Correspondence to Arnoud Sonnenberg: a.sonnenberg{at}nki.nl
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Abbreviations used in this paper: ABD, actin-binding domain; BPAG1, bullous pemphigoid antigen-1; cDNA, complementary DNA; EBS-MD, epidermolysis bullosa simplex with muscular dystrophy; FC, focal contact; FNIII, fibronectin type III; IF, intermediate filament; INM, inner nuclear membrane; KASH, Klarsicht/ANC-1/Syne-1 homology; MACF, MTactin cross-linking factor; MEF, mouse embryonic fibroblast; MT, microtubule; NE, nuclear envelope; nesprin, nuclear envelope spectrin repeat; ONM, outer nuclear membrane; pAb, polyclonal antibody; PA-JEB, pyloric atresia associated with junctional epidermolysis bullosa; RACE, rapid analysis of cDNA ends; mRFP, monomeric red fluorescent protein; RIPA, radioimmunoprecipitation; siRNA, small interfering RNA; SMART, simple modular architecture research tool; SR, spectrin repeat; TM, transmembrane domain; VSV, vesicular stomatitis virus; WCL, whole cell lysate; Y2H, yeast two-hybrid.
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Introduction |
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Plakins can also mediate the binding of the cytoskeleton to membrane-bound protein complexes within a cell (Fuchs and Karakesisoglou, 2001). The NH2-terminal sequences of the 11 known plectin isoforms, which result from alternative splicing, are important for the cytoplasmic localization of these molecules (Fuchs et al., 1999; Rezniczek et al., 2003). For example, the actin-binding domain (ABD) of plectin-1A and -1C can bind to the cytoplasmic domain of the integrin ß4 subunit at the plasma membrane to initiate the formation of hemidesmosomes, which are adhesion structures that allow for the firm attachment of the basal epithelial cells to the underlying basement membrane (Borradori and Sonnenberg, 1999; Koster et al., 2004a). In addition, plectin-1F can become localized at focal contacts (FCs), whereas plectin-1B is associated with proteins of the mitochondria (Rezniczek et al., 2003). The binding properties of the latter two isoforms suggest that plectin can mediate the linkage of the IF system to proteins in lipid bilayers other than those of the plasma membrane. BPAG1-e (also known as BP230) also links the IF cytoskeleton to hemidesmosomes, and there is evidence that BPAG1 can associate with transmembrane glycoproteins on the plasma membrane (Guo et al., 1995; Hopkinson and Jones, 2000; Nakayama et al., 2002; Koster et al., 2003). MACF, on the other hand, has been reported to be localized at the plasma membrane at cellcell contacts and, recently, to membranes of the Golgi apparatus (Karakesisoglou et al., 2000; Kakinuma et al., 2004; Lin et al., 2005). Together, the evidence shows that the plakin family members, in addition to their role as cross-linkers of the cytoskeleton, can form a bridge between membrane-bound proteins and the various cytoskeletal systems. However, no plakin family members have been reported to associate with proteins of the nuclear envelope (NE).
The NE separates the nucleoplasm from the cytoplasm and consists of two lipid bilayers, the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). The INM contains a distinctive set of integral membrane proteins that interact with chromatin through associated proteins such as the nuclear lamins, which is a meshwork of IF proteins that underlies the INM (Burke and Stewart, 2002). Lamins themselves can also bind chromatin and play an essential role in maintaining the structural integrity of the NE (Hutchison, 2002; Gruenbaum et al., 2005). The proper position of the nucleus in a cell is thought to be maintained through the linkage of ONM proteins to the different cytoskeletal systems. In vertebrates, the only ONM proteins known to make associations with the cytoskeleton are the related integral membrane proteins NE spectrin repeat (nesprin)1 and 2, which bind to actin through their NH2-terminal ABDs (Zhang et al., 2002). However, in Caenorhabditis elegans, UNC-83 can link the nucleus to the MT cytoskeleton (Starr et al., 2001). This suggests that in higher organisms there are ONM proteins that can link the nucleus not only to the actin cytoskeleton but also to MTs.
