has also been implicated in the etiology of certain l aminopathies. It is known that fibroblasts from Lmna null mice, which display an EDMD like syndrome, feature changes in cytoplasmic and cytoskeletal mechanics. Furthermore mice that contain mutated Lmna or lack Lmna exhibit mislocalization of
35 S un 2 and N esp 1 in synaptic nuclei, leading to the mispositioning of synaptic nuclei and the structural disorganization of the NMJ (Mjat et al., 2009) I n AD EDMD patients similar defects in NMJ were demonstrat ed and thus were proposed to be a part of the disease mechanism in AD EDMD (Mjat et al., 2009) T he role of the LINC complex in EDMD was further supported by discovery of mutations in Nesp 1 and 2 in patients with EDMD phenotype (Zhang et al., 2007a) Mutations in Nesp 1 are also identified as a causative mechanism for autosomal recessive cerebellar ataxia (Gros Louis et al., 2007) In addition to mutati ons in LMNA abnormalities in other NE constituents are linked to a variety of unusual human diseases. D uplication of LMNB1 encoding lamin B1 is associated with adult onset autosomal dominant leukodystrophy characterized by loss of myelin in the central ne rvous system O verexpression of lamin B1 was detected in brains of affected individuals (Padiath et al., 2006) Overexpression of lamin B1 in D. melanogaster eye leads to a degenerative eye phenotype (Padiath et al., 2006) O verexpression in cultured cells induced morphological abnormalities similar to those in cell s with abnormal A type lamins (Padiath et al., 2006) Nucleotide variations in LMNB2 are associated with a form of lipodystrophy called Barraquer Simons syndrome (Hegele et al., 2006) However t here are little data showing how mutations in B type lamins cause this disease. Lamin B receptor (LBR) interacts with lami n B, DNA and chromatin proteins (Worman et al., 1988; Ye and Worman, 1994; Ye and Worman, 1996) and is responsible for multiple defects. Heterozygous mutations in LBR lead to Pelger Hut anomaly characterized by a bnormal ities in the nuclear shape of granulocytes (Hoffmann et al., 2002) H omozygous LBR mutations cause severe developmental
36 abnormalities or are lethal in utero (Hoffmann et al., 2002; Waterham et al., 2003) Heterozygote loss of function mutations in MAN1 have been identified in bone dysplasia called osteopoikilosis Defects in bone composition is likely caused by enhanced transforming growth factor c protein signaling by compromised activity of regulatory S mads mediated by a ltered associat ion with mutant MAN1 (Hellemans et al., 2004) Mutations in the gene DYT1 encoding torsinA lead to the movement disorder DYT1 dystonia (Ozelius et al., 1997) TorsinA is an ATPase expressed in the ER thought to participat e in a diverse range of biological functions (Ozelius et al., 1997) Tra nsgenic mice expressing a pathogenic torsinA exhibit disordered NE in neurons, suggesting a potential disease mechanism of DYT1 dystonia (Goodchild et al., 2005) Mutant DYT1 is known to interact with the luminal domain of LAP1, an abundant INM protein It is clear that human diseases linked the ab normalities in NE constituents cannot be explained by one simple model Indeed it would be nave to think that they could be M utations in three genes, encoding NE components known to interact, separately give rise to virtually the same disease. EDMD can b e linked to mutations in emerin A type lamins and the LINC complex (Bione et al., 1994a; Bonne et al., 1999; Fatkin et al., 1999; Taylor et al., 2005) On the other hand, some diseases result from a combination of several protein defects. MAN1 and emerin interact directly with lamin A and with each other Dislocation of MAN1 and emerin from the NE were observed when lamin A was lost from the NE (Clements et al., 2000; Liu et al., 2003; Mansharamani and Wilson, 2005; Ostlund et al., 2006) Taken together, evidence gleaned from human diseases and animal models suggests that there are complex interactions at the nuclear
37 periphery involving nuclear and NE proteins as well as cyt oplasmic components. My goal in this thesis has been to further explore these interactions. To do these I have taken a two pronged approach. The first of these was to identify and characterize novel LINC complex components while the second was to develop a new and universally applicable technique to identify interacting and neighboring proteins.
38 CHAPTER 2 DALEK 6 : A NOVEL MAMMALIAN MEIOSIS SPECIFIC KAS H DOMAIN PROTEIN REQUIRED FOR GAM E TOGENESIS Introduction The NE is a selective barrier that regulates mol ecul ar trafficking between the nucleoplasm and cytoplasm. INM and O NM separated by the PNS. The two membranes feature annular junctions where they are spanned by NPCs. Furthermore, the ONM displays multiple connections with the peripheral ER. Consequently, ONM, INM and ER represent separate domains within a single membrane system. Accordingly, the PNS represents a perinuclear extension of the ER lumen (Gerace and Burke, 1988) In metazoa, the nuclear membranes are with A and B type lamins, which assemble to form the underlying nuclear lamin a (Gruenbaum et al., 2005) This structure is considered to function as a scaffold for t he NE as a whole while at the same time playing an important role in defining interphase nuclear architecture. It is well established that nuclear structures, including the nuclear lamina, are mechanically coupled to the cytoskeleton. In the last few years the details of this nucleo cytoplasmic coupling has been defined at the molecular level (Crisp et al., 2006; Padmakumar et al., 2004; Starr and Han, 2002) These studies have revealed a pair protein families that t ogether assemble to form LINC complexes. These structures span both nuclear membranes thereby connecting nuclear and cytoskeletal components. LINC complexes play an essential role in nuclear anchoring and positioning in a variety of cell types and in th is way are facilitators of normal embryonic development. In addition it has become apparent that LINC complexes mediate cytoskeletal dependent changes in nuclear organization findings that are at heart of this chapter.
39 LINC complex es consist of INM SUN domain proteins and ONM KASH domain proteins. SUN and KASH domain proteins form translumenal links that span the PNS. SUN domain proteins typically interact with the nuclear lamina and chromatin proteins via nucleoplasmic N terminal domains. KASH domain family members, in contrast, bind various cytoskeletal components via their cytoplasmical l y oriented N terminal domains. Thus SUN and KASH domain proteins represent a pair of links in a molecular chain that couples nuclear and cytoplasmic structures (Crisp et al., 2006; Padmakumar et al., 2005; Starr and Han, 2002) While there are two somatic cell SUN domain proteins in mammals, Sun1 and Sun2, diversity in LINC complex function is provided primarily by the KASH domain p roteins. At the time that I began this thesis research, four KASH domain proteins, Nes p 1 4, had been identified. Giant isoforms of Nesprins 1 and 2 (1,000 and 800kDa isoforms respectively) couple the actin cytoskeleton to the NE (Padmakumar et al., 2004; Zhen et al., 2002) via actin binding calponin homology domains at their amino termini At least in the case of Nesp1, t h is interaction is thought to function, in the anchor ing of synaptic motor nuclei at the NMJ (Grady et al., 2005; Zhang et al., 2007a; Zhang et al., 2007b) Nesp 1 and 2 are also involved in nuclear movement during neurogenesis and neural migration (intra kinetic nuclear movement and nucleo kinesis respectively) (Schneider et al., 2011; Zhang et al., 2009) In this case it is the interaction with cytoplasmic dynein for Nesp1 and 2, and with kinesin 1 for Nesp2 that are thought to regulate nuclear dynamics. Nesp 3 functions to anchor intermediate filament to the NE. This is mediated by its interaction with the cytolinker protein, plectin (Ketema et al., 2007; Wilhelmsen et al., 2005) The phenotype of Nesp3 deficient mice remains
40 unrep orted. In zebrafish the loss of Nesp3 leads to a reduced association of intermediate filaments with the NE, yet these fish exhibit no evidence of an y functional consequences for this deficiency (Postel et al., 2011) Unlike the more ubiquitous expression of Nesp1 3, expression of Nesp 4 appears restricted to a subset of highly polarized epithelial cells (Roux et al., 2009) including secretory epi thelia such as exocrine pancreas, salivary gland and mammary epithelia, as well sensory epithelial cells of the inner ear Inactivation of the Nesp4 gene in mice results in progressive hearing loss. While the function of Nesp 4 remains has yet to be fully u nderstood its capacity to bind k inesin 1 combined with clear morphological changes in inner ears of Nesp4 deficient mice, suggests that it has a key role in basal positioning of nuclei in certain polarized cell types We have recently identified two addi tional members of the KASH domain protein family. One of these, lymphocyte restricted membrane protein (LRMP, also known as JAW1) remains largely uncharacterized. In this study I have focused on the identification and characterization of the sixth member o f the mammalian KASH domain protein family. I have demonstrated that this protein, named Dalek6 (for d ynein a ssociated L INC e ngaged K ASH 6 ), is a functional mammalian KASH domain protein expressed in primary spermatocytes where it colocalizes with Sun1 and the dynein motor complex at NE telomere attachment sites during meiotic prophase I. Disruption of the Dalek6 gene in mice results in infertility in both male and female homozygotes. I show that this infertility is associated with meiotic arrest during earl y meiotic prophase I, likely during leptotene/zygotene. My data suggests that this protein, as a mediator of
41 telomere dynamics is essential for homologue pairing and successful completion of meiosis. Results To identify novel members of the KASH domain f amily of proteins, we performed a series of BLASTP search es using mouse LRMP as probe. This picked up a possible zebra fish LRMP homologue which was used for a second series of BLASTP searches. Visual inspection of the results revealed an uncharacterized m ouse protein named coiled coil domain protein 155 ( CCDC155) containing a cluster of three proline residues at the C terminus. Further analysis of this protein revealed a single putative transmembrane domain just upstream of the proline cluster Taken toget her, these sequence features are typical of a KASH domain, albeit a somewhat degenerate example. For reasons that will be expanded upon below, we chose to rename this protein Dalek6. Comparison with the Nesp1 4 KASH domain s suggests that the putative Dalek 6 KASH domain possesses only ~20 lumenal residues rather than the ~30 seen in Nesp1 4. Two potential sequence variants of 649 and 578 amino residues are evident for mouse Dalek6. These differ according to their predicted translation start sites. The shorte r variant with a calculated mass of 62,752Da has the better Kozak consensus sequence. Furthermore, only the short form is predicted in other mammals, including humans. This suggests that the short variant is likely to be the predominant if not the exclusiv e form of Dalek6 that is present in the mouse (Figure 2 1A) Based upon sequence characteristics alone, Dalek6 appears to be a tail anchored membrane protein. The protein lacks any sequence motifs that might provide a clue to its function. However, as its temporary designation implies, it contains a coiled coli domain of 155 (more likely ~200) amino acid residues suggesting that it might be
