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- The Company of Biologists
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- © 2021 The Company of Biologists. All rights reserved.
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- 0021-9533
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- 0021-9533
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- 10.1242/jcs.014191
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Abstract
3384 Research Article Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin 1 1 1 1 2 Mirjam Ketema , Kevin Wilhelmsen , Ingrid Kuikman , Hans Janssen , Didier Hodzic and 1, Arnoud Sonnenberg * Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA *Author for correspondence (e-mail: [email protected]) Accepted 30 July 2007 Journal of Cell Science 120, 3384-3394 Published by The Company of Biologists 2007 doi:10.1242/jcs.014191 Summary The outer nuclear membrane proteins nesprin-1 and dimers, and dimerization of plectin is required for its nesprin-2 are retained at the nuclear envelope through an interaction with nesprin-3 at the nuclear envelope, which interaction of their klarsicht/ANC-1/syne homology is mediated by its N-terminal actin-binding domain. (KASH) domain with Sun proteins present at the inner Additionally, overexpression of the plectin actin-binding nuclear membrane. We investigated the requirements for domain stabilizes the actin cytoskeleton and prevents the the localization of nesprin-3 at the outer nuclear recruitment of endogenous plectin to the nuclear envelope. membrane and show that the mechanism by which its Our studies support a model in which the actin localization is mediated is similar to that reported for the cytoskeleton influences the binding of plectin dimers to localization of nesprin-1 and nesprin-2: the last four amino dimers of nesprin-3, which in turn are retained at the acids of the nesprin-3 KASH domain are essential for its nuclear envelope through an interaction with Sun proteins. interaction with Sun1 and Sun2. Moreover, deletion of these amino acids or knockdown of the Sun proteins results Supplementary material available online at in a redistribution of nesprin-3 away from the nuclear http://jcs.biologists.org/cgi/content/full/120/19/3384/DC1 envelope and into the endoplasmic reticulum (ER), where it becomes colocalized with the cytoskeletal crosslinker Key words: Nuclear envelope, Nesprin, Sun, Plectin, Intermediate protein plectin. Both nesprin-3 and plectin can form filaments Introduction both Sun1 and Sun2, and the last four amino acids of the KASH The eukaryotic cell nucleus is surrounded by a nuclear domain (consensus sequence PPPX) are thought to be crucial envelope (NE), a double-layered membrane consisting of an for this interaction (Crisp et al., 2006; Padmakumar et al., outer nuclear membrane (ONM) and an inner nuclear 2005). Moreover, the binding of nesprin-2 to Sun proteins membrane (INM) separated by a lumen called the periplasmic connects the ONM to the INM and assists in the maintenance or perinuclear space (Gerace and Burke, 1988). The INM is of the integrity of the NE (Crisp et al., 2006). characterized by a specific set of proteins, such as the lamin B The N-termini of nesprin-1 and -2 extend into the cytoplasm receptor (LBR) and lamina-associated polypeptide 2 (LAP2) and can bind to actin filaments (Padmakumar et al., 2004; Zhen that can interact with lamins and/or chromatin structures et al., 2002). Hence, nesprin-1 and -2 connect the ONM to the (Dreger et al., 2001; Schirmer et al., 2003). By contrast, the actin cytoskeleton and might play a role in nuclear migration ONM is continuous with the rough endoplasmic reticulum or positioning. Sun1 and Sun2 extend their N-termini into the (RER) and shares most of its proteins with this compartment. nucleoplasm, where they can interact with A- and B-type The only proteins thus far identified as residing specifically at lamins (Crisp et al., 2006). However, the localization of Sun the ONM are members of the nesprin protein family proteins at the NE does not depend on the binding to lamins (Padmakumar et al., 2004; Wilhelmsen et al., 2005; Zhang et (Crisp et al., 2006; Haque et al., 2006). The interaction of Sun al., 2001; Zhen et al., 2002). proteins with both lamins and nesprin-1 and -2 establishes a The mammalian nesprin protein family initially comprised connection between the cytoplasmic actin cytoskeleton and the two members, nesprin-1 and -2, both of which have multiple nuclear intermediate filament system; this complex is referred isoforms (Padmakumar et al., 2004; Zhang et al., 2005). The to as the LINC complex (linker of nucleoskeleton and full-length isoforms of these proteins are characterized by an cytoskeleton). A similar complex exists in lower organisms and N-terminal actin-binding domain (ABD), a series of spectrin plays an important role in nuclear migration and positioning repeats and a C-terminal KASH domain (Padmakumar et al., (McGee et al., 2006; Starr and Han, 2002; Starr and Han, 2003; 2004; Zhen et al., 2002). Nesprin-1 and -2 are retained at the Starr et al., 2001; Tzur et al., 2006; Wilhelmsen et al., 2006; ONM by interactions between their KASH domains and Sun Zhang et al., 2002). proteins present at the INM (Crisp et al., 2006; Haque et al., We recently identified a third member of the nesprin protein 2006; Padmakumar et al., 2005). Nesprin-1 and -2 can bind to family, nesprin-3 (Wilhelmsen et al., 2005). Similar to the other Journal of Cell Science Binding of nesprin-3 to Sun and plectin 3385 nesprin proteins, nesprin-3 is characterized by a series of 3 can form dimers and that the dimerization of plectin is spectrin repeats and a C-terminal KASH domain. The protein required for the interaction with nesprin-3 at the NE. In is present at the ONM, and the KASH domain is both necessary addition, overexpression of the plectin ABD stabilizes the actin and sufficient for its localization at that site. Furthermore, cytoskeleton, which, in turn, influences the expression pattern nesprin-3 has two isoforms, nesprin-3 and nesprin-3, which of endogenous plectin. differ in their N-termini. Although the structure of nesprin-3 is quite similar to that of nesprin-1 and -2, there are two main Results differences. Nesprin-3 is much smaller, ~116 kDa, and lacks Nesprin-3 is retained at the NE in a manner similar to an N-terminal ABD. The N-terminus of nesprin-3, but not that retaining nesprin-1 and -2 that of nesprin-3, can interact with the cytoskeletal