Review TRENDS in Cell Biology Vol.16 No.7 July 2006

Current insights into the formation and breakdown of

Sandy H.M. Litjens1, Jose´ M. de Pereda2 and Arnoud Sonnenberg1

1Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 2Instituto de Biologia Molecular y Celular del Cancer, Centro de Investigacion del Cancer, University of Salamanca - CSIC, E-37007 Salamanca, Spain

Hemidesmosomes are multiprotein adhesion of a6b4 [5]. Stable attachment of basal to the complexes that promote epithelial stromal attachment through hemidesmosomes is of in stratified and complex epithelia. Modulation of their fundamental importance for maintaining skin integrity; function is of crucial importance in a variety of biological inherited or acquired diseases in which either of the processes, such as differentiation and migration of components are affected lead to a variety keratinocytes during and of skin blistering disorders [6–8]. invasion, in which cells become detached from the The a6b4 has been associated not only with substrate and acquire a motile . Although stable adhesion, but also with cell migration [3,9,10]. In all much is known about the signaling potential of the a6b4 studies investigating migration or carcinoma integrin in carcinoma cells, the events that coordinate cell invasion, hemidesmosomes are disassembled, their the disassembly of hemidesmosomes during differen- components are redistributed and a6b4 is no longer tiation and wound healing remain unclear. The binding exclusively concentrated at the basal cell surface [9,10]. of a6b4 to has a central role in hemidesmosome Here, we focus on recent findings on the structural assembly and it is becoming clear that disrupting this properties of hemidesmosomes and their constituents, interaction is a crucial event in hemidesmosome and the regulation of their assembly and disassembly. disassembly. In addition, further insight into the functional interplay between a3b1anda6b4has contributed to our understanding of hemidesmosome Hemidesmosome structure and assembly disassembly and cell migration. The absence of a6b4 in genetically modified mice results in the loss of hemidesmosomes, and hence of stable epidermal adhesion [11–13]. This is also observed in patients carrying in either the a6 or the b4 Introduction subunit of the integrin, which can result in devastating The keratinocytes of the basal layer of the skin are firmly diseases characterized by skin fragility and blistering [7]. attached to the underlying basement membrane through In affected patients, hemidesmosomes are less robust or complexes called hemidesmosomes (Box 1 provides completely absent. Notably, two mutations identified in further details of skin structure). Hemidesmosomes were the b4 (ITGB4) of patients with a nonlethal form of first defined at the ultrastructural level as small electron- junctional disrupt the binding to dense domains in the plasma membrane, connecting the plectin, indicating that the interaction of b4 with plectin is extracellular basement membrane to the intracellular crucial for the formation of stable hemidesmosomes intermediate filament system [1,2]. The hemidesmosomal [14–16]. These two interact at two levels; the protein complex provides stable adhesion of the primary interaction occurs between the plectin - to the underlying and ensures resistance to binding domain (ABD) and a region on b4 comprising the mechanical stress when applied to the skin. In the skin, first pair of fibronectin type III (FnIII) domains and a hemidesmosomes contain the integrin a6b4, the type XVII small region of the adjacent connecting segment, and the collagen BP180, the tetraspanin CD151 and the two secondary between the domain of plectin and a plakin family members plectin and BP230 [1–3]. It is the more C-terminal part of the connecting segment of b4, and a6b4 integrin that connects the cells to a major component the ultimate C-tail (i.e. the region following the fourth of the basement membrane, -5 (now also referred FnIII domain) [14,17–20] (Figure 1). Consistent with the to as laminin-332 [4]), whereas the cytoplasmic proteins idea that the a6b4–plectin connection is crucial for the plectin and BP230 connect to the filaments, mechanical stability of hemidesmosomes, plectin-deficient thereby creating a stable anchoring complex. Although mice show extensive epithelial detachment and die the binding of BP180 to laminin-332 has been shown 2–3 days after birth [21]. Patients with mutations in the in vitro, this interaction by itself is not sufficiently strong PLEC gene also suffer from skin fragility, although the to mediate adhesion of cells to laminin-332 in the absence symptoms are less severe than those observed in plectin-

Corresponding author: Sonnenberg, A. ([email protected]). deficient mice. Most of these mutations are located within Available online 6 June 2006 the rod domain (Figure 1), which is not present in www.sciencedirect.com 0962-8924/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2006.05.004 Review TRENDS in Cell Biology Vol.16 No.7 July 2006 377

Box 1. Structure of the skin

The skin, the largest organ of our body, is crucial for protecting the produce transit-amplifying cells that are destined to withdraw from underlying tissues and organs from the external environment. It is the cell cycle and terminally differentiate after a few rounds of composed of three layers [57]: the epidermis (keratinocytes in division [58,59]. During terminal differentiation, the keratinocytes various stages of differentiation and melanocytes), the dermis detach from the basement membrane, migrate upward in the (blood vessels, nerve endings, sebaceous glands and hair follicles, epidermis, gradually flatten, start producing more , lose surrounded by connective tissue and collagen fibers) and the their nucleus and ultimately die and flake off the skin [60]. The basal subcutaneous layer (connective tissue and fat). The basal layer of keratinocytes are attached to the basement membrane, a meshwork cells in the epidermis contains the least differentiated keratinocytes, of extracellular macromolecules separating the dermis from the and among these are the stem cells, which ensure the constant epidermis, through specialized adhesion structures called hemi- renewal of the skin by their unlimited dividing capacity. Stem cells (Figure 1). a smaller splice variant that is expressed at low levels in basal cell surface of human keratinocytes [26]. These cells [17,22]. It is conceivable that this variant can clusters might serve as a ‘nucleation site’ for the assembly partially compensate for the loss of the canonical plectin of hemidesmosomes by the integrin a6b4. In contrast to molecule in humans. Interestingly, neither BP180 nor CD151, which becomes a component of mature hemi- BP230 are efficiently recruited into hemidesmosomes desmosomes (via binding to a6), a3b1 is recruited into when b4 cannot bind to plectin [15,17,23]. In addition, focal contacts or redistributed to cell–cell contacts after simple epithelia (e.g. that of the intestine) contain hemidesmosomes have been assembled and cell– complexes consisting of only a6b4 and plectin, contacts established. Although a3b1 does not appear described as type II hemidesmosomes [24,25].This directly to participate in hemidesmosome assembly, it suggests that the a6b4–plectin interaction is one of the might, together with other b1-containing , first steps in the formation of hemidesmosomes and occurs contribute to the efficacy of their formation, possibly by before the recruitment of BP180 and BP230 [17]. affecting the localization of a6b4 at the basal membrane. CD151 is also an early constituent of the hemidesmo- Indeed, in b1-deficient mice, the number of hemidesmo- somal adhesion structures. It is strongly associated with somes is reduced, which can partially explain the skin another laminin-binding integrin on keratinocytes, a3b1, defects that are observed in these mice [27,28]. However, if with which it forms ‘pre-hemidesmosomal’ clusters at the only a3b1 is absent, as in a3-null mice, the number and

(a) (b) Integrin α6β4 Outside Inside

IF S1356 α6 N- SSÐ -C R1225H S1360 R1281 S1364 β4 N- Calx I FnIII-1 FnIII-2CS FnIII-3 FnIII-4 -C Inner Plakin repeat domains 1115-1355 1382-1436 1667-1752 plaque Rod Plectin Plakin Rod domain N- CH1-CH2 B1 B2 B3 B4 B5 C -C BP230 FnIII 3 ABDPlakin domain Coiled-coil rod domain Plakin repeat domains HD FnIII 4 Plakin domain (d) FnIII 2 CH2 ABD (c) CH1 FnIII 1 Keratin Direct Plectin Nucleus Outer sequence filaments plaque repeats CalX

IP Lamina OP lucida CD151 LL EM LD BP180 LD BM Anchoring α6β4 Lamina Subbasal dense plate filaments Dermis densa