42 capable of forming homo oligomers. In addition it contains an N terminal EF hand like sequence that is usually associ ated with calcium dependent protein protein interactions (Figure 2 1B) We first wished to determine whether Dalek6 is a functional KASH domain protein and consequently whether it will localize to the NE. To accomplish this we obtained a full length human Dalek6 (CCDC155) cDNA from Invitrogen to which we attached a GFP tagged human Dalek6 into HeLa cells by transfection, where it localized predominantly to the NE (Figure 2 2A) However, its distribution within the NE was not always uniform. Instead it was frequently observed to be concentrated towards one pole of the nucleus. Double immunofluorescence labeling revealed that Dalek6 always concentrated towards a region of the NE that was closest to the centrosome (Figure 2 2B) We had previously noted a similar phenomenon with Nesp4. In this case, however, Nesp4 concentrated at a nuclear pole that was furthest from the centrosome. We had demonstrated that this behavior of Nesp4 was a consequence of its kinesin 1 b inding function. Thus this behavior of Dalek6 led us to speculate that perhaps Dalek6 was a binding partner for a microtubule ( ) minus end motor protein, cytoplasmic dynein for instance. To determine whether Dalek6 localized to the INM or ONM we prepared HEK293 cells that stably express GFP Dalek6 (Adam et al., 1990; Crisp et al., 2006; Liu et al., 2007; Roux et al., 2009) These were then a nalyzed by immunofluorescence microscopy following formaldehyde fixation and differential permeabilization employing digitonin versus Triton X 100. Low concentrations of digitonin will permeabilize the plasma membrane but not the nuclear membranes. Thus Dalek6 should only be
43 detectable following digitonin permeabilization if it is localized to the ONM. As shown in Figure 2 2C this is indeed the case. Dalek6 must be localized to the ONM with its N terminal domain exposed to the cytoplasm. To further clarify the NE targeting behavior of Dalek6 I fused GFP to the putative Dalek6 KASH domain to yield GFP KASH6. This was then transiently expressed in HeLa cells. As shown in F igure 2 2D, GFP KASH6 localized efficiently to the NE. In this case however, its distribution was always uniform. The implication was that the proposed Dalek6 cytop lasmic domain was required for concentration close to the centrosome. Overexpression of GFP KASH6 was found to efficiently evict endogenous Nesp2 from the NE suggesting that they compete for the same NE binding partners (Figure 2 2E) Conversely overexpres sion of a Sun1 dominant negative mutant leads to loss of Dalek6 from the NE. The implication here is that Dalek6 functions as a KASH domain protein that interacts with SUN domain proteins at the nuclear periphery (Figure 2 2F) This observation was further confirmed by the experiment depleting Sun1 and Sun2. Compared to the cell constitutively expressing HA Dalek6, introducing Sun1 and Sun2 small interfering RNA leads to displacement of HA Dalek6 from the NE (Figure 2 2G). The conclusion to be drawn from th e data so far is that Dalek6 is a novel KASH domain protein that localizes to the ONM in a SUN dependent manner and in this way appears to define a novel LINC complex isoform. Our next question concerned the identification of cytoplasmic binding partners for Dalek6. Comparison with Nesp4 suggested that Dalek6 might function as a NE adaptor for a minus end motor protein One obvious candidate is cytoplasmic dynein. Evidence for an interaction with cytoplasmic dynein was provided by immunofluorescence
44 micros copy of HeLa cells expressing tagged versions of Dalek6. The full length protein, but not GFP KASH lead to the recruitment of both cytoplasmic dynein intermediate chain (DIC) and the dynactin subunit p150 to the NE. (Figure 2 3 A and 2 3 C ). This effect cou ld be enhance by co expression of Sun2, which increases the capacity of the NE for Dalek6 (Figure 2 3 B ). Confirmation of the association between Dalek6 and the dynein / dynactin complex es including DIC, heavy chain (DHC), and p150 was provided by co immun oprecipitation studies. For these experiments I expressed GFP tagged versions of the Dalek6 cytoplasmic domain GFP Dalek6 KASH (i.e. lacking the KASH domain ) HEK293 cells (Figure 2 3 D ). This truncated form of Dalek6 was used as it is more easily solubilize d than the membrane associated full length version. As revealed in Figure 2 3D GFP Dalek6 KASH, but not GFP alone, co immunoprecipitated with DHC, DIC and p150. Given its interaction with dynein and the fact that it is a novel LINC complex component we re named Dalek6 as D ynein A ssociated L INC complex E ngaged K ASH domain protein 6 To examine the expression pattern for Dalek6, we generated an antibody that detects murine Dalek6 by both immunofluorescence microscopy (Figure 2 4 A) and Western blot (Figure 2 4 B) Since Dalek6 was not detected in any common cell lines, we analyzed its expression in a variety of adult rat and mouse tissue s by immunofluorescence microscopy of cryosections These studies revealed that Dalek6 expression was apparently limited to the testis in adult animals (Figure 2 5 A) These findings were confirmed by RT PCR analysis of a variety of mouse tissues (Figure 2 5 B). Within the testis, Dalek6 expression was restricted largely to the NEs of meiotic primary spermatocytes. The distribution of Dalek6, however, was not uniform. Instead it was
45 concentrated in discrete foci (Figure 2 5A) A similar distribution had previously been described for Sun1 in spermatocytes. Subsequent double label experiments revealed that Sun1 and Dalek6 exactly co lo calize (Figure 2 6D) Previous mouse studies by Min Han and colleagues, including the derivation of Sun1 deficient mice, revealed that Sun1 defined attachment sites at the NE for telomeres during meiotic prophase I Indeed loss of Sun1 was associated with detachment of telomeres and failure to complete meiosis (Ding et al., 2007) Consistent with these findings Dalek6 similarly colocalizes with telomeres. I performed double immunofluorescence microscopy of both tissue sections and single cell spreads employing antib odies against Dalek6 and SCP3, a component of the axial element of synaptonemal complex (SC) (Figure 2 5 A ) (Figure 2 5 B, C ). Dalek6 clearly colocalizes with the tips of the SCs (Figure 2 6A) Similarly, Dalek6 precisely colocalizes with the telomere marker Rap1 (Figure 2 6B) Finally I performed fluorescence in situ hybridization as an alternative method to identify telomeres. As expected, the FISH signal precisely colocalized with Dalek6 in spermatocytes (Figure 2 6C ). The role of the LINC complex during meiotic prophase I is well characterized in yeast and C. elegans (Bhalla and Dernburg, 2008; Hiraoka, 1998; Marshall et al., 1996; Scherthan et al., 2000; Scherthan et al., 1996; Zickler and Kleckner, 1998) Prior to pairing and recombination of homologous chromosomes during meiotic prophase I, telomeres attach to the NE during the stage known as leptotene. These sites form a transient cluster at the one pole of the NE in zygotene that coincides with homolog pairing and initiation of synapsis, the physical connection of homolog ou s chromosomes During this meiotic chromosome movement widely conserved among eukaryotes
46 evidence indicates that chromosome movement is actively mediated by the LINC complex composed of tel omere associated SUN domain proteins In animals, clustering of telomeres, also known as bouquet formation, occurs at the pole of the nucleus closest to the centrosome. This suggests that telomere movement is driven by a microtubule minus end motor protein engaged with an ONM KASH domain protein. In C. elegans the KASH domain protein Zyg 12 provides exactly this function (Bhalla and Dernburg, 2008; Hiraoka, 1998; Marshall et al., 1996; Scherthan et al., 2000; Schertha n et al., 1996; Zickler and Kleckner, 1998) If the same is true in mice, then we would predict that dynein motor complexes should colocalize with Dalek6. This in fact turns out to be the case (Figure 2 7 ). In mammals there is no identifiable orthologue o f Zyg 12. Consequently, b ased on our observation s w e hypothesized that Dalek6 represents the previously unknown mammalian KASH domain protein of the meiotic LINC complex. Consistent with this suggestion none of the other known mammalian KASH domain protei ns Nesp1 4, could be detected in mouse spermatocytes, precluding any role in meiosis in the male germline. (Figure 2 8 ). Thus we propose that Dalek6 is a mammalian meiotic KASH domain protein that functions as an adapter for the dynein motor complex and w hich mediates telomere movement in primary spermatocytes Telomere clustering is believed to facilitate homologue pairing during meiotic prophase I This view is consistent with observations that Sun1 deficient mice are infertile. This infertility is assoc iated with telomere detachment and meiotic arrest. We predicted, therefore, that Dalek6 deficient mice should be similarly infertile although Sun1 mediated attachment at the NE should be unperturbed.
47 To test these hypotheses we generated Dalek6 null mice by conventional methods To facilitate derivation of these mice we obtained mouse ES cells from KOMP that harbored a f loxed allele of the Dalek6 gene. Exposure to Cre recombinase should cause the elimination of exons 5 8 and the introduction of a frame sh ift at the exon4/9 boundary. Mice harboring the floxed allele were produce in the Mouse Core Facility of the Institute for Medical Biology, Singapore. Deletion of the Floxed exons was induced by mating with Zp3 Cre transgenic mice (Lewandoski et al., 1997) This results in the deletion of the Dalek6 in the female germline. Thus an additional mating cycle is required to obtain Dalek6 heterozygotes. Mating of heterozygotes yielded apparently healthy male and female Dal ek6 null animals. Loss of Dalek6 in the male mice was confirmed by qRT PCR (Figure 2 9A), Western blot (Figure 2 9B), and IF (Figure 2 9C) Dalek6 null mice are viable and develop normally with no obvious difference in general physical appearance when comp ared to their littermates However, additional mating cycles revealed that both male and female Dalek6 null animals are sterile. The testes of adult Dalek6 null mice are significantly smaller than those of wild type (WT) littermates (Figure 2 10 A). Histolo gical examination of testes from Dalek6 null mice reveals that germ cells are largely absent from the seminiferous tubules, which were noticeably smaller (Figure 2 10 B). More specifically, there is a complete lack of post meiotic spermatids and spermatozoa (Figure 2 10 C). To address the fate of these cells, TUNEL labeling reveals many prominent positive signals in a subset of the seminiferous tubules from Dalek6 null mice a s compared to the WT (Figure 2 11 ). The TUNEL positive labeling suggests that cell de ath of meiotic germ cells likely accounts for the absence of post meiotic cells in Dalek6 null mice. However, one must be aware that TUNEL
48 labeling detects double stranded DNA breaks. Such breaks are normal aspect of meiotic recombination. Consequently the TUNEL labeling could in part be a reflection of meiotic arrest where double stranded breaks remain unrepaired The ovaries of adult Dalek6 null mice are also smaller than WT littermates (Figure 2 1 2 A) and lack developing follicles (Figure 2 1 2 B) indicatin g a complete deficiency of mature germ cells. Thus it appears that Dalek6 is required for germ cell development in mice To investigate the stage of meiotic prophase I that is affected by a loss of Dalek6, we investigated the localization of Sun1 in Dalek 6 null primary spermatocytes. We observed that Sun1 remains associated with the NE at discrete foci in spermatocytes of Dalek6 null mice, suggesting that the association between Sun1 and telomeres is unimpaired (Figure 2 1 3 ) and that these cells have enter ed into leptotene. To further examine the impact of loss of Dalek6 on spermatocyte development, we examined the expression and distribution of SCP3 that forms on chromosomes during the transition between the leptotene and zygotene stages. As compared to WT mice SCP3 labeling is diminished and discontinuous in Dalek6 null spermatocytes (Figure 2 1 4 ). These data suggest that defects in Dalek6 null mice, in part, occur during the early stages of meiotic prophase I. Discussion In this study, we have identifie d a novel mammalian KASH domain protein, Dalek6. When introduced into tissue culture cells, Dalek6 localizes to the ONM and when overexpressed will displace other members of the KASH domain protein family from the NE. Localization of Dalek6 is mediated ent irely by its KASH domain and is dependent upon the presence of INM SUN domain proteins, which function as translumenal tethers.