To determine whether nesprin-3 is retained at the ONM by a crosslinker protein plectin (Wilhelmsen et al., 2005). mechanism similar to that retaining nesprin-1 and -2, we Plectin is a multi-domain protein of the plakin family that is investigated the effect of overexpression of nesprin-1 and -2 on characterized by an N-terminal ABD, a plakin domain, a the localization of endogenous nesprin-3. It was demonstrated coiled-coil rod domain and a C-terminal segment containing previously that overexpression of a GFP-tagged nesprin-1 plakin repeats (Elliott et al., 1997; McLean et al., 1996; construct, containing the two C-terminal spectrin repeats and Sonnenberg and Liem, 2007; Wiche, 1998). The protein can the KASH domain (GFP-dnNesprin-1), had a dominant- interact with intermediate filaments, actin stress fibers and negative effect on the amount of endogenous nesprin-2 microtubules (Foisner et al., 1988; Nikolic et al., 1996; localized at the NE (Libotte et al., 2005). A similar construct Sanchez-Aparicio et al., 1997; Seifert et al., 1992) and has also been described for nesprin-2 (GFP-dnNesprin-2) mediates interactions between these three cytoskeletal systems (Libotte et al., 2005). To investigate the effect of these (Seifert et al., 1992; Svitkina et al., 1996). Furthermore, plectin constructs on the localization of nesprin-3, NIH3T3 cells were anchors intermediate filaments at cell-cell and cell-matrix transiently transfected with constructs encoding GFP- junctional complexes, such as hemidesmosomes, desmosomes dnNesprin-1 or GFP-dnNesprin-2 and stained to determine the and focal contacts (Eger et al., 1997; Hieda et al., 1992; Seifert levels of endogenous nesprin-3. As shown in Fig. 1A, the et al., 1992; Wiche, 1998). In this way, plectin influences the amount of endogenous nesprin-3 at the NE of transfected cells structural organization and integrity of the cytoskeleton and is decreased in comparison with that in untransfected cells. thought to assist in the mechanical strengthening of cells Quantification of this effect shows a decrease in the average (Andra et al., 1997; Wiche, 1989). gray value of nesprin-3 at the NE from 170±3 in untransfected Plectin has a strong tendency for self-association (Foisner cells to 80±3 or 98±3 in cells transfected with constructs and Wiche, 1987) and forms parallel dimers by means of its encoding GFP-dnNesprin-1 or GFP-dnNesprin-2, respectively coiled-coil rod domain (Foisner et al., 1991; Green et al., 1992; (P<0.001) (Fig. 1B). This indicates that nesprin-3 is retained Uitto et al., 1996; Wiche, 1998; Wiche et al., 1991). Higher at the NE by a mechanism similar to that retaining nesprin-1 oligomeric states of plectin are also formed and might involve and -2. interactions of its globular end domains (Foisner and Wiche, 1987; Weitzer and Wiche, 1987). Indeed, it has been shown Nesprin-3 interacts with Sun proteins through its PPPX that the ABD of plectin can dimerize and cause the bundling consensus sequence or crosslinking of actin filaments (Fontao et al., 2001). A role Nesprin-1 and -2 are localized at the NE through an interaction for plectin in the regulation of actin dynamics has also with Sun proteins present at the INM (Crisp et al., 2006; Haque been demonstrated: plectin is required for short-term et al., 2006; Padmakumar et al., 2005). Moreover, the last four rearrangements of the actin cytoskeleton in response to amino acids of the nesprin-2 KASH domain (consensus extracellular stimuli that activate the Rho, Rac and Cdc42 sequence PPPX) are essential for the interaction with Sun1 and GTPase signaling cascades (Andra et al., 1998). the localization of nesprin-2 at the NE (Padmakumar et al., The interaction of plectin with nesprin-3 is mediated by the 2005). To investigate whether the last four amino acids of plectin N-terminal ABD (Wilhelmsen et al., 2005). We nesprin-3 (PPPT) are likewise required for its retention at the previously suggested that nesprin-3 and actin filaments NE through an interaction with the Sun proteins, a mutant of PPPT compete for binding to the plectin ABD. This was supported nesprin-3 lacking these amino acids, nesprin-3 , was by our observation that disruption of the actin network by generated. PA-JEB keratinocytes stably expressing GFP- PPPT latrunculin B led to an increased concentration of plectin at the nesprin-3 or GFP-nesprin-3 were stained for NE. Moreover, this effect was dependent on the presence of endogenous Sun1 or Sun2 and analyzed by confocal nesprin-3, as shown by the siRNA-mediated knockdown of microscopy. GFP-nesprin-3 colocalizes almost completely nesprin-3 (Wilhelmsen et al., 2005). As plectin binds to with both Sun1 and Sun2 (Fig. 2A), suggesting that it interacts nesprin-3 through its ABD and to intermediate filaments with these proteins at the NE. By contrast, GFP-nesprin- PPPT through its C-terminus, we hypothesized that the interaction 3 is largely absent from the NE and instead distributes between nesprin-3 and plectin links the nucleus to the throughout the cytoplasm. Consequently, there is only limited PPPT intermediate filament system. with Sun1 and Sun2 colocalization of GFP-nesprin-3 In the present study, we further investigated the requirements (Fig. 2A). This indicates that the last four amino acids of necessary for the localization of nesprin-3 and plectin at the nesprin-3 are indeed essential for its localization at the NE. NE. We show that nesprin-3 is retained at the NE by an To determine the intracellular localization of GFP-nesprin- PPPT interaction with Sun proteins and that the last four amino acids 3 , PA-JEB cells stably expressing this tagged nesprin of its KASH domain (PPPX consensus sequence) are involved mutant were stained for plectin and several cellular PPPT in this interaction. Furthermore, we demonstrate that nesprin- no longer concentrates at components. GFP-nesprin-3 Journal of Cell Science 3386 Journal of Cell Science 120 (19) PPPT expressing either GFP-nesprin-3 or GFP-nesprin-3 and the precipitates were probed for the presence of GFP-nesprin- 3, Sun proteins and lamins A and C. Sun1 and Sun2 were co- precipitated with GFP-nesprin-3 but not with GFP-nesprin- PPPT 3 (Fig. 2D). This effect was specific for the Sun proteins, as LAP1 and LAP2, two other INM proteins, did not co- precipitate with either form of nesprin-3 (data not shown). Furthermore, we were unable to co-precipitate lamin A and lamin C from cells expressing GFP-nesprin-3 and GFP- PPPT nesprin-3 (Fig. 2D). Taken together, these