The type II ONM proteins nesprin-1 and -2 (also known as Myne-1 and -2; Mislow et al., 2002), Syne-1 and -2 (Apel et al., 2000), or Enaptin and Nuance (Zhen et al., 2002; Padmakumar et al., 2004) contain a central rod domain with a variable number of spectrin repeats (SRs), flanked by an NH2-terminal ABD and a conserved COOH-terminal domain (Zhang et al., 2001, 2002). The NH2-terminal ABD is of the type found in ß-spectrin and consists of two calponin homology domains. The conserved COOH-terminal domain, which includes the transmembrane domain (TM) and the COOH-terminal amino acids present in the periplasmic space of the NE, shares a homologous region with the Drosophila melanogaster protein Klarsicht and is therefore referred to as the Klarsicht/ANC-1/Syne-1 homology (KASH) or Klarsicht-like domain. However, apart from this domain there are no other features shared with the D. melanogaster protein (Mosley-Bishop et al., 1999). In humans, both nesprin-1 and -2 are giant proteins with molecular masses of 976 and 764 kD, respectively. Because of the presence of an ABD, it has been suggested that nesprins play an important role in nuclear positioning. Indeed, the single homologue of nesprin in C. elegans, ANC-1, is required for the proper anchorage of nuclei to actin in syncytial cells (Starr and Han, 2002, 2003) and recently, overexpression of the KASH domain in mice highlighted the importance of Syne-1 for the proper positioning and localization of synaptic nuclei (Grady et al., 2005).
We have identified a new member of the nesprin family that can bind to the plakin family member plectin at the ONM. This protein was isolated in a yeast two-hybrid (Y2H) screen using the ABD of plectin-1C as bait. Like the other two known nesprins, nesprin-3 is a type II transmembrane protein of the ONM and contains a COOH-terminal KASH domain and a series of SRs related to those in nesprin-1 and -2. Unlike the other two full-length nesprins, nesprin-3 lacks an ABD and is therefore not able to associate with actin, but instead may associate with the IF cytoskeleton through plectin. Nesprin-3 is ubiquitously expressed in all cell lines and tissues tested. Expression of nesprin-3 strongly recruited endogenous plectin to the ONM in cells, where it was colocalized with both keratin-6 and -14 filaments. Nesprin-3 is the first ONM protein with an established link to the IF cytoskeletal system, and it seems certain that its discovery will increase our knowledge of the function of the NE during mitosis and proper nuclear positioning during quiescence and cell migration.
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Results |
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To determine if longer variants of nesprin-3 exist, we performed 5'rapid analysis of cDNA ends (RACE) analysis on mRNA from the Rac-11P and mTSC cell lines. The results indicate that the original nesprin-3 Y2H sequence contains the true start site of translation for nesprin-3 because the longest transcript detected containing exon 1a did not have any methionines that are in-frame with the nesprin-3
Y2H polypeptide (Fig. S1 A). Although exon 1a was the most frequently detected first exon in both cell lines, we did identify multiple other exon 1 splice variants of nesprin-3 (exons 1c, 1c', 1d, 1e, and 1f; Fig. S1 B). Three of the alternative exon 1 sequences, exons 1c', 1d, and 1e, contain upstream in-frame stop codons that preclude the possibility of any additional start sites for translation, but two exon 1 sequences, exons 1c and 1f, contain other potential start sites of translation that would encode for an additional 38 and 30 amino acids upstream of the initiation methionine of nesprin-3
, respectively (Fig. S1 B). Longer nesprin-3 variants in the nesprin-3ß reading frame are not possible because of a stop codon in exon 2 just upstream of the starting methionine (Fig. S1 A). The results of the 5'-RACE analysis suggest that transcript variants equivalent to the long isoforms of nesprin-1 and -2 do not exist of nesprin-3.
Distribution of nesprin-3 in cell lines and tissues
To further characterize the nesprin-3 protein, rabbit polyclonal antibodies (pAbs) were generated against the seventh SR of murine nesprin-3 and, subsequently, the pAbs were tested for their specificity to nesprin-3 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200506083/DC1). We first tested the expression of nesprin-3 in 14 different mouse-derived cell lines. A band of 110 kD, corresponding to nesprin-3
, was detected in all 14 cell lines tested, suggesting that this variant of nesprin-3 is ubiquitously expressed (Fig. 3 A). We did not detect any higher molecular mass bands in any of these cell lines, which is consistent with the notion that we are dealing with the full-length nesprin-3 protein.
We next tested the expression of nesprin-3 in several different mouse tissues and in all of these tissues we detected the 110-kD protein (Fig. 3 B). A 97-kD protein reacting with the nesprin-3 antibody is likely a degradation product of the nesprin-3 protein because this band is also present in the lysates of the vesicular stomatitis virus (VSV)-nesprin-3
transfected COS7 cells. These results indicate that nesprin-3 is ubiquitously expressed in tissues and most likely represents the largest and most widely expressed splice variant of nesprin-3.