Laminin-332

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Figure 1. Components and structure of hemidesmosomes. (a) A schematic drawing of the hemidesmosome, showing its six components (a6b4 heterodimer, CD151, BP180, BP230 and plectin) together with its ligand laminin-332 and intracellular intermediate filament partners keratin-5 and -14 (IF). (b) The structures of a6b4 and plectin. The various domains in the proteins are indicated, as well as the residues in the b4 cytoplasmic domain that are important for plectin binding and regulation of this interaction. (c) An electron micrograph of several hemidesmosomes. (d) An enlargement of the indicated area in the middle panel. Abbreviations: BM, basement membrane; CH, calponin homology domain; HD, hemidesmosome; IP, inner plaque; LD, lamina densa; LL, lamina lucida; OP, outer plaque. www.sciencedirect.com 378 Review TRENDS in Cell Biology Vol.16 No.7 July 2006 size of hemidesmosomes appears to be normal [29]. Role of laminin-332 in the formation of Similarly, no deficiencies in hemidesmosomes were hemidesmosomes observed in CD151 null mice, and these mice also have In several reports, data have been presented suggesting no signs of skin fragility [30]. Moreover, in cells expressing that the processing of laminin-332 is important for the b4-chimeras that are unable to associate with a6 and thus formation of hemidesmosomes. In quiescent epidermis, the CD151, hemidesmosome-like structures, containing plec- a3 chain of laminin-332 is cleaved, securing the formation tin, BP180 and BP230, are still formed [31,32]. Thus, in of stable a6b4-containing hemidesmosomes; however, mice and in human cultured keratinocytes, CD151 is not when keratinocytes need to migrate [e.g. following skin required for the formation of hemidesmosomes, and its wounding (Box 2)], unprocessed laminin-332 is secreted at precise function with a3b1 in the ‘pre-hemidesmosomal’ the leading edge, which stimulates migration through the clusters remains unknown. a3b1 integrin [36,37]. It is generally assumed that when Interestingly, wound healing was defective in CD151- the a3 chain of laminin-322 has not been processed, deficient mice; this was found to be the result of an keratinocytes switch from a6b4-mediated adhesion to impaired deposition of laminin-332 into the wound bed one mediated by a3b1, because the latter binds more and a failure to upregulate a6b4 [33]. Impaired organiz- efficiently to unprocessed laminin-332. Conversely, a6b4 ation of the basement membrane has also been described might bind less efficiently to this unprocessed laminin-322 in a3-null mice, which hints at an important function of than to the cleaved form, which might contribute to a the complexes of a3b1 and CD151 in this process [29]. higher turnover of hemidesmosomes. In addition, there is Intriguingly, although the absence of CD151 in mice has evidence that matrix metalloproteinase 19-dependent no effect on epidermal integrity, a nonsense in processing of the laminin-g2 chain can effectuate the the CD151 gene in a patient, resulting in a truncated above switch, leading to increased migration of keratino- protein, and loss of the integrin binding site, leads to skin cytes [38]. Therefore, whether laminin-332 is processed or blistering, albeit less severe and more localized than in not determines which of the two laminin-binding integrins patients with mutations in either a6orb4 [34]. Moreover, mentioned above becomes engaged, and consequently the it has been shown that the tetraspanin TSP-15, which is turnover of hemidesmosomes. Interestingly, in a recent similar to mammalian CD151, is required for epidermal report it was shown that unprocessed laminin-332 (a3 integrity in Caenorhabditis elegans [35]. It is possible that chain) does indeed prevent the formation of mature the truncated CD151 molecule acts in a dominant- hemidesmosomes but not the nucleation of hemidesmoso- negative fashion, with its presence being more deleterious mal plaques [39]. Furthermore, it showed that keratino- for tissue integrity than is the total lack of CD151. cytes migrate equally well on both processed and Alternatively, the absence of a functional CD151 molecule unprocessed laminin-332, whereas mature hemidesmo- in mice, but not in humans, might be compensated by somes can only be formed after the a3 chain of laminin-332 another tetraspanin [30]. Together, these findings indicate is processed. Nevertheless, mainly unprocessed laminin- that the binding of b4 to plectin represents the first step in 332 is present underneath the leading edge of keratino- the formation of hemidesmosomes, and that complexes of cytes within a skin wound. It is possible that in the case of a3b1 and CD151 might facilitate this step by providing a excessive production of laminin-332 following wounding, platform at the basal surface of keratinocytes through the efficiency of processing will be reduced and the which a6b4 becomes localized and can interact formation of mature hemidesmosomes inhibited, favoring with plectin. a migratory phenotype.

Box 2. The role of epidermal integrins in wound healing

Injury to the skin causes the transition of quiescent adherent subsequent spreading [64,65]. The initial spreading induced by keratinocytes on laminin-332 into migrating cells on the newly a3b1 is followed by the activation of RhoA, which in in vitro formed provisional matrix containing fibrin, fibronectin and vitro- experiments was found to be required for the adhesion of leading nectin. During this process, various growth factors and cytokines are keratinocytes to collagen by a2b1 [66] However, more recent studies secreted to induce healing of the wound [61]. Transforming growth have questioned the importance of a2b1 in wound healing because factor b is one of these and contributes to re-epithelialization of skin wound closure is normal in a2-deficient mice [67,68]. The wounds by modulating integrin expression, thereby stimulating fibronectin- and vitronectin-binding integrins a5b1, avb5andavb6 keratinocyte migration towards the provisional wound matrix. As probably have a more important role in wound healing because described in this review, stable adhesion of keratinocytes by a6b4to these integrins are receptors for the various components of the the basement membrane is lost and the integrin is redistributed provisional matrix and their expression is induced or strongly from hemidesmosomes to lamellipodia, which might facilitate their increased. Of these integrins, only avb5 is expressed at low levels on stabilization. In addition, the expression of the integrins a2b1 and proliferating keratinocytes, whereas a5b1andavb6 are normally a3b1, mostly restricted to the lateral membranes of basal keratino- absent [69,70]. (Increased) expression of these three integrins results cytes in normal epidermis, becomes extended throughout the entire in a more migratory phenotype of the keratinocytes. Interestingly, epidermis and their concentration at the basal membrane of basal upregulation of avb6 causes a fibronectin-dependent upregulation of keratinocytes increases [61–63]. After binding of keratinocytes by matrix metalloproteinase 9, which might partly be responsible for a3b1 to newly deposited laminin-332 at the wound edge, the T the increased migration [71]. Although not well documented, a role lymphoma invasion and 1 (TIAM1)–Rac1 signaling of the fibronectin- and tenascin-binding integrin a9b1 in epidermal pathway is activated to promote lamellipodia formation and wound repair has been proposed [72,73]. www.sciencedirect.com Review TRENDS in Cell Biology Vol.16 No.7 July 2006 379