49 The conclusion is that Dalek6 represents a new LINC component, effectively defining a new LINC complex isoform. Observations on the behavior of recombinant Dalek6 expressed in HeLa and HEK293 cells suggested that Dalek6 had characteristics consistent with a role in dynein binding. This notion was based in part upon comparisons with Nesp4, a mammalian KASH domain protein that functi ons as an ONM adapter for conventional Kinesin 1. A dynein binding function for Dalek6 was confirmed by both immunofluorescence microscopy and co immunoprecipitation. The implication was that Dalek6 migh t be functionally related to C. elegans Zyg 12 or Dro sophila Klarsicht, both metazoan dynein binding KASH domain proteins. Zyg 12 is expressed in C. elegans germ line cells where it known to function in meiotic progression (Malone et al., 2003; Zhou et al., 2009) Pos t fertilization, Zyg 12 is required for pronuclear movement. Zyg 12, including a KASH less Zyg 12 isoform has a related role in centrosome attachment to the nuclear periphery (Malone et al., 2003) A KASH less isofo rm of Drosophila Klarsicht is also found in germ line cells where it is required for the microtubule dependent movement of lipid droplets. Full length (i.e. KASH containing) Klarsicht is expressed primarily in the eye where it is essential for the movemen t and position of nuclei within photoreceptor cells. In the absence of Klarsicht, eye development is significantly compromised (Fischer et al., 2004) Studies on a wide variety of tissue culture cells revealed no evidence of Dalek6 expression. Subsequent immunofluor escence analyses of adult mouse tissues indicated that this protein was uniformly absent from somatic tissues. Instead it could be detected only within the testis where its expression was limited to primary spermatocytes. We
50 now know that Dalek6 is also pr esent in embryonic oocytes at 13.5 days of gestation In mammals the oogonia divide to form a limited number of egg precursor cells. At mouse embryo nic day 8, thousands of germ line stem cells, called oogonia divide rapidly Most oogonia die during this period, while the remaining oogonia enter the first meiotic division at around 13 days of gestation. These cells, called the primary oocytes, progress through the first meiotic prophase until the diplotene stage, at which point they are maintained until se xual maturity. While Dalek6 localization in spermatocytes was restricted to the NE, rather than displaying a uniform distribution it was restricted to discrete patches or foci. In addition to Dalek6, these foci contained Sun1 and featured associated telom eres. Given that Sun1 is known to be essential for the association of telomeres with the nuclear periphery, our conclusion is that these Dalek6 positive foci represent attachment sites at the NE for telomeres. Attachment of telomeres to the NE during meios is is a widely conserved phenomenon during eukaryote evolution. This telomere attachment is zygotene phase of prophase I luster at one pole of the nucleus closest to the microtubule organizing center. This event is thought to facilitate faithful homologue pairing and successful completion of meiosis. In Sun1 deficient mice telomere attachment sites at the NE are lost and hom ologue alignment is impaired, resulting in meiotic arrest Needless to say, Sun1 mice are infertile (Ding et al., 2007) Since Dalek6 is localized to the telomere attachment sites, it was our view that Dalek6 could provide a n effective mechanism for the unique chro mosome movement
51 that occurs during meiotic prophase I. The major theme in chromosome dynamics during meiosis, from yeast to mammals, is that it is LINC complex mediated and in the large part is dependent on microtubules and cytoplasmic dynein (Bupp et al., 2007; Conrad et al., 2007; Penkner et al., 2007) In fission yeast for instance, the prototype SUN domain protein, Sad1 is required for telomere attachment. This mediated by a pair of soluble proteins Bqt1 and Bqt2 which form a molecular bridge between the nuc l eoplasmic domain of Sad1 and the telomere associated protein Rap1 (Chikashige et al., 2006) The nature of the telomere Sun1 interaction in mammalian cells has yet to be elucidated. Kms1 is the ONM KASH domain protein that is tethered by Sad1. Kms1 functions as an ONM adaptor for the dynein motor complex which drives telomere clustering at the spindle pole body (Miki et al., 2004; Niwa et al., 2000) In mammals, while Sun1 was recently identified as an essential component for telomere NE association (Chi et al., 2009; Ding et al., 2007; Schmitt et al., 2007) T he corresponding KASH domain protein remained unknown. Our identification of Dalek6 as a meiocyte specific KASH domain protein that colocalizes with Sun1 and functions as a dynein adapter, suggested that it was likely the missing LINC complex component. Confirmation for this role came from our derivation of Dalek6 deficient mice. The Dalek6 knockout mice provide the first genetic model to provide function al insight into the role of mammalian KASH proteins during meiosis. Deletion of Dalek6 in mouse prevented the development of haploid cells. However, the S un 1 telomere association does not appear to be perturbed by loss of Dalek6. Although Dalek6 does not s hare homology to any other proteins, ou t side of its KASH domain, the phenotype from the Dalek6 knockout mice is generally comparable to other model systems
52 deficient in meiotic KASH domain proteins. In C. elegans homozygous deletion of, Zyg 12 led to comp lete sterility due to failure, at least in part, to defects in chromosome synapsis during pachytene (Malone et al., 2003; Sato et al., 2009) In this model, Sun1 remains associated with the NE, indicating that chrom osomes are tethered to the NE (Sato et al., 2009) In S. pombe perturbation of Kms1 leads to a significantly reduced recombination rate between homologou s chromosomes and impaired telomere clustering. As in other model systems, the association of the NE and telomeres was maintained despite mutation of Kms1 (Niwa et al., 2000) The coupling between the NE and microtubules appears to play a key role in telomere mediated meiotic chromosome dynamics in a wide variety of organisms, including S. pombe C. elegans rye, and wheat (Corredor and Na ranjo, 2007; Cowan and Cande, 2002; Cowan et al., 2002; Ding et al., 1998; Tepperberg et al., 1997; Yamamoto et al., 2001) Genetics studies in C. elegans revealed that a Zyg 12 mutant lacking both a self association and dynein interaction domain exhibit discontinuous synaptonemal complex formation between homologous chromosomes during meiotic prophase I, coincident with severely reduced pairing. However, a Zyg 12 mutant lacking only the self associating domain exhibited milder defects during this process (Sato et al., 2009) These observations were supported by knockdown experiment in wild type C. elegans Depletion of either dynein heavy chain or light ch ain by RNA interference led to a failure in synaptonemal complex formation and homolog pairing, suggesting dynein participates in coordinating events early in meiosis (Sato et al., 2009; Zhou et al., 2009) As in th e C. elegans model lacking dynein, primary spermatocytes from Dalek6 null mice, presumably deficient in dynein NE association, cannot progress to the pachytene
53 stage and fail to properly load the synaptonemal complex onto the chromosomes. However the preci se role of dynein in mammalian pairing and synapsis remains largely unclear. Both male and female Sun1 null mice are sterile due to disruption of the association between the NE and telomeres. In developing spermatocytes from Sun1 null mice, non homologous synapsis in pachytene was observed, resulting in apoptosis at the pachytene stage (Ding et al., 2007) As in Sun1 null mice, deletion of Dalek6 led to complete sterility in both male and female mice with evidence of cell death in developing germ cells. However, th e lack of normal SCP3 labeling in Dalek6 null mice indicates that defects occur at an earlier stage of meiosis than that observed in Sun1 null mice. It remains unclear at which specific stage of meiotic prophase I the loss of Dalek6 impacts germ cell devel opment. We can observe that primary spermatocytes from Dalek6 null mice fail to load synaptonemal complexes onto the chromosomes, a process initiated between the leptotene and zygotene stage. I t is possible that Dalek6 is a part of checkpoint mechanism for leptotene zygotene transition to permit pairing and synapsis of homologous chromosomes. One model for the early checkpoint involves mobility of chromosomes during meiotic prophase I. As shown in Figure 2 13, at the NE of Dalek6 null primary spermatocytes, Sun1 appears as distinct foci and evenly distributed indicating that the association between the NE and condensed chromosomes are not only affected by loss of Dalek6 but also remained immobilized at the entry of meiosis. Limiting chromosome movement in Da lek6 null primary germ cells induced by loss of connection to the cytoplasmic motor protein complex is likely to be associated with significantly reduced chance s of synapsis and pairing between
54 homologous chromosomes and thus leads to germ cell death. In c ontrast to Dalek6 null mice, absence of the association between the NE and chromosomes in Sun1 null primary germ cells permit s an incomplete but higher rate of synapsis and paring of freely diffus ing homologous chromosomes than that of Dalek6 null primary germ cells. Incomplete but synaps ed chromosomes appears to be sufficient to pass the early check point but trigger s pachytene arrest as shown in Sun1 null mice (Ding et al., 2007). Another model for the mechanism for the early checkpoint induced cell death involves altered NE elasticity. During leptotene and zygotene, mechanical strain can be generated by tethered condensed chromosomes at the both ends of NE but immediately reduced by moving chromosomes. This mechanical stress on the NE is further resolved during random movement of bivalent chromosomes during the pachytene stage. Prolonged attachment of condensed chromosomes to the both poles of the NE likely generates abnormally increased mechanical stress that is a proposed mechanism of decreased viability and increased apoptosis in HGPS patient cells (Valerie et al., 2008 ). In this model, primary germ cells with reduced NE stiffness caused by loss of Sun1 is permissive to the early check point. Signaling cascades are also involved in monitoring early meios is progression. In yeast, meiotic recombination is initiated by programmed double strand breaks catalyzed by a topoisomerase II like enzyme Spo11 (Keeney et al., 1997). S ubsequent homologous recombination is established by regulatory proteins associated wi th chromosome axis structures, including Hop1 and Mek1 that are regulated by ATM kinase dependent phosphorylation (Carballo et al., 2008). I t has been recently reported in a study using C. elegans that p rogression through meiosis involve s the checkpoint p rotein kinase CHK 2 at the