results indicate that nesprin-3 interacts with Sun1 and Sun2 through its C-terminal four amino acids (PPPT). Sun proteins are required for proper localization of nesprin-3 at the NE PPPT The localization of nesprin-3 at the ER suggests that the interaction with Sun proteins is essential for the proper retention of nesprin-3 at the NE. Sun proteins were previously reported to interact with lamin A (Crisp et al., 2006; Haque et al., 2006). To investigate whether Sun proteins and lamin A are indeed required for the localization of nesprin-3 at the NE, we performed siRNA knockdown studies. PA-JEB cells stably expressing GFP-nesprin-3 were mock transfected or transfected with siRNAs directed against the expression of lamin A/C (LmnA), Sun1, Sun2 or a combination of Sun1 and Sun2. Knockdown efficiency was tested by western blot (Fig. 3A) and the effect on the localization of GFP-nesprin-3 was analyzed by immunofluorescence. The siRNA-mediated Fig. 1. Nesprin-3 is displaced from the NE upon overexpression of knockdown is specific as it has no effect on the expression of nesprin-1 and nesprin-2. (A) NIH3T3 cells were transiently other INM proteins or lamin B (Fig. 3B). The decrease in the transfected with constructs encoding GFP-dnNesprin-1 or GFP- dnNepsrin-2, fixed in paraformaldehyde and stained for endogenous amount of lamins A and C has no, or only a limited, effect on nesprin-3. Representative confocal images are shown. Bars, 10 m. the localization of Sun1 and Sun2 at the INM (data not shown). (B) Quantitative analysis of the effect demonstrated in (A). The Similarly, knockdown of Sun2 or lamins A and C has no effect average gray value of endogenous nesprin-3 at the NE was measured on the localization of GFP-nesprin-3 at the ONM (Fig. 3B,C). for individual transfected and untransfected cells. Results are shown However, knockdown of Sun1 or a combination of Sun1 and as the mean gray value±s.e.m. (n=50). *, P<0.001. Sun2 leads to a decrease in the amount of GFP-nesprin-3 at the NE of 27% and 40%, respectively (P<0.001) (Fig. 3B,C). This suggests that expression of Sun1, but not that of Sun2, is the NE but is primarily found on membrane structures in the required for proper retention of nesprin-3 at the ONM. cytoplasm, where it colocalizes with endogenous plectin (Fig. PPPT does not colocalize The fact that no effect of Sun2 knockdown was observed on 2B). Furthermore, GFP-nesprin-3 with markers for mitochondria (data not shown) or the Golgi the localization of GFP-nesprin-3 could be attributable to an complex (Fig. 2B) and only partially colocalizes with protein incomplete knockdown of Sun2. To investigate the interaction disulphide isomerase (PDI), a marker for the ER (Fig. 2B) between Sun2 and nesprin-3 in more detail, PA-JEB cells (Luz and Lennarz, 1996). As the expression pattern of stably expressing GFP-nesprin-3 were transfected with a PPPT GFP-nesprin-3 resembled that of PDI, a possible construct encoding the lumenal domain of Sun2, encompassing PPPT localization of GFP-nesprin-3 at the ER was further the nesprin interaction site, fused to an ER retention signal. In investigated by electron microscopy. In PA-JEB cells stably untransfected cells, the localization of GFP-nesprin-3 at the PPPT expressing GFP-nesprin-3 or GFP-nesprin-3 , wild- NE was normal (Fig. 3D). However, overexpression of the type GFP-nesprin-3 is mainly found at the ONM and to Sun2 lumenal domain results in a relocalization of GFP- some extent at the ER (Fig. 2C). By contrast, although some nesprin-3 away from the NE and into the ER, where it PPPT GFP-nesprin-3 still occurs at the ONM, the majority becomes colocalized with the Sun2 lumenal domain (Fig. 3D). PPPT of the GFP-nesprin-3 associates with the ER (Fig. 2C). Based on this finding, we conclude that nesprin-3 is not only Hence, deletion of the last four amino acids of nesprin-3 retained at the NE through an interaction with Sun1 but also prevents its retention at the NE and leads to a dispersion of with Sun2. the protein over the ER. The results thus far show that nesprin-3 is retained at the NE PPPT The dispersion of GFP-nesprin-3 by the same mechanism as that responsible for the retention of over the ER might nesprin-1 and -2. Specifically, nesprin-3 interacts with both be caused by its inability to interact with Sun proteins. Sun1 and Sun2 through its PPPX consensus sequence. This Therefore, we investigated, by co-immunoprecipitation, PPPT interaction is required for the proper localization of nesprin-3 whether GFP-nesprin-3 , in contrast to GFP-nesprin-3, at the NE as deletion of the consensus sequence, knockdown is unable to interact with Sun proteins. GFP-nesprin-3 was of the Sun1 proteins and overexpression of a Sun2 lumenal- precipitated from whole-cell lysates of PA-JEB cells stably Journal of Cell Science Binding of nesprin-3 to Sun and plectin 3387 Fig. 2. Nesprin-3 interacts with Sun proteins through its PPPX consensus sequence. (A) PA-JEB cells stably expressing GFP-nesprin- PPPT 3 or GFP-nesprin-3 were fixed in paraformaldehyde, stained for endogenous Sun1 and Sun2 and analyzed by confocal microscopy. Bar, 10 m. (B) PA-JEB cells stably PPPT expressing GFP-nesprin-3 were fixed in paraformaldehyde, stained for endogenous plectin (HD- 121 antibody), the ER (PDI antibody) or the Golgi complex (Golgin97 antibody) and analyzed by confocal microscopy. Bar, 10 m. (C) Ultrathin sections of PA- JEB cells stably expressing GFP- PPPT nesprin-3 or GFP-nesprin-3 were labeled with a pAb against GFP, followed by an incubation with 10 nm colloidal-gold-conjugated protein A. Nesprin-3 associates with the ONM (arrowhead) and the ER (arrow). N, nucleus; C, cytoplasm; NE, nuclear envelope; ER, endoplasmic reticulum. Bars, 200 nm. (D) Whole-cell lysates (WCLs) from PA-JEB cells stably expressing GFP-nesprin-3 or GFP- PPPT nesprin-3 were immunoprecipitated with the nesprin-3 mAb Nsp3. Immunoprecipitates (IPs) and WCLs were probed with antibodies directed against Sun1, Sun2, nesprin-3, and lamins A and C. KDEL construct result in displacement of nesprin-3 from the molecular-mass variant of nesprin-3, double the size of NE to the ER. nesprin-3, is occasionally observed in western blots prepared from non-denaturing gels (data not shown), independently Nesprin-3 can form dimers showing that nesprin-3 can exist as a dimer. Proteins containing spectrin repeats are thought to form dimers by means of an intermolecular interaction between their Plectin requires dimerization for its interaction with spectrin