Although the above studies suggest an important suggest that Erbin might have a general role in the function of laminin-332 in the formation and stabilization establishment and maintenance of cell–cell and cell– of hemidesmosomes, studies from several laboratories basement membrane adhesions. have indicated that the formation of hemidesmosomes can be driven entirely from within the cell through interaction Hemidesmosome regulation of the cytoplasmic domain of b4 with plectin [31,40–42]. Although nothing is known about the dynamics of Keratinocytes expressing a b4 subunit that is unable to hemidesmosomes in the epidermis, in cultured keratino- interact with its ligand form structures that contain all of cytes these structures appear continuously to renew the components of type I hemidesmosomes. Although themselves [32,51]. This dynamic state is thought to be a these data came as a surprise at the time, they do not prerequisite to enable hemidesmosomes to be quickly imply that laminin-332 does not have a role in nucleating disassembled when required – for example, for cell hemidesmosomes. Indeed, hemidesmosomes can also be migration or differentiation, when cells detach from the initiated through an interaction of a6b4 with clustered underlying basement membrane. Although the role of b4 laminin-332 that has been deposited by the cells in the migration of keratinocytes, and particularly of themselves. Furthermore, ligation of the integrin clearly carcinoma cells, has been studied extensively, only a few affects the organization and density of the formed clusters models for hemidesmosome disassembly have been pro- [32,36,38]. When a6b4 is not bound to laminin-332, plectin posed [52,53]. The regulatory requirements for each clusters the integrin into tight complexes. However, upon assembly and disassembly step are not yet clear. It is ligation to laminin-332 in the matrix, the dynamics of known that the interaction between plectin and b4is a6b4 are reduced and, therefore, the degree of intra- regulated by serine phosphorylation of residues 1356, cellular clustering by plectin is restricted [32]. Thus, the 1360 and 1364 in the connecting segment of b4. formation of hemidesmosomes is driven both from within Phosphorylation of these residues by PKCa, and possibly the cells through an interaction of the cytoplasmic domain other enzymes, results in partial hemidesmosome disas- of b4 with plectin and from outside the cell through sembly owing to loss of interaction with plectin [53]. Most binding of a6b4 to laminin-332. likely, activation of a phosphatase is required to induce binding of plectin and reassembly of hemidesmosomes. Basolateral targeting of hemidesmosomal components This putative phosphatase might also be activated upon in polarized epithelia ligation of a6b4 to processed laminin-332. Evidence is emerging that hemidesmosomes are also The cytoplasmic domain of b4 can adopt a folded affected by mechanisms that control polarized vesicular conformation by binding of the C-tail to the connecting transport in cells. In polarized epithelia, where tight segment [54] (Box 3). Interestingly, these same sequences junctions prevent the free diffusion of plasma membrane in b4 are involved in binding to the plectin plakin domain proteins between the apical and basolateral membranes, [17,20,54]. Therefore, the intramolecular folding of the b4 membrane trafficking pathways are used to transport cytoplasmic domain might also regulate the interaction proteins to their respective domains. These trafficking with plectin. Posttranslational modifications, such as mechanisms are not only important for maintaining, but phosphorylation, might determine the conformation of also for establishing epithelial cell polarity. One of the the b4 cytoplasmic domain and thereby affect its affinity proteins that has been implicated in directing protein for other hemidesmosome components. Based on the trafficking to the basolateral membrane is the cytoskeletal limited data available, several mechanisms for phos- protein lethal giant larvae 2 (Lgl2). Indeed, this protein is phorylation-induced hemidesmosome disassembly are crucial for correct hemidesmosome formation in zebrafish conceivable (Figure 2). [43]. Lgl2 functions in polarized exocytosis by regulating First, phosphorylation of the connecting segment might the function of target membrane soluble N-ethylma- disrupt the interaction between b4 and the plakin domain leimide attachment protein receptors (t-SNAREs) at the of plectin and/or the intramolecular interaction, thereby basolateral plasma membrane [44,45]. At the subapical destabilizing the interaction with plectin (Figure 2a). region, its activity is inhibited as result of phosphorylation Second, phosphorylation of b4 might increase its affinity by atypical protein kinase C (PKC), which is a member of for a third protein, which subsequently competes with the Par protein complex [46,47]. plectin for binding to the connecting segment and/or Another protein that might have a role in the interferes with binding of plectin with the first pair of basolateral targeting of hemidesmosomal proteins is FnIII domains in the integrin C-tail (Figure 2b,c). Indeed, Erbin, which binds to both b4 and BP230 [48]. Erbin was macrophage-stimulating protein-induced serine phos- originally identified as an ErbB2-associated protein phorylation of the b4 connecting segment by PKC-a, involved in the targeting of this to the basolateral leads to hemidesmosome disassembly and relocation of membrane. It is a member of the LAP [leucine-rich repeats a6b4 to lamellipodia at the leading edge of migrating (LRR) and postsynaptic density protein-95, Discs-large keratinocytes via binding of 14-3-3 proteins to the and zonula occludens-1 (PDZ)] family of proteins, impli- phosphorylated connecting segment [52]. Interestingly, cated in establishing epithelial cell polarity [49,50].In macrophage-stimulating protein is one of the growth addition to the hemidesmosomal proteins b4 and BP230, factors secreted in the wound fluid and might, by inducing Erbin interacts with components of adherens junctions the disassembly of hemidesmosomes, contribute to wound and desmosomes, both of which are cell–cell adhesion repair [55,56] (Box 2). Third, phosphorylation of the structures at the lateral membrane. These observations connecting segment might induce a significant www.sciencedirect.com 380 Review TRENDS in Cell Biology Vol.16 No.7 July 2006

Box 3. Structure of hemidesmosomal components

Knowledge of the structure of the protein components of hemi- However, a validated model of this binary complex has been desmosomes has contributed to our understanding of the structure, proposed in which the N-terminal CH domain contacts b4atthe assembly and disassembly of these adhesion complexes. The inter-FnIII domain area [78]. cytoplasmic domain of b4isw1000 residues long and contains The PLEC gene contains many alternative first exons, resulting in 11 four FnIII domains arranged in two tandem pairs separated by a plectin splice variants differing in their N-terminal sequence [79]. connecting segment (Figure I). The knowledge of the structure of b4 Although it has been shown that both plectin-1A and plectin-1C can is limited to the crystal structure of the first pair of FnIII domains interact with b4 in vitro, only plectin-1A is expressed in basal [74]. This region and the first 35 residues of the adjacent connecting keratinocytes and is localized in hemidesmosomes [14,77,79,80]. The segment (1321–1355) constitute the primary plectin binding site in N-terminal sequence in plectin-1A might be important for regulating b4, a region essential for targeting plectin to hemidesmosomes the affinity of the ABD for b4 and F-actin [77]. [14,17,18]. Residues important for the interaction with plectin map The cytoplasmic domain of b4 might adopt a closed conformation onto a continuous area of b4 extending over the first and second owing to the interaction between its connecting segment and its C- FnIII domains and define the binding interface (Figure I). Plectin terminal tail, which might affect the affinity for plectin [54]. For the binds to the first pair of FnIII domains of b4 via its N-terminal ABD. folded state of b4, a strong bending of the second pair of FnIII domains The ABD is formed by two CH domains arranged in a closed is probably required and might be favored by the 20-residue linker conformation, as shown in the crystal structure [75,76]. Residues connecting the third and fourth FnIII domains. The b4 regions involved crucial for binding to b4 are clustered on the surface of the N- in the intramolecular interaction contain binding sites for the plakin terminal CH domain, and overlap partially with one of the actin- domain of plectin located adjacent to the ABD [17,54]. Similarly to b4, binding sequences essential for binding to F-actin, providing a plectin contains a binding site for BP180, and together these proteins structural basis for the competitive nature of plectin binding to b4 recruit BP180 into hemidesmosomes [23]. Ultimately, BP230 becomes and F-actin [14,77,78]. The structure of the complex of the first pair recruited into hemidesmosomes by binding to both b4 and BP180, and of FnIII domains of b4 and the ABD of plectin is not known. thus type I hemidesmosomes are formed.

Connecting segment

E A C’ B FnIII-2 C F G 2 Arg121 Arg1225 Arg123 Lys126 Arg1281 Arg98 F E D Pro1243 Glu95 Ala1244 A C C B G Tyr1187 E F G Cys1190 A

C F E G Arg138 2 Gln131 FnIII-1 C′ Asn149 CH1 CH2

N TRENDS in Cell Biology

Figure I. Structure of the binding interfaces of integrin b4 and plectin. Schematic representation of a model of the complex between the first pair of FnIII domains of b4and the plectin ABD obtained by computational docking methods [78]. The Ca atoms of residues whose mutation affects binding are shown as red spheres. The b4 residues that are important for binding to plectin are located in the loop EF of the first FnIII domain and in loops BC, EC0 and the region preceding strand A of the second FnIII domain. The initial residues of the connecting segment are necessary for plectin binding but are not present in the crystal structure, yet they are likely to be orientated towards the plectin ABD in the model (dashed line). Residues in the plectin ABD relevant for binding to b4 are located in a continuous area of the first calponin homology (CH) domain (orange) and include the loop preceding the a-helix C and the a-helices E and F. The second CH domain (pink) does not contain particular residues crucial for binding but its presence is essential for the interaction. Abbreviation: N-term, amino terminus. conformational change of the b4 cytoplasmic domain that the binding of the plectin ABD is prevented and the would distort the binding sites for the plectin ABD and/or affinity of the plakin domain of plectin for b4 is not strong plakin domain (Figure 2d). In the first two models, binding enough to induce binding. In these models, the interaction of the plectin plakin domain is lost, whereas binding of the between plectin and b4 has a key role. Obviously, other plectin ABD to b4 is still possible. In the latter two models, hemidesmosomal interactions might be subject to www.sciencedirect.com Review TRENDS in Cell Biology Vol.16 No.7 July 2006 381