55 meiotic entry. CHK 2 is responsible for the polarization of the chromatin and is involved in induction of double strand break s and proper SC polymerization, as well as the phosphorylation of S un 1 (Penkner et al., 2009) However t he early checkpoint for mammals is still unknown. T he large numbers of spermatozoa produced in humans exhibit a reduced survival rate during development that is at least 10 fold lower than that calculated for oocytes (Hassold and Hunt, 2001; Pacchierotti et al., 2007). T he oocytes in Dalek6 null mice are also susceptible to the loss of Dalek6 most likely due to cell death in the early stage of oocyte development. Seminiferous tubules of Dalek6 null mice are largely devoid of post meiotic cells but maintai n their tissue shape. As mentioned before, in mammals, spermatocytes are continuously produced from spermatogonia that reside at the periphery of the seminiferous tubule. The existence of spermatogonia contributes to the organization of seminiferous tubule s of Dalek6 null mice. Loss of primary oocytes seems to cause more severe effect s on ovaries than spermatocyte loss on testes in Dalek6 null mice. Primary oocytes are differentiated from oogonia during embryo genesis and stored until sexual maturity M assiv e cell death of primary oocytes, combined with absence of oogenic stem cells in the ovaries, likely results in the severely disorganized ovaries that are apparent in Dalek6 null mice. Th ese anatomical analys es clearly support the essential role suggested f or Dalek6 in primary germ cell development. In summary, our results reveal that the mammalian outer nuclear membrane KASH domain protein, Dalek6, is required for germ cell development. We propose that
56 Dalek6 mediates chromosome movement during meiotic pr ophase I by linking telomere associated Sun1 to cytoplasmic dynein (Figure 2 1 5 ). Materials and Methods Generation of Dalek6 null M ice ES cells harboring the floxed allele of Dalek6 was obtained from Kno ck out Mouse Project (KOMP). Generating Dalek6 null mice using the ES cells was carried out in the Mouse Core Facility of the Institute for Medical Biology, Singapore. Deletion of the Floxed exons was induced by mating with Zp3 Cre transgenic mice (Lewandoski et al., 1997) Plasmids M ouse Dalek6 cDNA (clone ID 30008752) was obtained through Invitrogen For N terminal tagging amplified by PCR. The PCR product was inserted downstream of an H A tag and G FP tag in p c DNA3.1 Cell Lines D oxycycline inducible HEK293 cells were transfected with pTight plasmids containing a puromycin resistance gene (Clontech) Twenty four hours post transfection, these cells were selected with 0.25 g/mL puromycin. Growth med ium was replaced with fresh medium containing puromycin every 2 to 3 days to maintain the concentration of active puromycin. After 10 to 12 days of puromycin selection, expression of Dalek in individual surviving clones were isolated and checked by fluores cence microscopy, in which clones that expressed moderate levels (Crisp et al., 2006; Liu et al., 2007; Roux et al., 2009)
57 Antibodies T h e following antibodies were used in this study: the monoclonal anti lamin A/C (XB10), anti Nup153 (SA1), anti HA (12CA5, Covance), anti tubulin (GTU 88, Sigma) anti DIC(MMS400R, Covance), p150 (610474, BD bioscience); polyclonal rabbit anti HA (ab9110, Abcam), anti GFP (ab290, Abcam), anti SCP3 ( ab85621 Abcam), anti Rap1 ( ab111 91 Abcam), anti Sun1 (HPA008346, Sigma), anti Sun2 (HPA001209, Sigma), anti DHC ( sc9115, Santa Cruz) Polyclonal anti Nesp3 was previously described (Hodzic et al., 2004) R abbit polyclonal mouse Dalek6 was raised by Yenzym. Immunofluorescence Specimens fixed and permeabilized with 3% PFA and 0.4% TX 100 were labeled with appropriate primary and secondary antibodies. Images were obtained using a microscope (model DMRB; Leica) and a CDC camera (CoolSNAP HQ; Photometrics) linked to a Macintosh G4 computer running IPLab Spectrum software (Scan alytics). I mmunoprecipitations For immunoprecipitations, HEK293 cells transiently expressing GFP Dalek6 KASH were lysed in 1 mL of lysis buffer containing 50 mM NaCl, 50 mM Tris pH, 2.5 mM MgCl2, 0.5% TX 100, 1 mM DTT and proteinase inhibitor (Thermo Scientific) Lysates were passed through a 21 gauge needle (10) and centrifuged 16,000 g for 10 min at 4 C. The supernatants were rotated for 4 h at 4 C with protein A Sepharose beads (Sigma) coupled to rabbit anti GFP. Samples were analyzed by SDS/PAGE as previously described (Liu et al., 2007) Isolation of M ouse S permatocytes S preading of spermatocytes was performed as previously described (Scherthan et al., 2000)
58 TEL FISH Combinatorial immunostaining and telomere FISH were carried out as previously described (Scherthan, 2009) TUNEL Assay TUNEL Assay s were performed following a protocol described previously (Tornusciolo et al., 1995)
59 Figure 2 1. Ident ification of Dalek6. (A) KASH domain of Dalek6 has homology with other mammalian KASH domain proteins. (B) Dalek6 contains putative EF hand and coiled coil motif s
60 Figure 2 2. Dalek6 is a genuine KASH family member. (A) In HeLa cells, GFP Dalek6 targets to the NE, colocalizing with NPC constituent, Nup153. (B) GFP Dalek6 expressed in HeLa is polarized to the centrosome. (C) After digitonin or Triton X 100 permeabilization, the GFP tagged N terminus is readily detected while lamin C is only detected with Triton X 100 permeablization (D) The KASH domain of Dalek6 (GFP KASH) is sufficient for NE targeting ( E ) The KASH domain of Dalek6 displaces endogenous Nesprin s from the NE. ( F ) Dominant negative S un 1 (SS HA Sun1L KDEL) perturbs the interaction between S UN domain proteins and Dalek6. (G) NE targeting of Dalek6 is dependent on SUN proteins. Sun1/2 RNAi dislocates HA Dalek6 from the NE of the cells stably expressing HA Dalek6. Bar, 10 m.
61 Figure 2 3 Dalek6 recruits cytoplasmic d ynein to the NE (A) GFP Dalek6 expressed in HeLa re cruits DIC to the NE (B) which is enhanced by co expression of myc Sun2. (C) Expression of GFP Dalek6 in HeLa cells recruits p150 to the NE Bar, 10 m. (D) HEK293 cells expressing GFP Dalek6 KASH or GFP were processed for immu noprecipitation with anti GFP. Immunoprecipitated samples were analyzed by Western Blot using anti GFP, anti DIC, anti DHC and anti p150. DIC, DHC, and p150 were clearly evident in IPs containing GFP Dalek6 KASH as shown in total lysate.
62 Figure 2 4 Dalek6 antibody (anti Dalek6) specifically recognizes murine Dalek6 (MmDalek6) in immuno fluorescence and Western blot. (A) In HeLa cells, anti Dalek6 recognized transiently expressed m urine Dalek6. A nti Dalek6 did not detect transiently expressed GF P tag ged m urine Nesprin 4 (Mm Nesprin 4). Pre immune serum was not reactive to GFP MmDalek6. Bar, 10 m. (B) HeLa cells expressing GFP MmDalek6 and GFP Mm Nesprin 4 were analyzed by Western Blot using anti GFP and anti Dalek6. Anti GFP detected GFP Mm Nesprin 4 and GF P MmDalek6. MmDalek6 was specifically detected only by anti Dalek6.
63 Figure 2 5 Dalek6 is predominantly expressed in the adult testis. (A) Immunofluorescence microscopy of adult mouse testis using rabbit anti mouse Dalek6. Dalek6 is prominent at distin ct punctae at the NE in primary spermatocytes and to a lesser extent at one pole of post meiotic spermatids Bar, 10 m. ( B ) RT PCR analysis of major mouse tissues. Dalek6 message was detected only in adult mouse testis.
64 Figure 2 6 Dalek6 is localized at the meiotic attachment plates in primary spermatocytes. (A) Immunolabeling of isolated primary spermatocytes reveal s colocalization of Dalek6 with the end of condensed chromosomes indicated by S cp 3. (B) Dalek6 is colocalized with telomere binding prote in, Rap1. (C) Dalek6 colocalizes with telomeres labeled by T el FISH. (D) Immunolabeling of cryosectioned mouse testis reveal s colocalization of Dalek6 with S un 1. Bar, 10 m.
65 F igure 2 7. Dalek6 colocalizes with cytoplasmic dynactin in primary spermatoc ytes. Immunofluorescence microscopy of adult mouse testis using anti Dalek6 and anti p150 detects Dalek6 colocaliz ation with p150, a major subunit of dynactin. Bar, 10 m.
66 Figure 2 8 Dalek6 appears to be the only KASH domain protein expressed in pri mary spermatocytes Nesprins 1 3 are not detected in primary spermatocytes that are co immunolabeled with either Dalek6 or Scp 3. Bar, 10 m.
67 Figure 2 9. Validation of Dalek6 null mice Absence of Dalek6 message is confirmed by qRT PCR (A) and Western blot (B). Dalek6 is not detected in the Dalek6 null primary spermatocytes labeled by SCP3 (C).
68 Figure 2 10. Spermatogen e sis is largely disrupted in the seminiferous tubule of Dalek6 null mice. (A) The testes from adult Dalek6 nul l mice are smaller than WT littermates (B) Hematoxylin and eosin stained histological cross sections of testes from adults Dalek6 nul l mice (C) Enlarged images of (B) reveal a lack of germ cells in the seminiferous tubules.
69 Figure 2 11. Cell deat h was occurred in Da l ek6 null mice. The seminiferous tubules from Dalek6 null mice contain many TUNEL positive cells as compared to WT littermates.
7 0 Figure 2 12 Oogenesis is disrupted in Dalek6 null mice. ( A ) The ovaries from Dalek6 null mice are sma ller than WT littermates ( B ) Dalek6 null ovaries are devoid of developing follic les. Arrows indicates the ovaries. Arrowhead indicates f allopian tube s.
71 Figure 2 13. Sun1 remains associated with the NE in spermatocytes of Dalek6 null mice In primar y spermatocytes from Dalek6 null mice, i mmunolabeling with anti Sun1 and anti SCP3 reveal s that Sun1 is expressed as distinct punctae and is associated with the NE. Bar, 10 m.
72 Figure 2 14. Loss of Dalek6 does not prevent entry into meiotic prophase Immun ofluorescence microscopy with anti Dalek6 and anti SCP3 detects expression reveals expression of SCP3 is diminished and discontinuous in primary spermatocytes from Dalek6 null mice. Bar, 10 m.