repeats (Djinovic-Carugo et al., 2002; Djinovic- nesprin-3 at the NE Carugo et al., 1999; Imamura et al., 1988; Pascual et al., 1997; We previously demonstrated that overexpression of nesprin- Yan et al., 1993). As nesprin-3 contains a series of spectrin 3, but not of nesprin-3, results in the recruitment of repeats, the ability of nesprin-3 to self-associate was endogenous plectin to the NE (Wilhelmsen et al., 2005). This investigated by co-immunoprecipitation. COS7 cells were interaction is mediated by the plectin ABD and requires the transiently transfected with constructs encoding nesprin-3 presence of the first spectrin repeat in nesprin-3 (Wilhelmsen tagged with vesicular stomatitis virus (VSV) glycoprotein et al., 2005). Like nesprin-3, plectin is also thought to form (VSV-nesprin-3) and hemagglutinin (HA)-tagged HA- dimers, which is achieved by means of an intermolecular nesprin-3, HA-plectin-1C ABD or HA--actinin ABD. The interaction of the rod domain (Foisner et al., 1991; Green et ABDs of plectin-1C and -actinin served as a positive and a al., 1992; Wiche, 1998). To test whether dimerization of plectin negative control, respectively, for nesprin-3 binding. The HA is necessary for the interaction with nesprin-3 at the NE, PA- precipitates were subsequently analyzed for the presence of JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin- VSV-nesprin-3. In support of our previous study (Wilhelmsen 3 were transiently transfected with HA-tagged plectin et al., 2005), the ABD of plectin-1C, but not that of -actinin, constructs encoding full-length plectin or C-terminal deletion could co-precipitate VSV-nesprin-3 (Fig. 4). Furthermore, mutants that either lack (plectin 1-399 and 1-606) or contain VSV-nesprin-3 co-precipitates with HA-nesprin-3 (Fig. 4), (plectin 1-2532) the rod domain (Fig. 5A). The localization of indicating that nesprin-3 can self-associate. Moreover, a high- these plectin proteins was subsequently analyzed by confocal Journal of Cell Science 3388 Journal of Cell Science 120 (19) Fig. 3. Nesprin-3 is dependent on Sun1 and Sun2 for its localization at the NE. (A) PA-JEB cells stably expressing GFP-nesprin-3 were mock transfected or transfected with siRNA directed against lamin A/C (LmnA), Sun1, Sun2 or a combination of Sun1 and Sun2. 72 hours after transfection, cells were lysed and WCLs were analyzed for expression of the indicated proteins. (B) PA-JEB cells stably expressing GFP- nesprin-3 were mock transfected or transfected with siRNAs directed against LmnA, Sun1, Sun2 or a combination of Sun1 and Sun2. 72 hours after transfection, cells were fixed in paraformaldehyde, stained for the indicated proteins and analyzed by confocal microscopy. Bar, 10 m. (C) Quantitative analysis of the effect shown in (B). The gray values of GFP-nesprin-3 at the nucleus and the complete cell were determined for individual siRNA- and mock-transfected cells. Results are shown as the mean ratio of the gray value in the nucleus to the gray value in the total cell±s.e.m. (n=50). *, P<0.001. (D) PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with a Myc- tagged Sun2 lumenal domain construct carrying an ER-retention signal (Sun2L-ER). After 72 hours, cells were fixed in paraformaldehyde and stained for Myc. Representative confocal images are shown. Arrows indicate a displacement of GFP-nesprin-3 over the ER. Bar, 10 m. microscopy. While full-length plectin and plectin 1-2532 patient with muscular dystrophy-associated epidermolysis colocalized with nesprin-3 at the NE, plectin 1-339 and bullosa simplex (MD-EBS), who was homozygous for an 8- plectin 1-606 did not (Fig. 5B). As expected, none of the HA- base-pair duplication in exon 31 of plectin (Smith et al., 1996). tagged plectin proteins colocalized with nesprin-3 at the NE As a consequence, the MD-EBS-1 cells do not have full-length (Fig. 5B). As only those proteins containing the rod domain plectin, but express a variant that lacks the rod domain (Fig. were localized at the NE, the results suggest that only plectin 5C). By contrast, MD-EBS-2 cells completely lack expression dimers are efficiently recruited by nesprin-3. of plectin (Fig. 5C). To investigate the influence of the rod To further confirm the essential role of the rod domain and domain on the recruitment of plectin to nesprin-3 at the NE, the dimerization of plectin in the recruitment of plectin to the PA-JEB cells expressing GFP-nesprin-3 and MD-EBS-1 cells NE, we made use of the MD-EBS-1 cell line (Geerts et al., stably expressing either GFP-nesprin-3 or GFP-nesprin-3 1999; Koster et al., 2004). These cells were established from a were stained for plectin and analyzed by confocal microscopy. Journal of Cell Science Binding of nesprin-3 to Sun and plectin 3389 To investigate a potential effect of the plectin ABD on the localization of endogenous plectin, PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with E95S constructs encoding HA-plectin ABD or HA-plectin ABD , a mutant of the plectin ABD that binds less strongly to nesprin- 3 (see supplementary material Fig. S1). The cells were subsequently stained for endogenous plectin. Interestingly, while untransfected cells clearly show staining of endogenous plectin at the NE, plectin is absent from the NE in transfected cells (Fig. 6A). This dominant-negative effect on the localization of endogenous plectin is observed, to a similar extent, with both the wild-type and the E95S plectin ABD (Fig. 6A). These results indicate that the dominant-negative effect is not caused through competition for nesprin-3 binding between endogenous plectin and the plectin ABD. As plectin has been implicated in the regulation of actin dynamics (Andra et al., 1997), we wondered whether the dominant-negative effect of the plectin ABD could be mediated through a stabilizing effect on the actin cytoskeleton. This was tested with cytochalasin D, a reagent known to disrupt actin filaments through the inhibition of actin polymerization. PA- JEB cells stably expressing GFP-nesprin-3 were transiently transfected with a construct encoding the HA-plectin ABD, treated with cytochalasin D and stained for either F-actin or endogenous plectin. In untransfected cells, actin filaments are lost upon cytochalasin D treatment and endogenous plectin is found associated with nesprin-3 at the NE (Fig. 6B). By Fig. 4. Nesprin-3 can form dimers. COS7 cells were transiently contrast, when cells are transfected with a construct encoding transfected with a construct encoding VSV-nesprin-3 (lanes 