N

FnIII-1 A B D FnIII-2 Integrin Plectin P CS D C FnIII-4 FnIII-3 R o d B B B B B Serine/threonine kinase C Phosphatase C

(a)N (b)N (c) (d) 1 FnIII-1 A ? FnIII-1 A 3 ? FnIII-1 FnIII-1 B B 2 N FnIII-2 N FnIII-2 D FnIII-2 D 1 FnIII-2 P CS P 1 PD PD 2 A A CS P P P B B X C D P 1 CS P X 2 CS P D C FnIII-3 R R PD FnIII-4 PD o o d C d FnIII-4 FnIII-3 B FnIII-3 B FnIII-3 B B B B R R FnIII-4 B B o o B FnIII-4 B d d C C B B B B C C B B B B B B C C C C

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Figure 2. Four putative models for phosphorylation-induced hemidesmosome disassembly. Upon phosphorylation of the b4 connecting segment, the interaction with plectin is lost. (a,b) Binding of plectin might disappear by interfering with binding of the plakin domain, thereby destabilizing the interaction between plectin and b4. Binding of the plakin domain can be (a) prevented directly by phosphorylation, or (b) prevented indirectly by the binding of a third protein to the phosphorylated connecting segment. (c,d) Alternatively, binding of the plectin ABD might be lost owing to phosphorylation of b4. Phosphorylation might (c) indirectly lead to loss of plectin ABD binding through competition with a third protein, or (d) directly lead to loss of binding by inducing a large conformational change. Only the cytoplasmic domain of b4 is shown for simplicity. Abbreviations: CS, connecting segment; PD, plakin domain. regulation, and their posttranslational modifications (de-)phosphorylation of the b4 cytoplasmic domain is might contribute to the balance between hemidesmosome required for plectin binding. Furthermore, it is not clear formation and disassembly. whether serine phosphorylation of b4 is sufficient to induce hemidesmosome disassembly. The answers to these questions will help to understand the switch in Summary function of the a6b4 integrin between the formation of Although research on carcinoma cell lines has informed us stable adhesion structures and induction of migration. about possible pathways for hemidesmosome disassembly, it should be kept in mind that cancer cells have developed Acknowledgements mechanisms enabling them to escape pathways that We would like to thank Drs K. Wilhelmsen, E. Roos and L. Borradori for normally regulate proliferation and migration. Therefore, critical reading of the manuscript. The research of Arnoud Sonnenberg is in physiological conditions in the skin, the pathways supported by grants from the Dutch Cancer Society and Dystrophic leading to the disassembly of hemidesmosomes might be Epidermolysis Bullosa Research Association (DEBRA, Crowthorne, UK). entirely different. It is clear that serine phosphorylation of the b4 cytoplasmic domain is involved in the disassembly References of hemidesmosomes in keratinocytes to initiate migration. 1 Jones, J.C. et al. (1998) Structure and assembly of hemidesmosomes. Bioessays 20, 488–494 However, it is not yet known which signals induce 2 Borradori, L. and Sonnenberg, A. (1999) Structure and function of clustering of a6b4, or subsequent incorporation hemidesmosomes: more than simple adhesion complexes. J. Invest. of other hemidesmosome components, and whether Dermatol. 112, 411–418 www.sciencedirect.com 382 Review TRENDS in Cell Biology Vol.16 No.7 July 2006

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KASH-domain proteins in nuclear migration, anchorage and other processes

Kevin Wilhelmsen, Mirjam Ketema, Hoa Truong and Arnoud Sonnenberg*

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

*E-mail address for the corresponding author: [email protected]

Short title: Nuclear Migration and Anchorage

Key words: nuclear envelope, outer nuclear membrane, KASH domain, nesprin

1 Summary

The nucleus in eukaryotic cells can move within the cytoplasm, and its position is critical

for many cellular events, including migration and differentiation. Nuclear anchorage and

movement can be achieved through association of outer nuclear membrane (ONM)

proteins with the three cytoskeletal systems. Two decades ago studies described C.

elegans mutants with defects in such events, but only recently it has been shown that the strategies for nuclear positioning are indeed conserved in C. elegans, as well as in

Drosophila, mammals and potentially all eukaryotes. The integral ONM proteins implicated in these processes thus far all contain a conserved Klarsicht/ANC-1/Syne homology domain at their C-terminus that can associate with Sad1p/UNC-84-domain proteins of the inner nuclear membrane within the periplasmic space of the nuclear envelope (NE). The complex thus formed is responsible, not only for association with cytoplasmic elements, but also for the integrity of the NE itself.

2 Introduction

The contents of the nucleus are enclosed by two lipid bilayers, the inner (INM) and the outer (ONM) nuclear membrane, which together form the nuclear envelope (NE). The

INM and the ONM fuse at the nuclear pore complexes (NPCs) and the lumen region between these membranes is called the periplasmic (or perinuclear) space (PS) (Gerace and Burke, 1988). Beneath the INM is the nuclear lamina, a web of

(IF) proteins composed of A- and B-type . These proteins give shape and stability to the NE, in addition to associating with proteins bound to chromatin (Gruenbaum et al.,

2005). The ONM, by contrast, is continuous with the endoplasmic reticulum and, in fact, these two structures share a set of proteins as well as ribosomes (Gerace and Burke,

1988).

The nucleus and other organelles can move within the cytoplasm. The position of the nucleus is important for processes such as mitosis, meiosis, cell migration and polarization, and nuclear positioning occurs in the cells of most eukaryotes (Morris,

2000). Interactions with the three cytoskeletal filament systems (i.e. F-actin, IFs and (MTs)) anchor the nucleus or allow it to move in the cytoplasm. The different filament systems, in turn, are connected to each other through members of the plakin family of cytoskeletal cross-linkers (Fuchs and Karakesisoglou, 2001; Leung et al.,

2002). Although defects in nuclear positioning have been observed for a relatively long time, the mechanisms responsible are only now becoming evident.

3 Nuclear Positioning in Caenorhabditis elegans

Nuclear migration and anchorage were initially studied in C. elegans. The positions of the nuclei within the developing embryo can be mapped in great detail because C. elegans has a transparent body (Sulston and Horvitz, 1977; Sulston et al., 1983). Nuclear migration has been extensively studied in hyp7 precursor and P cells within the embryo and larvae, respectively. Prior to the cell fusion events that form the large, multi- nucleated hypodermal syncytium (the hyp7 syncytium), the hyp7 precursor cells go through a series of elongation and nuclear migration events, whereby the nuclei move past the dorsal midline to the opposing lateral side (Sulston et al., 1983; Williams-

Masson et al., 1998). Similarly, the P cells, which ultimately develop into motor neurons and epithelial cells, have nuclei that migrate from the lateral sides of the newly hatched larva to form a single row in the ventral cord (Sulston, 1976; Sulston and Horvitz, 1977).

The first worm strains shown to have defects in nuclear migration and anchorage in these two cell types, which result in uncoordinated movement, were unc-83 and unc-84

(Horvitz and Sulston, 1980; Sulston and Horvitz, 1981; Malone et al., 1999). The unc-83, and some unc-84, worms also display defective nuclear migration in cells of the intestinal primordium (Starr et al., 2001). In the majority of the unc-84 mutants, the nuclei move slowly and do not migrate past the dorsal midline in the hyp7 precursor cells and float freely within the hyp7 syncytium. However, some unc-84 worms only display the migratory defects. In P cells, the nuclei fail to migrate to the ventral cord and the cells ultimately apoptose (Malone et al., 1999). The unc-83 worms have the same phenotype,

4 except that the nuclei have never been observed to float freely within the cytoplasm of

the hyp7 syncytium (Starr et al., 2001) (Fig. 1A).