73 Figure 2 1 5 Model of Mammalian LINC complex functio n during meiotic prophase I. (A) A model for the mammalian LINC complex consisting of telomere associated Sun1 and dynein engaged Dalek6. (B) After tethering chromosomes to the NE in leptotene, (C) ( )end directed motor proteins direct the chromosome ends towards the MTOC in zygotene, an event that coincides with initial pairing and sy n apsis. (D) Based on studies in C. elegans during pachy t ene, chromosomes are tugged by motors to license synapsis, thus providing quality control to recombination.
74 CHAPTE R 3 IDENTIFICATION OF TH E NUCLEAR ENVELOPE P ROTEINS BY A NOVEL M ETHOD USING A MUTATED BIOT IN LIGASE Introduction Elucidation of protein protein interactions represents a significant barrier to the understanding of many biological processes. Biochemical and genetic techniques, including affinity capture complex purification and yeast two hybrid strategies provide powerful tools in the search for unknown associations. However, these methods also display fundamental limitations. For many high throughput geneti c approaches, protein interactions are commonly assessed in a cellular environment unlike that in which they would normally occur, resulting in incomplete or erroneous data sets. Biochemical approaches suffer loss of candidates through protein insolubility and transient or weak interactions. These issues are more relevant than ever, as we collectively look to the E. Coli BirA is a 35kDa DNA binding protein and biotin protein ligase (BPL) BirA mediate s the biotinylation of a subunit of acetyl CoA carboxylase, while at the same time functioning as transcriptional repressor of the biotin operon (Chapman Smith and Cronan, 1999) BirA has been harnessed fo r experimental applications, including use in eukaryotic cells. The BirA acceptor peptide system takes advantage of the extreme specificity of BirA in biotinylating its single physiological substrate (Beckett et al., 1999) With this system, a minimal recognition sequence, a biotin acceptor tag (BAT), is fused to a protein of interest and co expressed with BirA. This leads to the biotinylation of the BAT sequence permitting one step high affinity (K d =10 14 M) (Green, 1963) avidi n/streptavidin mediated purification of the tagged protein. Since biotinylation is a rare modification, in mammalian cells it is restricted primarily to only a few carboxylases
75 (Chapman Smith and Cronan, 19 99) BAT independent binding is minimal. Biotinylation by BirA is a two step process. The first combines biotin and ATP to form biotinoyl AMP (bioAMP) and pyrophosphate (Lane et al., 1964) This activated biotin is held within the BirA active site until it reacts with a specific lysine residue of the BAT sequence. Certain BirA mutants forgo this second step and instead release the highly reactive yet labile bioAMP (Kwon and Beckett, 2000; Streaker and Beckett, 2006) One such BirA mutant (R118G, hereafter called BirA*), that is defective in both self association and DNA binding (Kwon et al., 2000) displays an affinity for bioAMP two orders of magnitude less than that of the wild type enzyme (BirA WT). In E. coli BirA* expression results in promiscuous protein biotinylation since free bioAMP will readily react with any available primary ami nes. More significantly however, it has been demonstrated in vitro that BirA* will promiscuously biotinylate proteins in a proximity dependent manner (Choi Rhee et al., 2004; Cronan, 2005) R esults We explored the possibility that BirA* would promiscuously biotinylate proteins in live mammalian cells. To this end we generated myc epitope tagged humanized BirA WT and BirA*, each of which we expressed in HEK293 cells. Western blot analysis employing HRP streptavidin y ielded qualitatively similar yet limited spectra of biotinylated proteins with both BirA WT and BirA* (Figure 3 1A). However, addition of 50M biotin to the tissue culture medium resulted in a massive stimulation of apparently promiscuous biotinylation by BirA* but not BirA WT (Figure 1A). Fluorescence microscopy revealed that the distribution of biotinylated proteins was virtually identical to that of Myc BirA* itself (Figure 3 1B). The effect of exogenous biotin can be accounted for by the fact that the d issociation equilibrium constant (K D ) of BirA* for biotin is about
76 2M while the intracellular concentration of biotin is likely less than 10nm. Clearly biotinylation by BirA* is limited by availability of biotin, a finding that allows us to manipulate Bir A* activity at will. We next wished to determine whether BirA* could be used as a tool to identify interacting or nearby proteins in vivo To this end we fused Myc BirA* to the N terminus of human A type lamin (LaA). LaA is a well characterized constituent of the nuclear lamina, a structure that underlies the INM LaA has a relatively restricted distribution within the cell (Goldman et al., 2002a) and a number of LaA interacting proteins, both soluble and membrane associated, have been described To provide consistent and controllable expression levels, HEK293 cells were generated that stably and inducibly express mycBirA*LaA. Like endogenous LaA, Myc BirA*LaA localizes predominantly to the nuclear envelope and displays similar solubility propert ies. Biotinylation of proteins in cells expressing Myc BirA*LaA, either in the presence or absence of exogenous biotin, was followed by western blot analysis. As was the case with Myc BirA* alone, the presence of 50M biotin in the culture medium was found to strongly stimulate biotinylation of a broad range of endogenous proteins (Figure 3 2A), in addition to Myc BirA*LaA itself. Fluorescence microscopy employing streptavidin Alexa revealed that the bulk of these biotinylated proteins must reside at the NE and co localize with Myc BirA*LaA detected with an anti Myc antibody (Figure 3 2B). The implication is that proteins in the vicinity of Myc BirA*LaA are preferentially biotinylated. These results suggest that BirA* can be targeted to a specific cellular l ocation and will biotinylate proteins in a proximity dependent manner. Moreover the requirement for exogenous biotin suggests a means to modulate BirA* activity. To explore this further, HEK293
77 cells expressing myc BirA* LaA were processed for both Western blot analysis (Figure 3 3 A) and fluorescence microscopy (Figure 3 3 B) at various times following addition of 50M biotin to their culture medium. Both methods reveal a time dependent accumulation of biotinylated proteins. The effect reaches saturation wit hin 6 24 h. These studies indicate that by controlling access to biotin we can temporally regulate biotinylation by BirA*. We next set out to test our hypothesis that proteins biotinylated by Myc BirA* LaA should be enriched with known interactors of LaA as well as with near neighbors within the nuclear lamina and inner nuclear membrane. To accomplish this we induced Myc BirA*LaA expression in HEK293 cells in the presence of 50 lysed the cells under denaturing conditions. Parental HEK293 cells, processed in parallel were used as controls. For these experiments 4.0X10 7 cells (four confluent 10cm dishes) were analyzed. Biotinylated proteins were captured with streptavidin immobilized on paramagnetic beads, rigorously washed, and bound proteins analyzed by mass spectrometry. Proteins that were unique to the BioID LaA (mycBirA*LaA) sample were categorized based on localization and function (Figure 3C). The relative abundance of the identified proteins within each category is given as a percentage of the total. Identities of these proteins can be found in Table 3 1 The bulk of the proteins identified by BioID LaA are lamin associated, constituents of the INM NE associated or nucleoplasmically oriented nuclear pore complex (NPC) components. Proteins associated with DNA repair, transcription, chromatin regulation and RNA processing are also well represented among the BioID LaA candidates, albeit at lower level s.
78 An uncharacterized protein of 75kDa, FAM169A (KIAA0888), featured prominently in the BioID LaA data set. FAM169A has no predicted transmembrane domain and lacks any sequence motifs that might provide a clue to its function. To test the possibility that FAM169A is a novel, likely soluble, NE constituent we examined the localization of the endogenous protein in HEK293 cells by immunofluorescence microscopy Figure 3 4 clearly shows that FAM169A is concentrated at the NE (Figure 3 4A) We also introduced hu man HA epitope tagged FAM169A into HeLa cells, which do not normally express this protein. Consistent with the findings in HEK293 cells, recombinant FAM169A localized predominantly to the NE (Figure 3 4 B).Differential permeablization of HEK293 cells with d igitonin versus Triton X 100 indicates that FAM169A must reside on the nuclear face of the NE ( Figure 3 5) At the same time we could see no obvious association with NPCs. These observations indicate that FAM169A is a novel NE component that must be locali zed to the nuclear lamina or to the interface of the lamina and IMN. We therefore propose to name this protein, SLAP75 for s oluble l amina a ssociated p rotein of 75 kDa. As far as we are aware, SLAP75 is the only soluble protein, other than the nuclear lamin s themselves, to be enriched at the nuclear lamina. Our identification an entirely new constituent of the well characterized NE highlights the use of BioID as a new tool in defining protein protein interactions and protein proximity. D iscussion We have de vised a simple and rapid technique, BioID, which provides a means of identifying interacting and neighboring proteins in vivo The method takes advantage of BirA*, a highly promiscuous form of the E. coli BirA biotin protein ligase. BirA* may be targeted t o specific sub biotinylated by BirA*, can then be recovered in a single step on streptavidin coated
79 beads and identified by mass spectrometry. The only requirement for BioID is the expressi on of a single fusion protein. Consequently, BioID should be applicable to map protein associations in essentially any cell type, mammalian or otherwise. There are currently two strategies that are widely used to detect protein interactions. The first of t hese involves the yeast two hybrid (Y2H) system and takes advantage of the ability of hybrid transcription factor domains to functionally associate, thereby driving expression of reporter genes. The second strategy is based upon co immunoprecipitation or p ull down, frequently involving expression of single or double precipitated proteins are then identified by mass spectrometry. A significant advantage of the Y2H approach is that since it is based on a cDNA library screen it is more likely to detect weak or rare interactions. On the other hand it is contingent upon proteins, or protein fragments maintaining their ability to associate when removed from their normal cellular environment. By definition these interactions must tak e place within yeast nuclei. In many situations this may present a significant problem, especially when membrane proteins enter the equation. The other with th eir cognate partners may display other spurious interactions and hence give rise to false positives. The pull down approach has provided valuable data in a variety of systems. However, it has two limitations. The first of these is the problem of scale when dealing with low abundance proteins. The second and more serious limitation concerns with preserving interactions with partner proteins. This becomes especially signi ficant
80 when considering weak interactions. In the case of lamin A, a highly insoluble protein, this has proved to be a serious stumbling block in the reliable identification of interacting proteins. Recently, however, Misteli and colleagues have used chemi cal cross linking to stabilize complexes prior to solubilization and pull down (Kubben et al., 2010) Significantly, this approach detected many of the same putative LaA interactors that we have identified using BioID LaA. While cross linking represents a valuable enhancement to the pull down strategy, it may in turn create additional artifacts due to aggregation. These will be apparent as false positives. We believe that BioID provides a useful complement to these more established approaches in the charact erization or protein protein interactions and near neighbor analyses. BioID uniquely combines two important attributes. The first of these is that it detects potential interactions in their normal cellular context. The second is that it occurs prior to solubilization it should detect both weak and transient interactions. Both of these features are highlighted in our BioID LaA data where both soluble and membrane proteins were efficiently detected. As with any method, BioID has certain limitations that must be appreciated. Foremost among these is that it relies on fusion of the protein under investigation to BirA*. Clearly, it is essential that the fusion protein should di splay the same targeting properties as its wild type counterpart. Given that BirA* is only a little larger than GFP, there is at least precedent that many BirA* fusion proteins will exhibit functional interactions and dynamics that are similar to wild type There is also the possibility that biotinylation of the fusion protein itself could modify its own behavior. Obviously these