1-3, 5) the plectin ABD, actin fibers are still present after cytochalasin and expression constructs for HA-nesprin-3 (lanes 1 and 4), HA- D treatment and endogenous plectin is found in the cytoplasm plectin 1C ABD (lanes 2 and 6) or HA--actinin ABD (lanes 3 and associated with them (Fig. 6B). This indicates that the plectin 7). The cells were lysed in RIPA buffer, and HA precipitates were ABD stabilizes the actin cytoskeleton. probed for VSV glycoprotein (upper panel) and HA (third panel). To confirm the effect of actin stabilization on the localization WCLs were probed for the expression levels of VSV-nesprin-3 of endogenous plectin independent of the expression of the (second panel) and total nesprin-3 (lower panel). plectin ABD, we made use of the actin-stabilizing and polymerizing drug jasplakinolide. PA-JEB cells stably As the HD-121 plectin antibody applied in the other expressing GFP-nesprin-3 were treated with jasplakinolide experiments recognizes the rod domain, this antibody could not and stained for F-actin and endogenous plectin. In untreated be used for the MD-EBS-1 cell line (Okumura et al., 1999). cells, endogenous plectin is found associated with GFP- We therefore used a plectin antibody (clone 31) that recognizes nesprin-3 at the NE (Fig. 6C). Treatment with jasplakinolide an epitope in the plakin repeats. However, this antibody gives results in the formation of peripheral actin aggregates that do a considerable background staining of the nuclei, which not stain for phalloidin (Fig. 6C) as jasplakinolide competes renders an accurate identification of plectin at the NE more with phalloidin for the same binding site (Bubb et al., 1994). difficult. As shown in Fig. 5D, PA-JEB cells that lack GFP- Moreover, jasplakinolide causes a change in the distribution of nesprin-3 only show nuclear background staining, whereas endogenous plectin, away from the NE and into the peripheral PA-JEB cells expressing GFP-nesprin-3 demonstrate an actin aggregates (Fig. 6C). Hence, stabilization of F-actin by additional rim of plectin around the nucleus, which indicates jasplakinolide influences the localization of endogenous that full-length plectin is recruited to the NE. By contrast, plectin and results in a redistribution of plectin to the actin although nuclear background staining could still be observed cytoskeleton. This observation supports the idea that the in the MD-EBS-1 cells, rod-less plectin only very limitedly dominant-negative effect of the plectin ABD is caused by a colocalizes with GFP-nesprin-3 and is absent from the NE in stabilizing effect on F-actin structures. cells expressing GFP-nesprin-3 (Fig. 5D). This supports our notion that only plectin dimers can efficiently interact with Discussion nesprin-3 at the NE. We have shown that nesprin-3 is retained at the ONM through an interaction of its PPPX consensus sequence with the INM Overexpression of the plectin ABD stabilizes the actin proteins Sun1 and Sun2. This mechanism is identical to the one cytoskeleton and has a dominant-negative effect on the previously observed for nesprin-1 and -2 (Crisp et al., 2006; localization of endogenous plectin at the NE Padmakumar et al., 2005), indicating that all nesprin family Although the results in Fig. 4 indicate that the plectin ABD and members localize at the NE in a similar way. Furthermore, nesprin-3 interact at the NE, this does not occur upon nesprin-3 can form dimers, and dimerization of plectin is transient overexpression of the plectin ABD in cells (Fig. 5B). required for its interaction with nesprin-3 at the NE. We also Journal of Cell Science 3390 Journal of Cell Science 120 (19) Fig. 5. Plectin dimers are required for the interaction with nesprin-3 at the NE. (A) Schematic domain structure of a plectin molecule, containing an ABD (purple), a plakin domain (green), a rod domain (yellow) and a series of plakin repeats (blue). The length of the different plectin constructs used and their respective domains are indicated below. (B) PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were transiently transfected with HA-tagged constructs encoding plectin 1-399, plectin 1- 606, plectin 1-2532 or full-length plectin. 48 hours after transfection, cells were fixed in paraformaldehyde, stained for HA and analyzed by confocal microscopy. Bar, 10 m. (C) PA-JEB, MD-EBS-1 and MD-EBS-2 keratinocytes were lysed and WCLs were analyzed by western blot for the expression of plectin (clone 31). Myosin heavy chain (MHC) served as a loading control. (D) PA-JEB cells expressing GFP-nesprin-3 and MD-EBS-1 cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were fixed in paraformaldehyde, stained for plectin (clone 31) and analyzed by confocal microscopy. Arrows indicate the presence of plectin at the NE. Bar, 10 m. demonstrated that overexpression of the plectin ABD results in In this study, we demonstrate that nesprin-3 binds to Sun1 stabilization of the actin cytoskeleton, which in turn influences and Sun2 and that these proteins are required for its the localization of endogenous plectin. This suggests that actin localization at the NE. Knockdown of Sun1 or overexpression dynamics can regulate the interaction between plectin and of a Sun2 lumenal-KDEL construct result in a reduced amount nesprin-3. of GFP-nesprin-3 at the NE. A similar observation was made Nesprin-1 and -2 are localized at the NE through a previously for nesprin-2, which was found to be dependent on mechanism involving the INM proteins Sun1 and Sun2 (Crisp Sun1 for its localization at the NE (Crisp et al., 2006; et al., 2006; Padmakumar et al., 2005). Overexpression of one Padmakumar et al., 2005). In addition, our results are in line of these two nesprin proteins influences the localization of the with those of a previous study, showing that depletion of both other and causes its displacement from the NE (Padmakumar Sun1 and Sun2 in HeLa cells causes a loss of full-length et al., 2005; Zhang et al., 2007). We now extend these nesprin-2 from the NE (Crisp et al., 2006). observations by demonstrating that nesprin-3 is also displaced Sun1 can bind to lamin A and, more specifically, to the from the NE upon overexpression of nesprin-1 or -2. Hence, unprocessed form of lamin A (Crisp et al., 2006; Haque et al., identical protein interactions are responsible for the 2006). However, Sun1 and Sun2 do not require functional A- localization of all nesprin family members at the NE. or C-type lamins for their localization at the INM (Crisp et al., Furthermore, the