The unc-83 and unc-84 genes encode an ONM protein, UNC-83, and an INM protein,

UNC-84, respectively. UNC-83 has a conserved C-terminal Klarsicht/ANC-1/Syne-1 homology (KASH) domain, which contains a transmembrane region and residues that lie within the PS, and a cytoplasmic N-terminus that does not show sequence similarity to any known protein (McGee et al., 2006). UNC-84 contains several potential transmembrane domains and a C-terminal region that is homologous to the yeast protein

Sad1p and, accordingly, is called the Sad1p, UNC-84 (SUN) domain (Malone et al.,

1999; McGee et al., 2006). The localization of UNC-84 to the INM is not dependent upon its SUN domain but rather on the presence of nuclear lamins, which probably interact with its N-terminus (Lee et al., 2002). Deletion of UNC-84, mutations in the UNC-84

SUN domain or mutations within the UNC-83 KASH domain can prevent the localization of UNC-83 at the ONM (McGee et al., 2006; Starr et al., 2001), presumably because of a loss of direct interaction between the UNC-83 KASH and UNC-84 SUN domains within the PS (McGee et al., 2006) (Fig. 2). This explains why the nuclear migration defects in unc-83 and unc-84 mutant worms are very similar (Malone et al., 1999). Curiously,

UNC-83 is present at the NE in a limited number of cell types (including P, hyp7, intestinal, pharyngeal and uterine cells), unlike UNC-84, which is localized at the NE in nearly all cells (Starr et al., 2001).

5 UNC-83 and -84 were originally proposed to tether the nucleus to centrosomes, which

was hypothesized to drive nuclear migration (Malone et al., 1999; Reinsch and Gonczy,

1998); however, later studies showed a normal association between the centrosomes and

nuclei in unc-83 and unc-84 mutant cells whose nuclei fail to migrate (Lee et al., 2002;

Starr et al., 2001). The proteins that interact with the N-terminus of UNC-83 to facilitate

nuclear migration have not been identified. Recently, another ONM protein, ZYG-12,

was shown to mediate an association between the centrosomes and the nucleus in C.

elegans. In zygote defective (zyg)-12 mutant worms, the centrosome detachment defect in

the developing embryo results in death as a consequence of segregation

defects (Fig. 1B). ZYG-12 is a member of the Hook family and is localized at both the

centrosomes, which is MT-dependent, and the NE. Interestingly, its recruitment to the NE

is dependent upon the expression of the only other SUN-domain-containing protein in C.

elegans, Matefin/SUN-1 (Fridkin et al., 2004; Malone et al., 2003). This is consistent with the results of the experiments showing that in unc-84 null cells the centrosomes are correctly localized at the NE (Lee et al., 2002). Unlike for UNC-84, nuclear lamins are not involved in the retention of Matefin/SUN-1 at the INM (Fridkin et al., 2004). There are three splice variants of ZYG-12 (A, B and C), two of which contain a KASH domain

(B and C). Localization of these two variants at the ONM probably depends upon an

association between their C-termini and that of Matefin/SUN-1 (Fig. 2).

Defects in nuclear anchorage were first detected in the hyp7 syncytium. Five mutant

alleles of the anchorage defective (anc)-1 gene were isolated from mutant worms in

which unanchored nuclei float freely in the cytoplasm of the syncytium (Hedgecock and

6 Thomson, 1982). In these worms, mitochondria also appear unanchored, which suggests

that the same mechanism is responsible for the anchorage of the nuclei and mitochondria.

The ANC-1 protein contains two N-terminal calponin homology (CH) motifs, which

together comprise an actin-binding domain (ABD), and a C-terminal KASH domain

(Starr and Han, 2002). The presence of a cytoplasmic ABD indicated that the defects in

nuclear and mitochondrial anchorage might be due to a loss of interaction with the actin

cytoskeleton. Indeed, a direct association with F-actin has been demonstrated in vitro and

an N-terminal fragment of ANC-1, containing the ABD, specifically decorates F-actin in

cells (Starr and Han, 2002). ANC-1 is an enormous molecule of ~950 kDa. This size is

due to a region of repetitive structural elements between the ABD and KASH domains.

Therefore, it seems that a relatively large distance needs to be maintained between the

nucleus and F-actin. The KASH domain of ANC-1 is necessary for its localization at the

ONM and mutations within the SUN domain of UNC-84 prevent the proper localization

of ANC-1 to the NE (Starr and Han, 2002). Additionally, overexpression of a construct

containing the KASH domain alone results in nuclear anchorage defects in the

hypodermal syncytium, probably due to a competition with endogenous ANC-1 for a

limited number of UNC-84 binding sites (Starr and Han, 2002). Interestingly, an unc-84 null mutation has no effect on mitochondrial anchorage in muscle cells (Starr and Han,

2002). The SUN domain of UNC-84 is thus also responsible for the recruitment of ANC-

1 to the ONM through its KASH domain, although note that in this case a direct interaction has not been detected (Starr and Han, 2002) (Fig. 2).

7 Worms with mutations in both anc-1 and unc-83 have a compound phenotype. The nuclei within the syncytium are misplaced as well as unanchored because they fail to migrate prior to fusion and are unable to interact with the actin cytoskeleton (Hedgecock and

Thomson, 1982). Most of the unc-84 mutant alleles similarly produce nuclear migration and anchorage defects, but some only lead to defects in nuclear migration (Fig. 1A). The worms that show both defects either lack UNC-84 or have mutations in regions in or near the SUN domain; those that only display defects in migration have mutations within the nucleoplasmic N-terminus of UNC-84 (Malone et al., 1999), indicating that anchorage may not only depend upon an interaction with the nuclear lamins.

The data thus support a model in which UNC-84 becomes localized at the INM by binding to the nuclear lamins with its N-terminus, which leaves its periplasmic SUN domain available for making associations with the KASH domain of either ANC-1 or

UNC-83 to retain these proteins at the ONM. ANC-1 is responsible for nuclear anchorage through interactions with the actin cytoskeleton, whereas UNC-83 is necessary for nuclear migration, although how this is achieved is currently unknown (Fig. 2).

Nuclear Positioning in Drosophila melanogaster

Two KASH-domain proteins have been identified in Drosophila, Klarsicht and Msp-

300/nesprin. Klarsicht, originally called Marbles, is important for the development of the compound eye in Drosophila. Mutation of the klarsicht gene results in the failure of the nuclei in photoreceptors to migrate to the apex of the developing eye imaginal disc and, therefore, most nuclei remain at the basal side, which results in oddly shaped

8 photoreceptors (Fischer-Vize and Mosley, 1994). Importantly, this nuclear migration defect coincides with the detachment of the centrosome from the nucleus (Patterson et al.,

2004) (Fig. 3A). Klarsicht is also involved in the transport of lipid droplets in Drosophila embryos (Welte et al., 1998), but this involves a different C-terminal splice variant

(Klarsicht-β, rather than Klarsicht-α and -γ) (Guo et al., 2005). Interestingly, klarsicht- null mutants are viable and fertile and only display major defects in eye morphology

(Mosley-Bishop et al., 1999).

Klarsicht-α is ~251 kDa, whereas Klarsicht-γ is ~62 kDa. Both contain a C-terminal

KASH domain and are localized at the NE in several cell types (Klarsicht-β does not have a KASH domain) (Guo et al., 2005). Klarsicht also associates with MTs in photoreceptors (Fischer et al., 2004; Patterson et al., 2004); the N-terminal region of

Klarsicht, which is not present in Klarsicht-γ and shows no sequence similarity to other known proteins, is responsible (Fischer et al., 2004). Analyses of fly associated with mutations in the Drosophila B-type Dm0 indicate that Klarsicht and lamin Dm0 are part of the same pathway (Patterson et al., 2004). Lamin Dm0 may therefore be required for the localization of an UNC-84-like protein to the INM, and the consequent retention of Klarsicht at the ONM. The Drosophila genome encodes two uncharacterized SUN-domain proteins, although these are not predicted to have transmembrane regions (Malone et al., 2003; Starr and Han, 2003) (Fig. 2). Moreover, overexpression of the Klarsicht KASH domain in photoreceptors does not result in a mutant phenotype (Fischer et al., 2004). This suggests that they do not compete with the

9 endogenous protein for a limited number of binding sites at the NE, in contrast to the

ANC-1 KASH domain (Starr and Han, 2002).