81 are issues that must be addressed on a case by case basis. As is the situation with the co immunoprecipitation and pull down stra tegies described above, BioID may also be limited by scale and may miss very low abundance proteins. Because biotinylation requires the availability of primary amines (most commonly on lysine residues), the absence of biotinylation does preclude an interac tion. Equally important, the presence of biotinylation cannot be used to validate an interaction. Given the mechanisms that underlie BioID, biotinylated proteins can be placed into three categories: 1) those that form direct interactions, either transient or stable, with the protein of interest; 2) form indirect interactions; 3) proximate proteins that do not interact either directly or indirectly. As shown in Figure 2 3B, mycBirA* LaA and biotinylated proteins are concentrated at the NE. This observation is consistent with the mass spectrometry data containing very low abundan ce cytoplasmic protein s among th ose isolated by BioID LaA. Further analysis on the proteins identified by BioID LaA suggests a n approximate activity radius or radius of biotinylation. A majority of identified proteins (> 5 0%) are nuclear proteins associat ed with the nuclear lamina a nd with the nucleoplasmic fac e of NPCs. Given what is known of the disposition of these proteins, the implication is that the majority of biotinylation event s occur within 20 nm of mycBirA* LaA ( Hppener et al., 2005). In other words, the radial distance in which biotinoyl AMP decays by 50% is likely in the range of 10s of nanometers. Clearly it will be essential to better define the activity radius of BirA* s ince this will provide important information on the functional interactions of BirA* targets. It may be possible to examine the radius of BirA* biotinylation by using characteristic self biotinylation of a BirA* fusion protein F o r this purpose BirA* conj ugated recombinant protein that contains a series of unique tags upstream of target
82 modules, separated by rare proteolytic cleavage sites and containing acceptor lysine residues, could be expressed in cells or by in vitro translation. Self biotinylation on the specific lysine residue of the fusion protein might then be detected by Western blotting follow ing proteolytic digestion In this way, biotinylation of specific domains could be correlated with distance from the enzyme. Clearly one would have to emplo y protein modules with extended conformations such as large coiled coil domains A nother approach might be to use a BirA* tagged protein that localizes to the basal membrane of epithelial cells such as MDCK. Vertical section using confocal microscopy on th e MDCK cells expressing the fusion protein will track the fusion protein and biotin and thus will detect the distance of biotinylation from the basal membrane. Nevertheless it is obvious that biotinylation generated by BirA* is highly dependent on the mult iple factors such as accessibility of biotin, folding and structure of interacting proteins, number of lysine s, strength of interaction, cell types etc. Results would also be complicated by the mobilities of target proteins, which are rarely fixed in vivo. As such the best way to examine the radius of biotinylation of BirA* fusion protein will be identifying the known binding partners of well characterized bait protein s As such BioID does not specify direct interactions but instead identifies candidates t hat must be validated by alternative methods The strength of the BioID technique resides in the promiscuous biotinylation activity of BirA*. However, this also conceals a potential weakness. Biotinylation by BirA* is indirect and involves the release of a highly reactive species, BioAMP from the BirA* active site. There is currently little information on how long BioAMP may survive in vivo and how far it might diffuse. Based on our mass spectrometry results we see clear
83 evidence that a plurality of biotin ylated proteins identified by BioID LaA are well characterized NE proteins that are known interactors of LaA. These include LAP2 beta/gamma, LBR and the NPC protein, ELYS. Each of these proteins were recently identified as potential LaA interactors by the study using cross linking method (Kubben et al., 2010) In all, greater than 50% of peptides detected by MS identified proteins of the INM, lamina and nuclear face of NPCs. More than 80% of the peptides identified nuclear proteins (including the aforementi oned LaA interactors). Intriguingly, abundant nuclear proteins such as histones and nucleolar components were poorly represented, indicating that we were not seeing general or widespread biotinylation within the nucleus. This could also be inferred from ou r fluorescence microscopy data where the streptavidin labeling is restricted largely to the NE. The implication is that the biotinylation is most effective within the immediate vicinity of the Myc BirA* LaA fusion protein. However, it will be essential to better define the BirA* activity radius. A further validation of the BioID technique is the identification of SLAP75, a novel component of the NE in HEK293 cells but not in HeLa cells. Its lack of any obvious transmembrane domain combined with its concentr ation at the nuclear face of the NE implies that it is most likely contained within the nuclear lamina, perhaps in association with INM proteins. Certainly we can find no evidence that it is enriched at NPCs. Our preliminary data suggest that the localizat ion of SLAP75 is independent of LaA, indicating that it must interact directly with other NE components. SLAP75 appear to be conserved among vertebrates and has been identified in D. rerio X. laevis G. gallus B. t aurus M. musculus R. norvegicus and H sapiens. Expression databases predict that SLAP75 is concentrated in brain and spinal cord, suggesting that SLAP75 has functions
84 related to the nerv ous system. The role of the nuclear lamina and its associating proteins in neuronal cells is unclear. Alth ough mutations in A type lamins are associated with a myriad of human diseases, there are no obvious defects in the central nervous system in these patients. Duplication of LMNB1 encoding lamin B1 leading to increased expression of the protein causes adult onset autosomal dominant leukodystrophy, a slowly progressive neurological disorder characterized by symmetrical widespread myelin loss within the central nervous system (Padiath et al. 2006). La min B2 deficient mice exhibit severe brain abnormalities res embling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum (Coffinier et al., 2010). C omparing the location of SLAP75 in WT and lamin B2 null mice will provide helpful information to understand the role of SLAP75. In add ition BioID SLAP75 in MEF s from WT and lamin B2 null mice might identify binding partners that are coordinately involved in neuronal cell physiology. Less abundant biotinylated proteins that we identified include those that are associated with DNA repair, transcription, chromatin regulation and RNA processing. Since these proteins are not significantly enriched at the NE, this raises the question of how they were specifically biotinylated by BioID LaA. Our view is that these likely represent nuclear protei ns that transiently associate with LaA at the NE and/or were biotinylated by nucleoplasmic BioID LaA (Goldman et al., 2002a) Some of the identified proteins that are classified as cytoplasmic or ER resident proteins may reflect an unrecognized subpopulation of these proteins that transiently associate with LaA at the NE. Certainly ER proteins can access the INM. It is also possible that biotinylation of these proteins by BioID LaA might occur during mitosis when the NE and lamina are
85 disassemble d. Cytoplasmically oriented NPC proteins such including Nup214 and RanBP2/Nup358 were also specifically biotinylated. It is possible that this might occur during nuclear import of Myc BirA* LaA. However, the more abundantly biotinylated nucleoplasmic NPC p roteins Nup153, ELYS and TPR all have documented associations with the nuclear lamina (Al Haboubi et al., 2011) These interactions could explain the altered distribution of NPCs observed in LMNA deficient cells (Sullivan et al., 1999a) In summary, BioID as a technique should be accessible to a broad range of researchers comfortable working with conventional molecular and cell biology techniques, and does not require specialized equipment other than the proteomic analysis that has become a commonly available service. We propose that BioID provides a powerful new approach to probe protein interactions and proximity in a variety of cell types. We will continue to explore the advantages intrinsic to the BioID system. This includes its application in various subcellular compartments and the ability to monitor interactions of proteins at different time points following their synthesis, or at different stages of the cell cycle by regulating biotin availability. Materials a nd M ethods Plasmids H umanized BirA (Mechold et al., 2005) was mutated to R118G by overlap extension PCR. Products fo end, an XhoI, stop codon and AflII. These were digested with SalI and AflII and inserted into pcDNA3.1 C terminal to a myc epitope digested with XhoI and AflII. Human LaA was excised from pcDNA3. 1 by XhoI and AflII and inserted in frame with the mycBirA* in pcDNA3.1 using the same restriction sites. The entire mycBirA*LaA sequence was removed from pcDNA3.1 by NheI and AflII, bunted and inserted into pRetroX.Tight.puro
86 that was digested with EcoRI and blunted. Clones were screened for proper directionality. Cell C ulture and G eneration of S table C ell L ines pRetroX Tet ON Advanced HEK293 cells (Clontech) were transiently transfected with pRetroX Tight.puro mycBirA*LaA as previously described (Roux et al., 2009) 48 hrs after transfection the cells began selection with 1ug/m L puromycin. Upon colony formation, subclones were isolated and selected by immunofluorescence following the addition of 1ug/m L doxycycline for 24hrs. Immunofluorescence C ells were fixed with paraformaldehyde and imaged as previously described (Roux et al., 2009) Anti myc (9E10, Covance) and streptavidin 568 (Invitrogen) were used to identify mycBirA fusion proteins and biotinylated proteins, respectively. Other antibodies include anti FAM169A (Sigma) and anti HA (12CA5, Covance). Western B lotting C ells were lysed in Laemmli SDS sample buffer, separated by SDS PAGE and transferred to nitrocellulose (Liu et al., 2007) Immunoblotting was performed (Liu et al., 2007) with the following antibodies; rabbit anti myc (Abcam). Biotinylated proteins were detected similarly with t he following modifications. Membranes were blocked in 2.5% bovine serum albumin in PBS with 0.4% Triton X 100 and incubated in the same buffer with HRP conjugated streptavidin (Invitrogen). Affinity C apture of B iotinylated P roteins C ells were incubated 24 h in complete media supplemented with 1 g/m L doxycycline and 50 M biotin. After three PBS washes 4x10 7 cells were lysed at 25C in 1 mL lysis buffer (50 mM Tris (pH 7.4), 500 mM NaCl, 0.4% SDS, 5 mM EDTA, 1 mM
87 DTT, 1X Complete protease inhibitor (Roche)) and sonicated. Triton X 100 was added to 2% final concentration. Following further sonication, an equal volume of 4C 50 mM Tris (pH 7.4) was added before additional sonication (subsequent steps at 4C). Samples were diluted with an equal volume of 50 mM Tris (pH 7.4) and centrifuged 16,000 RCF. Supernatants were incubated with 600 mL Dynabeads (MyOne Steptavadin C1, Invitrogen) overnight. Beads were collected and washed twice for 8 min at 25C (all subsequent steps at 25C) in 1 m L wash buffer 1 (2% SDS i n dH2O). This was repeated once with wash buffer 2 (0.1% deoxycholate, 1% Triton X 100, 500 mM NaCl, 1 mM EDTA, 50 mM Hepes (pH 7.5)), once with wash buffer 3 (250 mM LiCl, 0.5% NP 40, 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8.1)) and twice with wash buffer 4 (50 mM Tris (pH 7.4), 50 mM NaCl). 10% of the sample was reserved for Western blot analysis. Bound proteins were removed from the magnetic beads with 5 minutes of Laemmli SDS sample buffer saturated with biotin at 98C. The 90% of the sample to be analyzed by mass spectrometry was washed twice in 50 mM NH 4 HCO 3 Protein I dentification by M ass S pectrometry O n bead tryptic digests were analyzed by 1D LC/MS/MS by the Sanford Burnham Proteomic Facility (La Jolla, CA). Tris(2 carboxyethyl)phosphine (TCE P) was added to 100 L of beads suspension mix and proteins were reduced at 37C for 30 min. Iodoacetamide was added (to 20 mM) and proteins were alkylated at 37C for 40 min in the dark. Mass spectrometry grade trypsin (Promega) was added (~1:50 ratio) fo r overnight digestion at 37C. Magnetic beads were removed by centrifugation. Formic acid was added to the peptide solution to (2%) prior to on line analysis of peptides by high resolution, high accuracy LC MS/MS, consisting of a Michrom HPLC, a 15 cm Mich rom Magic C18 column, a low flow ADVANCED Michrom MS source, and a LTQ
88 Orbitrap XL (Thermo Fisher). A 120 min gradient of 10 30%B (0.1% formic acid, 100% acetonitrile) was used to separate the peptides. The total LC time was 141 min. The LTQ Orbitrap XL wa s set to scan precursors in the Orbitrap at a resolution of 60,000, followed by data dependent MS/MS of the top four precursors. Raw LC MS/MS data was submitted to Sorcerer Enterprise (Sage N Research Inc.) for protein identification against the IPI human protein database, which contains semi tryptic peptide sequences with the allowance of up to 2 missed cleavages and precursor mass tolerance of 50.0 ppm. A molecular mass of 57 Da was added to all cysteines to account for carboxyamidomethylation. Differenti al search included 16 Da for methionine oxidation, and 226 Da on N terminus and lysine for biotinylation. Search results were sorted, filtered, statically analyzed and displayed using PeptideProphet and ProteinProphet (Institute for Systems Biology). The m inimum trans proteomic pipeline (TPP) probability score for proteins was set to 0.95, to assure TPP error rate of lower than 0.01.