dominant-negative effect of overexpression of 2006; Hasan et al., 2006; Padmakumar et al., 2005). In nesprin-1 or -2 indicates that the number of Sun docking sites addition, nesprin-1 binds to lamin A directly, and nesprin-2 is in the periplasmic space is limited. This warrants a careful dependent on lamin A for its localization at the NE (Libotte et interpretation of nesprin overexpression studies as the observed al., 2005; Mislow et al., 2002). However, these studies focused effects will be influenced by the presence or absence of other on smaller isoforms of nesprin-1 and -2 that are thought to nesprin proteins at the NE. reside at the INM instead of the ONM. In this study, we were Journal of Cell Science Binding of nesprin-3 to Sun and plectin 3391 INM. Hence, it is unlikely that nesprin-3 binds to lamin A or lamin C directly. However, this does not exclude the possibility of an indirect link with lamin A through the association with Sun proteins. This would require the formation of a protein complex containing nesprin-3, Sun proteins and lamin A that bridges the NE. However, although an interaction between lamin A and the Sun proteins might take place, lamin A and lamin C are not required for the localization of nesprin-3 at the NE. In the interaction between nesprin-3 and the Sun1 and Sun2 proteins, the PPPX consensus sequence present in the KASH domain is involved. Similarly, the conserved C- terminal four amino acids of nesprin-2, PPPT, are essential for the interaction of nesprin-2 with Sun1 (Padmakumar et al., 2005). The requirement of the PPPX consensus sequence for the localization of nesprin-3 at the NE is illustrated by the PPPT observation that nesprin-3 does not concentrate at the NE but is found instead dispersed over the ER. However, PPPT there is still some residual nesprin-3 at the ONM. This can be explained in two ways. First, as the ONM is PPPT continuous with the RER, the nesprin-3 molecules that are not able to bind to Sun proteins on the INM, but are synthesized on ribosomes associated with either membrane, will become distributed equally over the RER and the ONM. Second, as nesprin-3 can self-associate, GFP-nesprin- PPPT 3 might form heterodimers with endogenous nesprin- 3 at the NE. As the murine and human nesprin-3 spectrin repeats are highly homologous, we can reasonably conclude that they might form heterodimers. However, only the KASH domain of the wild type nesprin-3 in the dimer can bind to Sun proteins, and therefore their interaction with Sun proteins is likely to be weakened. Consequently, this should result in PPPT the amount of GFP-nesprin-3 retained at the ONM being reduced. Nesprin-3 can bind to the cytoskeletal crosslinker protein plectin. This interaction takes place between the N-terminal ABD of plectin and the first spectrin repeat of nesprin-3 (Wilhelmsen et al., 2005). The finding that some C-terminally truncated plectin fragments containing the ABD do not colocalize with nesprin-3 at first sight might seem to contradict the results from biochemical assays, in which Fig. 6. Expression of the plectin ABD stabilizes the actin nesprin-3 co-precipitates with the plectin ABD. However, it cytoskeleton and has a dominant-negative effect on the localization should be understood that, in biochemical assays, no F-actin is of endogenous plectin at the NE. (A) PA-JEB cells stably expressing present to which the plectin ABD can bind and that therefore, GFP-nesprin-3 were transiently transfected with constructs E95S under these circumstances, all the ABDs of plectin are free to encoding HA-plectin ABD or HA-plectin ABD . 48 hours after bind to nesprin-3. The plectin ABD can also bind to transfection, cells were fixed in paraformaldehyde, stained for HA and endogenous plectin (HD-121 antibody) and analyzed by monomeric actin, but this binding is relatively weak and confocal microscopy. Bar, 10 m. (B) PA-JEB cells stably apparently does not have an impact on the binding of the expressing GFP-nesprin-3 were transiently transfected with a plectin ABD to nesprin-3 (Fontao et al., 2001). However, in construct encoding the HA-plectin ABD. The cells were treated with cells, the plectin ABD can bind to F-actin. Moreover, in the co- cytochalasin D for 30 minutes, fixed in paraformaldehyde and immunoprecipitation experiments, the amount of the plectin stained for HA and either endogenous plectin (HD-121 antibody) or ABD could well exceed the amount that can bind to F-actin, F-actin. Bar, 10 m. (C) PA-JEB cells stably expressing GFP- and therefore the remainder will be available to bind to nesprin- nesprin-3 were treated with 100 nM jasplakinolide for two hours. 3. Interestingly, our data also indicate that the expression of Treated and untreated cells were fixed in paraformaldehyde and the plectin ABD exerts a dominant-negative effect on the stained for F-actin and endogenous plectin (HD-121 antibody). Bar, localization of endogenous plectin at the NE. This effect was 10 m. not caused by direct competition with endogenous plectin for nesprin-3 binding sites but by a stabilizing effect of the plectin ABD on the actin cytoskeleton. Stabilization of the unable to co-precipitate lamin A or lamin C with nesprin-3. actin cytoskeleton increases the amount of actin filaments, This is not unexpected as, by an extensive analysis, no nesprin- thereby creating more binding sites for endogenous plectin. As 3 isoforms were found in the nucleus or associated with the Journal of Cell Science 3392 Journal of Cell Science 120 (19) a consequence, plectin is no longer found at the NE associated with nesprin-3. Taken together, these results support previous observations that plectin is involved in the regulation of the actin cytoskeleton (Andra et al., 1998; Fontao et al., 2001) and suggest that actin dynamics are likely to influence the interaction between plectin and nesprin-3 at the NE. In this study, we were able to show that nesprin-3 is capable of dimerization. This was not unexpected as spectrin repeats have previously been shown to mediate protein-protein interactions as well as dimerization (Djinovic-Carugo et al., 2002; Djinovic-Carugo et al., 1999; Imamura et al., 1988; Pascual et al., 1997; Yan et al., 1993). Furthermore, one of the nesprin-1 isoforms was shown to self-associate. Mislow et al. showed, by yeast-two-hybrid studies and blot overlay assays, that nesprin-1 can self-associate through an interaction between the third and fifth spectrin repeats (Mislow et al., 2002). Based on the known