Although Klarsicht and ZYG-12 are both necessary for the association of the centrosome

with the nucleus, these two proteins are not related except in the KASH domain. In

Drosophila, only two proteins show some sequence similarity to ZYG-12. One is a member of the Hook family, dHk (Kramer and Phistry, 1996; Kramer and Phistry, 1999), and the other is a recently identified Hook-related protein, dHkRP (Simpson et al., 2005).

Both contain a putative N-terminal MT-binding domain, but do not contain a KASH domain or are localized at the NE. dHk is cytosolic and reported to play a role in endocytic trafficking (Kramer and Phistry, 1996; Kramer and Phistry, 1999). The Hook proteins and ZYG-12 therefore do not appear to have similar functions in the two species, although, Klarsicht and ZYG-12 seem to have acquired analogous functions in some cells, given their retention at the NE by a KASH domain and their ability to tether the nucleus to the centrosome.

Msp300/nesprin is another KASH-domain protein present in Drosophila. Msp-300

(muscle-specific protein 300 kDa) was originally identified in a search for muscle- specific genes expressed during embryonic myotube migration and attachment (Volk,

1992). Given its localization to actin-rich muscle-attachment sites, Z-lines and the leading edge of migrating myotubes, Msp-300 was hypothesized to be critical for muscle morphogenesis in the developing Drosophila embryo (Volk, 1992). Indeed, an embryonic lethal mutation in Msp-300 leads to defects in some of these processes. In the Msp-300SZ-

10 75 mutant flies, the myotubes cannot extend towards to the epidermal attachment sites

and, as a result, the ability of the embryonic somatic muscle cells to contract is severely

compromised (Rosenberg-Hasson et al., 1996).

Ten years after Msp-300 was characterized, a gene immediately downstream of Msp-300,

provisionally called nesprin (nuclear envelope -repeat), was shown to encode a

KASH domain (Zhang et al., 2002). Careful analysis revealed that Msp-300 and nesprin are in fact part of the same enormous gene spanning about 80 kb. Msp-300 encodes the

N-terminal portion of the full-length protein and nesprin encodes the C-terminal region.

A repetitive coding region lies between the two and, therefore, the complete gene can potentially encode a gigantic molecule of ~1300 kDa: Msp-300/nesprin (Zhang et al.,

2002). Like ANC-1, Msp-300/nesprin contains a C-terminal KASH domain and an N- terminal ABD, which binds directly to F-actin in vitro and decorates these filaments in vivo (Rosenberg-Hasson et al., 1996; Volk, 1992). The region in between comprises a series of spectrin-repeats (SRs) (Fig. 2) - three-helix bundles that can give proteins length and elasticity and mediate protein interactions, including homodimerization (Djinovic-

Carugo et al., 2002; Mislow et al., 2002a). Interestingly, the repetitive regions in ANC-1 are not related to SRs, although they apparently have an analogous function. Msp-

300/nesprin appears to be localized predominantly at the NE in nurse cells and the oocyte, but some also appears to be present in the cytoplasm (Yu et al., 2006). Curiously, expression of a GFP-KASH domain fusion protein did not have any obvious detrimental defects on the position of the nuclei in these cells. This suggests that the docking sites for

Msp-300/nesprin, like those of Klarsicht, are not limited on the NE.

11

During oogenesis in Drosophila, one of the cells in the egg chamber becomes the oocyte;

while the other 15 become the supporting polyploid nurse cells. Eventually, the nurse

cells “dump” their cytoplasmic contents into the oocyte through ring canals, and then

undergo apoptosis (Spradling, 1993). The positions of the nuclei are thought to be critical

during this process because if they would become detached they would block the canals

(Yu et al., 2006). Since actin structures are important for nuclear anchorage in the nurse cells during dumping (Guild et al., 1997), Yu et al. investigated whether Msp-300/nesprin plays a role in this process by generating flies carrying the Msp-300SZ-75 allele only in the

germ line (these flies are viable but do not contribute any wild-type Msp-300/nesprin to

the developing Msp-300SZ-75 egg chamber) (Yu et al., 2006). Indeed, the egg chambers in these flies exhibit defects in cytoplasmic dumping and nuclear positioning (Yu et al.,

2006) (Fig. 3B). Interestingly, they also have defective actin structures, which suggests that Msp-300/nesprin also has a role in actin organization (i.e. bundling and/or cross- linking), a role attributed to other ABD-containing proteins (Winder and Ayscough,

2005).

Interestingly, the Msp-300SZ-75 mutation is lethal, unlike the deletion of ANC-1 in C. elegans. Larvae die because they do not hatch from the chorion owing to the previously mentioned defects in muscle attachment and contraction and not of nuclear anchoring defects (Rosenberg-Hasson et al., 1996). In contrast, ANC-1 seems not to have critical functions besides those associated with nuclear and mitochondrial anchorage.

12 The Mammalian Nesprins

The mammalian nesprins were originally identified in yeast two-hybrid screens for binding partners of a tyrosine kinase of the postsynaptic membrane in muscle (MuSK)

(which yielded nesprin-1) (Apel et al., 2000) and for the cytoskeletal cross-linker protein plectin (which yielded nesprin-3) (Wilhelmsen et al., 2005). Nesprin-1, originally named synaptic nuclear envelope (Syne)-1 because of its presence at postsynaptic nuclei of neuromuscular junctions (Apel et al., 2000), is actually localized at the ONM of various cell types (Wilhelmsen et al., 2005; Zhang et al., 2001; Zhang et al., 2002). It is also referred to as myocyte nuclear envelope (Myne)-1 (Mislow et al., 2002b) or Enaptin

(Padmakumar et al., 2004). Nesprin-2 was identified in database searches for sequences related to nesprin-1 or the α- ABD (Apel et al., 2000; Zhen et al., 2002). It is also known as Syne-2 (Apel et al., 2000) or NUANCE (Zhen et al., 2002).

Nesprin-1 and nesprin-2 are giant proteins of ~976 kDa and ~764 kDa, respectively

(Zhang et al., 2002), and each contains an N-terminal ABD (Padmakumar et al., 2004;

Zhen et al., 2002). Nesprin-3 is much smaller, ~110 kDa, and instead binds to plectin at its N-terminus to connect it to IFs (Wiche, 1998; Wilhelmsen et al., 2005). All three contain a series of homologous SRs and a C-terminal KASH domain (Fig. 4). In vitro binding assays and localization studies with the isolated N-terminal regions indicate that the nesprins can indeed interact with actin filaments and IFs (Fischer et al., 2004;

Padmakumar et al., 2004; Starr and Han, 2002; Wilhelmsen et al., 2005; Zhen et al.,

2002). Interestingly, plectin is ~500 kDa and, when bound to nesprin-3, generates a bridge to IFs similar in size as the one that nesprin-1 and -2 provide for F-actin. This

13 suggests that it is important that there is a significant distance between the nucleus and both the IF and F-actin systems (Fig. 2).

Nuclear Functions

In transgenic mice overexpressing the nesprin-1 KASH domain in muscle cells, the nuclei do not aggregate beneath the postsynaptic membrane because endogenous nesprin-

1 is displaced from the NE (Grady et al., 2005). This implies that the number of KASH domain binding sites at the NE is limited (as is the case for ANC-1). How nesprin-1 tethers nuclei to the postsynaptic membrane is not known, although an association with actin structures or MuSK is probably required (Apel et al., 2000; Grady et al., 2005).

Surprisingly, although the nuclei are not properly localized, the neuromuscular junction functions normally.