89 Figure 3 1. BirA* promiscuously biotinylates endogenous proteins in mammalian cells. HeLa cells were analyzed 24 hours af ter transient transfection with mycBirA WT or myc BirA* (R118G). Following transfection, cells were grown either with or without supplemental biotin (50 M). ( A) By Western blot analysis similar levels of the exogenous BirA (asterisk) are detected in all s amples with anti myc. Biotinylated proteins, including both exogenous BirA (asterisk) and endogenous proteins, were detected with HRP streptavidin. Enhanced protein biotinylation is observed in the mycBirA* samples as compared to the WT isoform. This diffe rence is dramatically enhanced by the presence of excess biotin. (B) By fluorescence microscopy the BirA is predominantly nuclear as observed with anti myc (red). Biotinylated proteins were detected with fluorescently labeled streptavidin (green). Consider able biotinylation is only observed in cells expressing mycBirA* and supplemented with excess biotin. The biotin signal predominantly colocalizes with mycBirA*. DNA is labeled with Hoescht (blue). Scale bar is 4 m. These data indicate that BirA* is efficie nt at promiscuously biotinylating endogenous proteins in the presence of excess biotin.
90 Figure 3 2. Proximity dependent promiscuous biotinylation by BioID LaA. HEK293 cells inducibly expressing mycBirA*LaA were analyzed 24 hours following induction. Parental HEK293 cells were treated identically in parallel. Cells were grown either with or without excess biotin. (A) By immuno blot analysis the LaA fusion protein (asterisk) is detected with anti myc. Levels of endogenously biotinylated proteins, detect ed with HRP streptavidin, are unaffected by the supplemental biotin. However, the biotinylation of endogenous proteins by mycBirA*LaA is dramatically enhanced in the presence of excess biotin. (B) The mycBirA*LaA is detected at the nuclear rim and to a les ser extent in the nucleoplasm (red). Biotinylated proteins (green) colocalize with the LaA fusion protein. DNA is labeled with Hoechst (blue). Scale bar is 5 m. (C) The identity of the proteins biotinylated by BioID LaA was determined by mass spectrometry of proteins isolated with streptavidin coupled magnetic beads. The relative abundance (% of total identified peptides) and classification of proteins uniquel y biotinylated by mycBirA*LaA is depicted in the chart. These data support the concept that BioID biotinylates proteins in a proximity dependent manner and that this method can be used to screen for candidate proximate and interacting proteins.
91 Figure 3 3. Temporal regulation of access to excess biotin controls biotinylation by BioID. To monitor the relative rate of biotinylation by BioID, HEK293 cells were induced to express mycBirA*LaA and provided with 50 M biotin at different time points. Cells were simultaneously analyzed by Western blot and fluorescence microscopy. (A) The levels of mycBirA*LaA (asterisk in A) are similar for all conditions; however, the extent of biotinylation, detected with HRP strept avidin, increases in relation to the duration of biotin supplementation. This effect appears to be saturated by 24 h. (B) Similar results were observed by fluorescence microscopy. All images were taken at identical exposure times. MycBirA*LaA was detected with anti myc (red) and biotinylated proteins with fluorescently labeled streptavidin (green). DNA was labeled with Hoechst (blue). Scale bar is 30 m.
92 Figure 3 4 SLAP75 is a novel NE constituent identified with BioID LaA. To determine if FAM169A/SLAP75 is a novel NE constituent its subcellular localization was analyzed by immunofluorescence microcopy. (A) As detected with anti SLAP75 (red), the endogenous protein is co localized with LaA (green) at the NE of HEK293 cells. (B) Although endogenous SLAP75 is not detected in HeLa cells (not shown), transiently expressed HA SLAP75 (red) is localized to the NE, labeled with anti Nup153 (green). These d ata indicate that SLAP75 has NE targeting properties. DNA was labeled with Hoechst (blue). Scale bar is 10
93 Figure 3 5. SLAP75 is a constituent of the INM. To verify the topology of SLAP75 localization at the NE we performed immunofluorescence with selective permeabilization. HEK293 cells, permeabilized with Triton X 100 to permit access to the nuclear interior or digitonin to limit access to the cytoplasmic compartment, were immuno labeled with anti SLAP75. A NE distribution is observed with Triton X 100 but not digitonin permeabilization. Permeabilization of the plasma membrane with both methods is dem onstrated with anti tubulin. These results indicate that SLAP75 predominantly resides inside the nucleus, at the INM of the NE. DNA was labeled with Hoechst. Scale bar is 10 m.
94 CHAPTER 4 CONCLUSION Dalek6 Is an ONM P rotein R equired for E arly M eiotic P rogression in M ice Overview of F indings Dalek6 was originally identified by virtue of its C termin al KASH domain. This sequence, albeit somewhat degenerate, clearly marked Dalek 6 as a member of the KASH domain protein family, suggesting that it should be capable of interacting with SUN domain proteins and furthermore that it should be localized to the ONM. More extensive database analyses revealed that Dalek6 is unique to mammals However, with the exception of an EF hand motif there were few sequence clues as to its function. Experiments on recombinant Dalek6 expressed in mammalian cells in culture revealed that, as predicted, Dalek6 is indeed an ONM protein and that it has the c apacity to bind cytoplasmic dynein. In fact, Dalek6 will recruit dynein to the NE in a variety of tissue culture cells. In mice, Dalek6 has a highly restricted tissue distribution. In male animals, it is detected predominantly on the NE of primary spermato cytes during meiotic prophase I. In these cells Dalek6 colocalize s with both cytoplasmic dynein and dynactin at meiotic attachment plates These are discrete patches on the NE at which telomeres are anchored, via the SUN domain protein, Sun1 The implicat ion was that Dalek6 might have a role in meiosis, in particular, microtubule dependent telomere movement. To explore the role of Dalek6 in vivo mice were derived in which both copies of the Dalek6 gene were inactivated by homologous recombination Both m a le and female Dalek6 null mice developed normally with no o vert defects. However, it became clear that homozygous null animals of both sexes were infertile. In the males, testes from Dalek6 null animals are smaller and devoid of post meiotic haploid cells likely the result
95 of massive attrition of the meiotic germ cells that is apparent in the seminiferous tubules. Mature sperm cells are never observed in these animals. In adult female mice, the loss of Dalek6 is associated with small, almost unidentifiable ovaries that are uniformly devoid of growing follicles. The obvious implication here is that Dalek6 has similar roles in both male and female germ cells Significance B a sed on studies in several organisms both mammal and invertebrate a model for the me io t i c LINC complex ha s been generated in which SUN domain proteins tether chromosomes to the NE and anchor KASH domain proteins in the ONM. The KASH domain proteins in turn function as adaptors for motor proteins which can engage with elements of the cytos keleton. I t is known that Sun1 is required for telomere attachment to the NE during mammalian meiotic prophase I (Chi et al., 2009; Ding et al., 2007; Schmitt et al., 2007) However, the cognate KASH domain protein at the meiotic attachment plates remained unknown In this study I found a previously unidentified mammalian KASH domain protein which we named Dalek6 that functions as an ONM binding partner for the dynein motor complex. In primary germ cells Dalek6 is co ncentrated at meiotic attachment plates, suggesting that Dalek6 is the mammalian KASH component of the meiotic LINC complex that is responsible for dynein and microtubule dependent telomere movement. In invertebrate model systems including C. elegans and S. cerevisiae microtubule s and /or actin are required for movement of chromosomes during meiotic prophase I, indicating their physical association with the NE In C. elegans t elomere attachment sites are defined by the SUN domain protein matefin/Sun1. Thi s provides a tether for the KASH domain protein Zyg 12 which in turn functions as an adaptor for the
96 dynein motor complex at the nuclear surface. Zyg 12 is unique to C. elegans w ith the exception of its KASH domain it shares no sequence similarity with an y other proteins. Nevertheless, Dalek6 is clearly the mammalian functional homologue of Zyg 12, at least with respect to its role in meiotic progression. Whether Dalek6 might also share Zyg l that we cannot yet address in our mouse knockout model. The lack of pachytene germ cells observed in both male and female Dalek 6 null mice reveals the importance of the meiotic KASH domain protein to mammalian meiotic prophase I This is in contrast to l oss of the orthologous KASH domain proteins in yeast which lead to meiotic defects including delay of onset for the first meiotic division, a reduction in spore formation and viability, abnormal regulation of crossover number, and elevation of homolog nond isjunction (Joseph and Lustig, 2007) Loss of Zyg 12 in C. elegans leads to complete sterility, just as Dalek6 loss leads to complete infertility in mice. However, in Zyg 12 null C. elegans germ cells are able to progress to the pachytene stage of meiosis I, this is further than is observed in mice lacking Dalek6. This discrepancy suggests that there may be subtle differences in meiotic checkpoints in mammals versus nematodes, a finding that justifies further examination of the fu nctional consequences of meiotic LINC complex perturbation in mammals. My findings raise the possibility that certain LINC complex defects could be associated with human infertility, potentially as part of a broader syndrome. However, there are no reports of such defects in the literature as yet. Future D irections Although Dalek6 null mice are clearly defective in producing mature haploid cells, how germ cell development is perturbed in these animals is still unclear. However, there
97 seems little doubt that microtubule dependent telomere movement and clustering (bouquet formation) is a prerequisite for faithful and efficient homologue pairing Careful characterization of the functional consequences of the loss of Dalek6 through the analysis o f early meiotic events w ill enable us to elucidate the specific role of Dalek6 during meiotic prophase I. In order to define key meiotic events that are contingent upon Dalek6 analysis o f the first wave meiosis that initiates around 8.5 days after birth for male mice and 13.5 embryonic days for female mice will be helpful. The investigation of the consequences of Dalek6 loss during this first synchronous wave of meiosis should shed light on the nature of early meiotic check point (s) and how these are coupled to mechanism s causing germ cell death. At the same time this may provide additi onal insight into the processes involved in early meiotic progression, particularly those linked to meiotic checkpoints. Th ese issues will be initially addressed by examining specific marker s of homologue pairing, recombination and synapsis in developing germ cells from Dalek6 null mice. Such analyses may reveal how microtubule dependent chromosome movement facilitates timely progression through meiotic prophase I My data imply that cytopl asmic dynein engaged with Dalek6 at the nuclear periphery contributes to the chromosome reconfiguration during meiotic prophase I. Whether Dalek6 mediates the movement of chromosomes is still unclear. One potential approach to address this issue is to moni tor the movement of chromosomes Visualization of meiotic chromosomes with YFP SCP3 was previously reported (Li et al., 2004; Morelli et al., 2008) However, given the fact that SCP3 appears between zygotene and pac hytene, application of YFP SCP3 transgenic mice would be limited to late leptotene pachytene. Since the chromosome movement during meiotic prophase I