anti-parallel dimerization of other proteins containing spectrin repeats (Djinovic-Carugo et al., 1999; Imamura et al., 1988; Pascual et al., 1997; Yan et al., Fig. 7. Model illustrating the nesprin-3 LINC complex. Nesprin-3 1993), it has been suggested that the N-terminus of one dimers (blue) are retained at the ONM through an interaction with nesprin-1 molecule can interact with the C-terminus of Sun1 (brown) or Sun2 (green) protein dimers. The binding of another nesprin-1 molecule. This would result in the nesprin-3 to dimers of plectin (yellow) connects the nucleus to the intermolecular anti-parallel dimerization of nesprin-1 intermediate filament system. A potential interaction of Sun proteins (Mislow et al., 2002). Although the actual spectrin repeats with lamin A (red), indicated by a question mark, establishes an involved in the dimerization of nesprin-3 await further indirect link between the cytoskeleton and the nucleoskeleton: the nesprin-3 LINC complex. ONM, outer nuclear membrane; INM, identification, these previous observations suggest that inner nuclear membrane; PS; periplasmic space; IF, intermediate dimerization of nesprin-3 might also be anti-parallel. filaments. Previous studies have shown that plectin can form dimers in solution (Foisner and Wiche, 1987; Weitzer and Wiche, 1987; Wiche, 1998). In fact, the dimerization of plectin is thought to MACF to the NE, unlike the effect on plectin. Interestingly, be mediated by its coiled-coil rod domain (Foisner et al., 1991; MACF does not contain a coiled-coil rod domain but, instead, Green et al., 1992; Uitto et al., 1996; Wiche, 1998; Wiche et has a spectrin-based central domain. It is therefore tempting to al., 1991). Our results indicate that plectin requires speculate that the inability of nesprin-3 to recruit MACF to dimerization for its interaction with nesprin-3 at the NE. the NE is due to an inability of this protein to form parallel Shorter N-terminal plectin fragments, capable of binding dimers. This hypothesis will require further investigation. nesprin-3 in co-immunoprecipitation studies, were not found In addition to nesprin-3 and plectin, Sun proteins are also at the NE in cells overexpressing nesprin-3. Only plectin thought to form dimers. Padmakumar and colleagues have constructs containing the rod domain were found at the NE, mentioned yeast-two-hybrid data indicating that a region of indicating that the rod domain is essential for localization of Sun1 containing two coiled-coil domains is capable of self- plectin at the NE. Additionally, GFP-nesprin-3 stably interaction, suggesting that Sun1 forms dimers or oligomers expressed in MD-EBS-1 cells, which only express a rod-less (Padmakumar et al., 2005). Furthermore, it has recently been variant of plectin, was unable to efficiently recruit this plectin shown that Sun2 can form homodimers as well as forming isoform to the NE. From these results, we conclude that only heterodimers with Sun1 (Wang et al., 2006). Together with the plectin dimers can efficiently interact with nesprin-3 at the dimerization of plectin and nesprin-3, this implicates a model NE. in which homo- or heterodimers of Sun proteins interact with In contrast to PA-JEB cells, which do not express the dimers of nesprin-3, which in turn can bind to dimers of integrin 4 subunit, MD-EBS keratinocytes show normal plectin (Fig. 7). As plectin binds to nesprin-3 by its N- expression of 4. Like its binding to nesprin-3, plectin binds terminal ABD, the C-terminus is free to interact with to 4 through its N-terminal ABD (Geerts et al., 1999). Hence, intermediate filaments in the cytoplasm. By contrast, Sun the absence of rod-less plectin from the NE in MD-EBS-1 cells proteins are thought to interact with nuclear lamins (Crisp et overexpressing GFP-nesprin-3 might be a consequence of a al., 2006; Haque et al., 2006). Taken together, this suggests that competition for plectin binding between nesprin-3 and intermediate filaments in the cytoplasm are indirectly linked to integrin 4. However, we do not consider this a likely the nuclear lamina (Fig. 7). Hence, the LINC complex not only explanation as we previously demonstrated that even if GFP- connects the nucleoskeleton with the actin cytoskeleton but nesprin-3 is overexpressed in PA-JEB–4 cells (i.e. PA-JEB also might connect it to the intermediate filament system. cells reconstituted with 4) full-length plectin is still recruited to the NE (Wilhelmsen et al., 2005). Materials and Methods Using pulldown assays, we have previously shown that the Constructs GFP-tagged dominant-negative nesprin-1 and -2 constructs were kindly provided ABD of MACF is also capable of interacting with nesprin-3 by Iakowos Karakesisoglou (University of Cologne, Germany) (Libotte et al., 2005). (Wilhelmsen et al., 2005). However, we were unable to show The lumenal domain of Sun2 that includes the whole C-terminal region downstream that overexpression of nesprin-3 causes a redistribution of of the transmembrane domain was amplified by PCR and cloned in the SalI-NotI Journal of Cell Science Binding of nesprin-3 to Sun and plectin 3393 restriction sites of pShooter/Myc/ER (Invitrogen). This cloning resulted in the 3 were depleted of Sun1, Sun2 or lamin A and lamin C using appropriate expression of a recombinant protein fused to an N-terminal signal sequence and a SmartPool oligonucleotide duplexes (Dharmacon). Cells were transfected with C-terminal Myc epitope, followed by a KDEL sequence for protein retention within siRNAs using DharmaFect1 transfection reagent (Dharmacon) according to the the ER lumen. The HA-tagged plectin constructs and the constructs encoding VSV- manufacturer’s instructions. Analysis took place after at least 72 hours of culture. nesprin-3, VSV-nesprin-3 and HA--actinin ABD have been described previously (Geerts et al., 1999; Litjens et al., 2003; Litjens et al., 2005; Wilhelmsen Cell lysis and immunoprecipitation et al., 2005). Nesprin-3 was subcloned into pcDNA3-HA, a derivative of the Cells grown to confluency in 10 cm tissue-culture dishes were lysed in 1 ml eukaryotic expression vector pcDNA3 (Invitrogen) that contains an extra sequence radioimmunoprecipitation (RIPA) buffer [10 mM sodium phosphate, pH 7, 150 mM 5 of the multiple cloning site encoding the HA tag, by first inserting the C-terminal NaCl, 1% Nonidet P40, 1% DOC, 0.1% SDS, 2 mM EDTA, 50 mM NaF, 100 M part using BamHI-XbaI restriction sites. The N-terminal part was subsequently sodium vanadate and protease inhibitor cocktail (Sigma-Aldrich)]. Lysates were inserted using a BamHI digest of nesprin-3 from pcDNA3-VSV. The VSV-nesprin- cleared by centrifugation at 20,000 g in a microcentrifuge at 4°C for 45 minutes. PPPT 3 construct was made by inserting a PCR fragment containing the deletion Nesprin-3 proteins were immunoprecipitated by incubation of cell lysates with 100 [using primers 5-GCTGGACTCGAGACGCTG-3 (forward) and 5-GGTCCT - l supernatant containing the nesprin-3 mAb Nsp3 for 1 hour on ice, followed by CTAGACTAGCCATTGTACCG-3 (reverse)] into the XhoI-XbaI restriction sites a subsequent incubation with 100 l of a 10% slurry of -bind-Sepharose (GE PPPT of pcDNA3-VSV-nesprin-3. Nesprin-3 was subcloned into pEGFP-C3 Healthcare) with rocking for 3 hours at 4°C. For immunoprecipitation with anti- (Clontech laboratories) using HindIII-XbaI sites. The Eco47III-XbaI sites of this HA, cell lysates were incubated by rocking for 2 hours at 4°C with 100 l of a 10% PPPT construct were used to obtain a GFP-nesprin-3 fragment for cloning into the slurry of anti-HA mAb prebound to -bind-Sepharose. Immunoprecipitates were SwaI-SnaBI sites of pLZRS-IRES-zeo. All constructs were verified by sequencing, washed four times with lysis buffer, boiled for 5 minutes in SDS-sample buffer (50 and protein expression and size were confirmed by western blotting. mM Tris, pH 6.8, 10% glycerol, 1% SDS, 0.1% -mercaptoethanol and 0.025% bromophenol blue) and resolved by SDS-PAGE. Antibodies The preparations of the rabbit polyclonal antibodies (pAbs) against nesprin-3 and Immunofluorescence Sun2 have been described previously (Hodzic et al., 2004; Wilhelmsen et al., 2005). Cells grown on glass coverslips were fixed in 1-3% paraformaldehyde in PBS for A murine nesprin-3 monoclonal antibody (mAb) Nsp3 was made by immunization 10 minutes and permeabilized with 0.5% Triton X-100 in PBS for 5 minutes. After of BALB/c mice with the GST fusion protein encoding the seventh spectrin repeat blocking with 2% BSA in PBS, the cells were incubated with the primary antibody of nesprin-3 (Wilhelmsen et al., 2005). After five injections, the mice were for 1 hour at room temperature. Cells were washed three times with PBS and sacrificed and isolated splenic lymphocytes were fused with Sp2/0 mouse myeloma incubated for 45 minutes at room temperature with the secondary antibody. After cells. Hybridoma supernatants were collected and screened for antibodies by ELISA three washes with PBS, coverslips were mounted in Mowiol-DAPCO and viewed and western blot. Selected hybridomas were cloned three times by limiting dilution. under a confocal laser scanning microscope (model TCS NT or SP-2 AOBS; Leica). The rabbit pAb directed against human Sun1 was kindly provided by Sue Data was analyzed using Adobe Photoshop and ImageJ software. Quantification, Shackleton (University of Leicester, UK). The mouse mAb HD-121 against based on maximal projection of z-stacks, was achieved by determining the gray plectin/HD1 was provided by Katsushi Owaribe (University of Nagoya, Japan) value of nesprin-3 at the NE and relating it to the total gray value of nesprin-3 in (Hieda et al., 1992). The mAb against lamins A and C (131C3) (MUbio Products individual cells. BV) was a gift from Frans Ramaekers (University of Maastricht, The Netherlands). The rabbit pAbs directed against PDI and GFP were kindly provided by Hidde Electron microscopy Ploegh (Whitehead Institute, Cambridge, MA) and Jacques Neefjes (The PPPT PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were fixed Netherlands Cancer Inst., Amsterdam, The Netherlands), respectively. Myc-tagged for 2 hours in a mixture of 2% paraformaldehyde and 0.2% glutaraldehyde in PHEM proteins were detected with the mAb 9E10. The mAb 12CA5 and pAb Y-11 buffer (60 mM PIPES, 25 mM HEPES, 2 mM MgCl , 10 mM EGTA, pH 6.9) and recognizing HA were purchased from Santa Cruz Biotechnology. The mAb directed processed for ultrathin cryosectioning, as described previously (Calafat et al., 1997). against actin was purchased from Chemicon International. The anti-VSV The sections were incubated with a pAb against GFP for 60 minutes, followed by glycoprotein mAb P5D4 was obtained from Sigma, the anti-Golgin-97 mAb CDF4 an incubation with 10 nm colloidal-gold-labeled protein A for 30 minutes. After from Molecular Probes, the anti-plectin mAb clone 31 from BD Biosciences immunolabeling, the cryosections were embedded in a mixture of methyl cellulose Pharmingen and the anti-lamin B goat pAb M-20 from Santa Cruz Biotechnology. and uranyl acetate and examined with a FEI Tecnai 12G2 electron microscope. Goat anti-rabbit and goat anti-mouse TexasRed-conjugated antibodies were purchased from Invitrogen. Goat anti-mouse and donkey anti-goat Cy5-conjugated Statistical analysis antibodies were obtained from Jackson ImmunoResearch. Alexa Fluor 568 The results are shown as the mean±s.e.m. Comparisons were made using the phalloidin and BODIPY 650/660 phalloidin were purchased from Molecular Probes. The donkey anti-rabbit and goat anti-mouse horseradish peroxidase-conjugated Student’s t test, assuming equal variances. Statistical significance was defined as antibodies were obtained from GE Healthcare. P<0.001. Cell culture We thank Sue Shackleton, Katsushi Owaribe, Frans Ramaekers, NIH3T3 and COS7 cells were grown in DMEM (GIBCO Life Technologies) Jacques Neefjes and Hidde Ploegh for providing antibodies, Els supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml Groeneveld for the generation of the nesprin-3 hybridoma and PPPT streptomycin. PA-JEB, PA-JEB-GFP-nesprin-3, PA-JEB-GFP-nesprin-3 , Iakowos Karakesisoglou for his gift of the nesprin-1 and -2 cDNA PA-JEB-GFP-nesprin-3, MD-EBS-GFP-nesprin-3 and MD-EBS-GFP-nesprin- 3 keratinocytes were grown in keratinocyte serum-free medium (GIBCO Life constructs. This work was supported by a grant form the Netherlands Technologies) supplemented with 50 g/ml bovine pituitary extract, 5 ng/ml EGF, Science Organization (NWO/ALW). 100 U/ml penicillin and 100 U/ml streptomycin. MD-EBS keratinocytes stably expressing GFP-nesprin-3 or GFP-nesprin-3 have been described previously PPPT (Wilhelmsen et al., 2005). Stable integration of GFP-nesprin-3 was References Andra, K., Lassmann, H., Bittner, R., Shorny, S., Fassler, R., Propst, F. and Wiche, performed as described previously (Sterk et al., 2000). Briefly, retrovirus carrying PPPT G. (1997). 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Journal of Cell Science – The Company of Biologists
Published: Oct 1, 2007