Nesprin-1 and nesprin-2 exhibit a variety of isoforms produced through the use of different translational initiation and stop sites and alternative splicing (Cottrell et al.,

2004; Mislow et al., 2002b; Padmakumar et al., 2004; Zhang et al., 2001; Zhang et al.,

2005). Interestingly, some of these do not contain an ABD and/or a KASH domain.

Several isoforms are localized and function at areas of the cell other than the ONM, such as the INM. For example, nesprin-1α might localize at the INM because it can associate with the INM protein emerin and lamin-A/C, although this has not been confirmed

(Mislow et al., 2002a; Mislow et al., 2002b). Nesprin-1 is also found within the nucleus, where it is associated with heterochromatin (Zhang et al., 2001). Additionally, expression of lamin A/C can influence the localization of certain nesprin-2 isoforms at the NE and in

14 vitro experiments suggested that these bind directly to lamin A/C and emerin within the nucleus. Indeed, ultrastructural analysis suggested that they are present at the INM

(Libotte et al., 2005; Zhang et al., 2005). Notably, no splice variants of nesprin-3 have been detected at the INM (Wilhelmsen et al., 2005). The function of the nesprins at the

INM is currently not known, although it is possible that they help to organize the nuclear lamina.

Membrane Trafficking and Organization

Nesprin-1 might also be involved in protein trafficking and it has been detected at the

Golgi (Gough et al., 2003). Moreover, expression of certain SR fragments of nesprin-1 in cells results in the collapse of the Golgi apparatus, prevents the recycling of protein disulfide isomerase and alters the localization of both the KDEL ER-retrieval receptor and β-COP (part of the COPI coat complex involved in ER-Golgi transport and NE breakdown (Liu et al., 2003; Nickel et al., 2002)) (Gough and Beck, 2004). The nesprin-1 splice variant Golgi-localized SR-containing protein (GSRP)-56 contains two SRs present in the central region of the full-length protein and lacks both the ABD and the

KASH domain. GSRP-56 is localized at the Golgi apparatus and interestingly, its overexpression results in expansion of the Golgi apparatus (Kobayashi et al., 2006). The brain-specific splice variant CPG2 is also derived from the SR-rich region (Cottrell et al.,

2004; Padmakumar et al., 2004). Detected in a screen for plasticity-related genes upregulated after neuronal excitation in rat brains (Nedivi et al., 1993), it is localized at the postsynaptic endocytic zones of excitatory synapses in neurons. CPG2 regulates the constitutive internalization of glutamate receptors and the activity-induced internalization

15 of α-amino-3-hydroxy-5-methyl-isoxazole-4-proprionic acid (AMPA) receptors (Cottrell et al., 2004), and is thereby proposed to modify synaptic strength. Variants of nesprin-1 might thus function in protein trafficking, control of Golgi morphology and, possibly, NE breakdown.

Other Functions

Several other roles of nesprin-1 isoforms have been described. For instance, nesprin-1α can recruit muscle A-kinase anchoring protein (mAKAP) to the NE in striated myocytes through an association between their SRs (Pare et al., 2005). mAKAP is a scaffold protein that forms signaling complexes containing protein kinase A (PKA), the ryanodine receptor (RyR) calcium release channel, protein phosphatase 2A and phosphodiesterase type 4D3 (Kapiloff et al., 2001). Phosphorylation of the RyR by PKA augments the release of calcium from the sarcoplasmic reticulum and other luminal areas associated with the NE (Bers and Perez-Reyes, 1999). Nesprin-1α might thus serve as a receptor on the nucleus.

Nesprin-1 has also been shown to be involved in cytokinesis. A non-SR central fragment of nesprin-1 interacts with the KIF3B subunit of the II motor. Overexpression of this fragment or the C-terminus of KIF3B prevents cytokinesis. Furthermore, the proteins are co-localized at the central spindle and midbody during cytokinesis. Their association might allow nesprin-1 to attach vesicles to the kinesin motor protein, bringing extra membrane to the cleavage site to increase the surface area available for the formation of the two daughter cells (Fan and Beck, 2004).

16

Finally, two splice variants of nesprin-3 have been characterized: nesprin-3α and -3β.

Interestingly, nesprin-3β is unable to associate with the plakin family members

(Wilhelmsen et al., 2005). This suggests that nesprin-3, like the smaller isoforms of nesprin-1 and -2, has functions in the cell other than tethering the nucleus to the cytoskeleton.

Links with Microtubules

Like the Drosophila proteins dHk and dHkRP, the mammalian Hook and Hook-related

(HkRP) family members share regions of similarity with ZYG-12, but lack a conserved

KASH domain (Malone et al., 2003; Simpson et al., 2005; Walenta et al., 2001) and do not appear to be responsible for the association of the centrosome with the nucleus.

Nesprin-3, however, has been shown to associate with the ABDs of the plakin family members MACF and BPAG1. These proteins interact with MTs (Wilhelmsen et al.,

2005) and, thus, Nesprin-3 might provide the necessary link between the nucleus and this cytoskeletal system. However, an interaction between these proteins has not yet been demonstrated in cells.

The Mammalian SUN-domain Proteins

Two mammalian UNC-84 orthologues were discovered through an EST database search for sequences that share similarity with unc-84: SUN1 (UNC84A) and SUN2 (UNC84B)

(Malone et al., 1999). SUN1 was independently discovered in a proteomic analysis of neuronal NE components (Dreger et al., 2001). Both SUN1 and SUN2 are INM

17 components. Two other mammalian proteins containing a SUN domain have been

identified, SUN3 and SPAG4, but they were instead primarily detected at the ER

membranes (Crisp et al., 2006; Hasan et al., 2006). In common with matefin/SUN-1, but

not UNC-84, SUN1 does not require lamin proteins for its localization at the INM,

although it does bind specifically to lamin A (Crisp et al., 2006; Haque et al., 2006;

Hasan et al., 2006; Padmakumar et al., 2005). Lamin A is, however, partially responsible

for the localization of SUN2. Interestingly, both SUN1 and SUN2 preferentially bind to

the unprocessed (i.e. non-cleaved) form of lamin A in vitro and in vivo (Crisp et al.,

2006), which suggests that they play a role in the recruitment and organization of these

proteins at the nuclear lamina.

SUN1 and SUN2 each contain a conserved SUN domain that extends into the PS and

interacts with the KASH domains of nesprin-1, -2 and -3 (Crisp et al., 2006; Haque et al.,

2006; Ketema and Sonnenberg, unpublished; Padmakumar et al., 2005). However, a

region just upstream of the SUN domain may mediate high-affinity binding to the KASH domain (Padmakumar et al., 2005). The importance of this interaction is highlighted in transgenic mice overexpressing the KASH domain of nesprin-1, which prevents the localization of endogenous nesprin-1 at the ONM in muscle cell nuclei at the neuromuscular junction (Grady et al., 2005). Additionally, RNAi-induced depletion of

SUN1 and SUN2 proteins in HeLa cells results in the mislocalization of nesprin-2γ away from the ONM. Importantly, this study also showed that the interactions of the nesprins with the SUN-domain proteins is critical for maintenance of the NE because the absence of SUN1 and SUN2 leads to an expansion of the PS (Crisp et al., 2006). The

18 evolutionarily conserved interactions that take place between the conserved SUN and

KASH domains within the PS are thus critical not only for associations between the nucleus and cytoplasmic proteins, but also for the integrity of the NE in mammalian cells.

Conclusions/Perspectives

It is now clear that SUN-domain proteins of the INM retain the KASH-domain proteins at the ONM and these proteins probably directly interact within the PS. This generates a continuous protein scaffold that physically links the nucleoskeleton to the cytoskeleton: the so-called LINC complex (Crisp et al., 2006) (Figs 2, 5). However, other proteins present in the PS might influence these interactions.

It is intriguing that the ONM proteins in C. elegans, although unrelated to those of

Drosophila and mammals except for their KASH domain, have evolved similar mechanisms for nuclear anchorage and migration. ANC-1 has a function analogous to that of Msp-300/nesprin and the mammalian nesprins in nuclear anchorage, whereas

ZYG-12 has a function analogous to that of Klarsicht in the attachment of the centrosome to the nucleus. Although no proteins related to UNC-83 have been identified, its role in migration is likely to be similarly conserved in higher organisms.