98 is thought to be mediated by the association of telomeres to the NE we will monitor telomere dynamics in W T and Dalek6 null m ouse meiocytes expressing GFP tagged telomere repeat binding protein (GFP Trf1) during meiotic prophase I. We predict that there will be a substantial decrease in telomere movement in the absence of Dalek6. The recent development of an i n vitro system based upon testis slices which can recapitulate spermatogenesis will provide an ideal foundation for these analyses (Sato et al., 2011) While I have demonstrated th at Dalek6 is capable of physical association with cytoplasmic dynein and is colocalized with the motor complex at the meiotic attachment plates, the nature of the meiotic NE cytoskelet al association in mammals has still to be fully resolved In non mammali an meiosis, there is a diversity of mechanisms to move meiotic chromosomes For instance, meiotic chromosome reconfiguration in S. cerevisiae involves actin, whereas in S.pombe and C. elegans movement is dependent on microtubules and dynein (Koszul et al., 2008; Trelles Sticken et al., 2000) It is quite possible that the role for actin in meiotic chromosome movement in budding yeast is a peculiarity that has been lost during evolution. In order to rule out (or impli cate) actin as a mediator of chromosome/telomere movement it would be valuable to determine whether it can occur in the presence of cytoskeleton modulating drugs such as latrunculin versus nocodazole As before the newly described in vitro spermatogenesis system would be ideal for such an analysis. During meiotic prophase I, homologous chromosomes must pair prior to recombination. Loss of fidelity in this process contributes to aneuploidy, a collective term describing diseases caused by abnormally segregat ed chromosomes. Aneuploidy
99 is one of the leading known causes of early termination of pregnancy, developmental disorders and mental retardation in humans. This occurs in over 5% of clinically recognized pregnancies, most of which are naturally prematurely terminated. However, approximately 0.3% of liveborns are aneuploid causing conditions such as Down syndrome (trisomy 21) or sex chromosome trisomies (XXY, XXX and XYY) (Hassold and Hunt, 2001) Despite the important public health outcomes of meiotic defects, there remain profound deficiencies in our understanding of the causes of aneuploidy, including the mechanisms and consequences of fundamental events in meiotic prophase I. However the mechanism (s) explaining abnormal segregation of meiotic chromosomes remain unclear The discovery of Dalek6 al low s us to begin to ask how chromosome movement is regulated in mammals by potentially demonstrating a mechanism and consequence of active chromosome movement in meiotic prophase I. To this end it w ill be in formative to determine possible consequences of D alek6 haplo insufficiency Heterozygote mice although obviously fertile, may have a more subtle phenotype with meiotic defects that could include a higher frequency of aneuploidy or failure to complete meiosis These are long term experiments that could re quire a considerable number of mice in order to obtain statistically significant results in terms of both litter size and frequency of chromosomal abnormalities. Nevertheless, such studies will advance our fundamental understanding of health issues that ma y result from defects in meiotic prophase I BioID Is a Novel Method to Identify Protein Protein Interactions Overview of F indings We have developed a n in vivo labeling method named BioID that utilizes a promiscuous mutant of the E. coli biotin protein l igase BirA, to identify proximate
100 proteins in mammalian cells. Fusion to a bai t protein or a simpler targeting motif will determine the location of the mutant ligase (BirA*) with in the cell. Endogenous proteins that interact with and/or reside in close pr oximity to the fusion protein are labeled by covalent attachment of biotin to primary amines. Proteins biotinylated in this way are selectively isolated for identification by conventional techniques including mass spectrometry and western blot. We believe that BioID represents a powerful new approach that is broadly applicable to the identification of interacting and proximate proteins in live cells In this study, we demonstrated the application of BioID to identify LaA associated proteins Proteins biotin ylated by BirA* LaA were isolated and identified with conventional biotin affinity capture methods in conjunction with mass spectrometry. This led to the identification of known LaA interactors and NE associated proteins. In addition we identified a previo usly uncharacterized NE component that we have named SLAP75. Significance There are two common strategies that are used to identify interacting proteins. The first of these is the yeast two hybrid system, including various modifications and enhancements. The second is based upon co immunoprecipitation or pull down. The yeast two hybrid system has proved to be a very valuable tool. However, it has a number of drawbacks. The most significant of these is that interacting proteins, or indeed more commonly pro tein fragments, are removed from their normal cellular context. This may be especially true of membrane proteins. Loss of appropriate cellular context introduces a range of complications and artifacts associated with altered post translational modification s and potentially with aberrant protein folding. By its very
101 nature, BioID is able to side targeted to their normal cellular locations The success of pull down methods to identify interacting proteins is at the mercy of preserve interactions upon cell extraction and solubilization. Similarly, transient and weak interactors are unlikely to be detected. These probl ems have been addressed in part by the use of reversible chemical cross linkers. However, this may introduce an additional layer of artifacts stemming from the formation of cross linked protein aggregates. BioID eliminates the solubility issue since biotin ylation of interacting and proximate proteins takes place in vivo prior to solubilization. As a consequence, the use of harsh solubilization conditions (which will reduce non specifc background) actually becomes an attribute rather than a drawback of the B ioID procedure giving the exceptionally high affinity for avidin/streptavidin for biotin. Thus when compared with pull down approaches, the strength or transitory nature of interactions becomes largely irrelevant. A fortuitous property of BirA* is its micr omolar affinity for biotin (this is presumably reflected in a similarly high K m ). The consequence of this is that BioID could be used to identify temporal differences in protein association The addition of biotin leads to a time dependent biotinylation of endogenous proteins by mycBirA* LaA. This observation indicates that biotin is a limiting factor for biotinylation, which can regulate the biotinylation activity of BioID. Cellular events such as mitosis are regulated by rapid and precise protein protein interactions. Studying protein interactions that occur during these
102 events will require new methods to capture transiently associating proteins. BioID has the potential to fulfill such a requirement. Future Directions In this study, BioID was performed in HEK293 cell lines stably and inducibl y expressing BirA* fusion proteins. However one of BioID is its versatility and applicability to a broad range of cell types, which permits detect ion of proteins in cellular environments that are most releva nt to the specific questions being explored. Indeed there is no reason (other than time and expense) that BioID could not be employed in transgenic animals. Initial experiments will probe for cell type dependent differences in BioID candidates identified b y BirA* LaA. We expect to detect both similarities and differences between cell type bases of variability in gene expression and composition/organization of the NE. Given that BioID can detect weakly and transiently associating proteins in a natural setti ng, it has the potential to be applied to identify disease mechanisms. For instance, as mutation s in LMNA lead to a myriad of diseases, the mechanism (s) of which remain largely unknown, comparison of BioID candidates between WT and mutant A type lamin s may shed light on potential disease mechanisms. We propose that BioID can also be used to identify constituents of distinct subcellular domains that have proven refractory to conventional biochemical isolation and proteomic analysis. By fusing a general tar geting motif to the promiscuous biotin ligase, instead of a protein with specific binding partners (as with BioID LaA ), the ligase is free to move around a distinct subcellular region labeling proximate proteins and thus mapping the protein constituency. T his application of BioID will be
103 sequences and/or ER retentio n motifs. BioID is accessible to a broad range of researchers comfortable working with co nventional molecular and cell biology techniques, and does not require specialized equipment. Proteomic analysis, as applied to identification of proteins labeled by BioID, has become a nearly universal service, and does not represent a barrier to most res earchers. Thus, BioID has the potential to transform the landscape of research as it relates to basic cell biology and human health and disease. For example, individual groups that focus on specific disease associated proteins or pathways can apply this me thod to their particular projects and overcome long standing roadblocks to their research. Essentially, BioID provides a new versatile tool for researchers seeking to characterize how proteins participate in functional networks necessary for life.
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122 BIOGRAPHICAL SKETCH Dae In Kim was born and raised in Seoul, Korea. He received his B.S at Seoul National University in 1999. Since then he worked for Berna Biotech until he joined the Ph.D. program at the University o f Florida College o f Medicine in 2007. Starting in 2008, Dae I n began studying the nuclear envelope and nuclear envelope related human diseases in the Department of Anatomy and Cell Biology at the UF. He received his Ph.D. from the UF in the fall of 2011. Currently, Dae In is a member of the Roux laboratory at Sanford Children's Health Research Center where he is characterizing a novel nuclear en velope protein required for meiosis.