For several reasons, few studies have examined the importance of the mammalian nesprins in nuclear positioning. First, the genes and their mRNAs are very large (Zhang et al., 2002), which makes molecular, biochemical and cellular studies difficult.

Secondly, there are many splice variants, some of which are cell-type specific, which

19 complicates the interpretation of the results. Thirdly, the three nesprins may be able to

compensate for one another if one is absent or disfunctional. Studies in which the

function of all three nesprins is disrupted may ultimately prove their importance in

nuclear positioning, but such studies are complicated by the fact that the nesprins have

other critical functions.

The only study to demonstrate that the mammalian nesprins are essential in nuclear

positioning depended on the overexpression of the nesprin-1 KASH domain in muscle

cells (Grady et al., 2005) and was based on similar experiments using the C. elegans

ANC-1 KASH domain (Starr and Han, 2002). Both studies suggest that the number of

SUN-domain binding sites in the PS is limited. However, overexpressed KASH domains

might displace other KASH-domain proteins from the ONM, such as nesprin-3 in

mammals, which is also expressed in muscle cells (Wilhelmsen et al., 2005).

Furthermore, overexpression of the Klarsicht and Msp-300/nesprin KASH domains in

Drosophila does not affect nuclear positioning, or produce other phenotypic defects, in

photoreceptors or the egg chamber, respectively (Fischer et al., 2004; Yu et al., 2006).

Therefore one should be cautious when interpreting results from studies using KASH

domain overexpression in higher organisms.

An intriguing theory called cellular tensegrity proposes that eukaryotic cells are pre-

stressed through the concerted action of all three (Ingber, 2003a; Ingber,

2003b). Importantly, it would explain how simultaneous changes in cytoskeletal, nuclear

and other cellular structures can occur in response to localized force in the absence of any

20 biochemical changes (Maniotis et al., 1997). We have known for a relatively long time that integrins can mediate cross-talk between the ECM and the different cytoskeletal systems (Geiger et al., 2001), and that integral INM proteins can mediate the interactions of the nuclear lamina with heterochromatin (Gruenbaum et al., 2005). The well-studied

NPCs are known to bridge the nucleoskeleton and the cytoskeleton and have been suggested to mediate the mechanotransduction of signals from the cytoskeleton to the nuclear environment (Ingber, 2006). KASH-domain proteins may provide an alternative mechanism to direct forces into the nucleus (Fig. 5). There is no doubt that future studies on the architecture and positioning of the nucleus and cellular mechanotransduction will reveal many novel, exciting functions for this family of proteins.

21 Acknowledgements

We would like to thank Drs. Rik Korswagen, Yosef Gruenbaum and Nick Brown for critical reading of the manuscript. Kevin Wilhelmsen and Mirjam Ketema are supported by grants from the Dutch Cancer Society (KWF) and the Netherlands Science

Organization (NWO/ALW), respectively.

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31 Figure Legends

Figure 1

Phenotypes of the C. elegans mutants anc-1, unc-83, unc-84 and zyg-12. A. In C. elegans, hyp7 precursor cells go through a series of elongation and alternating intercalation events on the dorsal side of the developing embryo. In the wild-type embryo, the nuclei (grey and black circles) then move to the opposite side of the embryo

(S3). After fusion of the hyp7 precursors into the large multi-nucleated hyp7 syncytium, the nuclei are evenly anchored near the dorsal midline (DM) (S4). Unc-83 and some unc-

84 mutant alleles result in failure of the nuclei to migrate past the DM (S3); these are therefore mispositioned in the syncytium, although they appear to be anchored (S4). In the anc-1 worms, the nuclei migrate normally (S3) but are unanchored and typically form clumps in the syncytium (S4). The majority of the unc-84 mutant alleles, and all the unc-

83 anc-1 double mutants, result in incorrect positioning and anchorage of the nuclei in the syncytium (S4). Only a few of the hyp7 precursor cells are depicted for simplicity. B.

In the single-cell embryo of C. elegans, MTs associated with the two centrosomes pull the two pronuclei (black ovals) together; these then fuse and the nuclear envelope breaks down. At the start of the first mitosis, MTs associated with the centrosomes pull the paired apart. In the zyg-12 worms, the centrosomes are detached from the pronuclei. As a result, the nuclei are not pulled together and do not fuse properly. The nuclear envelopes still break down and the chromosomes from each pronucleus still associate with MTs. Cytokinesis occurs but results in daughter cells that contain abnormal numbers of chromosomes.

32 Figure 2

Proposed protein complexes spanning the NE in C. elegans, Drosophila and

mammals. The nuclear lamins of several species are proposed to interact with the N-

terminal regions of the SUN-domain proteins of the INM. Within the PS, the SUN-

domains (or regions immediately upstream of the SUN-domains) associate with the

KASH domains of ONM proteins. Within the cytoplasm, the N-terminal regions of these

proteins associate with the microtubule organizing center (MTOC), MTs, F-actin or

plectin (which can directly bind to the IFs). Thus, there is a continuous protein scaffold

from the nuclear lamina, through the NE, to the different cytoskeletal systems in several

different species. The SUN-domain proteins are shown as dimers. SUN1 crosses the INM

three times, while the other SUN-domain proteins cross it at least once. The two

Drosophila SUN-domain proteins identified in database searches (labeled Dm-SUN in this depiction) do not contain any recognizable membrane spanning regions, although these may not represent full-length sequences.

Figure 3

Phenotypes of the Drosophila mutants klarsicht and Msp-300SZ-75. A. In the wild-type eye imaginal disc, passage of the morphogenetic furrow (MF) initiates the differentiation of photoreceptors. Normally, the nuclei (black circles) are pulled up to the apical side through an association with the centrosome and MTs. In the klarsicht mutant flies, the

centrosomes are no longer attached to the nuclei. As a result, the nuclei remain at the

basal side. B. At the start of stage 10B in wild-type Drosophila ovaries, the nurse cells

(NCs) start to dump their cytoplasmic contents into the oocyte through the ring canals

33 (RCs). By stage 13, most NCs, and their nuclei (large black ovals), have disappeared and

the dorsal appendages are extruded at the anterior end. In Msp-300SZ-75 flies, the NC nuclei become detached once cytoplasmic dumping occurs and either form multi- nucleated cells, enter the oocyte or become mispositioned within individual cells. The germinal vesicle (GV) (the oocyte nucleus) (grey oval) is also mispositioned. For simplicity, only some of the NCs are depicted. A-anterior; P-posterior.

Figure 4

Alignment of KASH domains from various proteins. The amino acid residues present in the predicted transmembrane regions are shown in bold. The Zyg-12B KASH domain sequence is used because the Zyg-12C KASH domain sequence in the database lacks 16 residues encompassing part of the neck and transmembrane regions. Dark green highlighting indicates conserved amino acids and light green highlighting indicates amino acids with similar chemical properties in more than half of the KASH domains. Mm-Mus musculus; Hs-Homo sapiens.

Figure 5

In mammals, a continuous protein scaffold connects the extracellular matrix to the nuclear lamina via IFs and F-actin. Integrin α6β4, present in hemidesmosomes (HDs), and β1/β3 integrins, present in focal contacts (FCs), connect the extracellular matrix

(ECM) to cytoplasmic IFs (through plectin) and F-actin (through ), respectively.

These two cytoskeletal systems are interconnected to each other via the plakins (orange circles) or to themselves via or α-actinin (red circles) and the plakins (grey

34 circles). F-actin associates with the ABDs of nesprin-1 and –2; IFs associate with plectin dimers bound to nesprin-3. In turn, the ONM nesprins associate with either SUN1 or -2 within the PS. The INM SUN-domain proteins associate with lamin A through their N- termini within the nucleus. ER-endoplasmic reticulum; INM-inner nuclear membrane;

ONM-outer nuclear membrane; PM-plasma membrane; PS-periplasmic space.

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