To verify this at the mRNA level, we used an RT-PCRbased method to detect the presence of either nesprin-3 or -3ß in three different cell lines. We used primers that anneal in exon 2 and 4, thus encompassing the nesprin-3
specific exon 3. A PCR band of 228 bp represents nesprin-3ß and a band of 401 bp represents nesprin-3
because of the inclusion of the 173 bp of exon 3. We also performed a control RT-PCR reaction that would amplify a region of nesprin-3 from exon 1518, which includes the KASH domainencoding sequence (Fig. 3 C). For both primer pairs, PCRs of the nesprin-3
and -3ß cDNAcontaining plasmids were included as controls (Fig. 3 C). The results showed that in three different mouse-derived cell lines, NMK-1, mTSC, and mBCC-1, the predominant nesprin-3 splice variant was 3
, although its expression level differed (Fig. 3 C). This is consistent with the results obtained from the Western blots of the same mouse cell lines (Fig. 3 A).
Nesprin-3 is localized at the ONM
To determine the subcellular location of nesprin-3, we transiently transfected pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes with constructs expressing GFP- or VSV-tagged nesprin-3 and -3ß proteins. The results indicate that GFP-nesprin-3
and -3ß are localized at the NE, although there is some staining within the nucleus, which is likely caused by the folding of the NE. The location of nesprin-3 was confirmed by colocalization studies with endogenous lamin A, which is a structural protein known to reside at the nucleoplasmic side of the INM (Fig. 4 A). Immunofluorescence studies of the VSV-tagged nesprin-3
and -3ß proteins and endogenous nesprin-3 also showed localization at the NE and, in some cells, at the RER (Fig. 5 B and not depicted). Together, these results definitively show that nesprin-3 is a NE component. The results also indicate that the GFP and VSV tags do not affect the localization of the nesprin-3 proteins. Because confocal microscopy is not sensitive enough to determine which lipid bilayer of the NE contains nesprin-3, we used EM analysis on PA-JEB cells stably expressing either GFP-nesprin-3
or -3ß. Using antibodies directed against the GFP moiety, we were able to show that, like nesprin-1 and -2, nesprin-3 is also present in the ONM (Fig. 4 B; Starr and Han, 2002; Zhen et al., 2002). Furthermore, some cells showed strong staining of nesprin-3 in the RER (Fig. 4 B), which was always correlated with a higher level of nesprin-3 expression in individual cells (Fig. 5 B).
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The results thus far indicate that nesprin-3 can directly associate with plectin at the ONM. To verify these results, we used immunofluorescence studies on these proteins in cells. We first looked at the location of endogenous nesprin-3 and plectin in mouse embryonic fibroblast (MEF) cells. The results show that nesprin-3 is indeed present at the NE, but that plectin is not obviously concentrated at the nuclear perimeter, although it is present throughout the cytoplasm (Fig. 5 B). One possible reason for the relatively low concentration of plectin at the NE may be the ability of the actin cytoskeleton to bind and sequester plectin (Wiche, 1998), thereby limiting the amount of plectin available for interacting with nesprin-3
. To test this notion, we stably overexpressed GFP-nesprin-3
or -3ß into PA-JEB or PA-JEB/ß4 cells and performed colocalization studies with plectin. We hypothesized that nesprin-3
, but not nesprin-3ß, when overexpressed, competes with actin for binding to plectin, resulting in the sequestration of plectin at the NE. To determine whether plectin, when bound to nesprin-3
, can also associate with IFs, we also labeled cells with antibodies that recognize keratin-6 and -14. The results show that nesprin-3
can strongly recruit plectin to the ONM in both the PA-JEB and PA-JEB/ß4 cells and, consistent with the findings shown in Fig. 5 A, nesprin-3ß did not recruit plectin to the NE. The keratin-6 and -14 IF proteins are also strongly stained at the nuclear perimeter, which infers that plectin may mediate the association of the IFs to nesprin-3
at the NE (Fig. 5 C). In addition, a higher magnification of the nucleus showed almost equally strong staining of nesprin-3
, plectin, and keratin-6 at the ONM (Fig. 5 C). We also performed EM studies to determine the exact location of plectin and GFP-nesprin-3
in PA-JEB cells. The results confirm that nesprin-3
and plectin are situated together at the ONM (Fig. 5 D). These findings clearly demonstrate that nesprin-3
can bind plectin at the ONM in cells. Intriguingly, the results also suggest that plectin may mediate the linkage of nesprin-3
with the keratin-6/16 and -5/14 IF systems.
We also decided to make use of a GFP-nesprin-3 construct, which lacks the KASH domain but contains the sequence present in the original Y2H clone (GFP-nesprin-3
KASH; Fig. 2 C). This clone retains the sequences necessary to bind plectin, but because of the absence of the KASH domain it is not localized at the ONM and instead displays a filamentous cytoplasmic and nuclear distribution (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200506083/DC1). We stained for plectin along with either keratin-5 or F-actin in PA-JEB cells transiently expressing GFP-nesprin-3
KASH to determine whether it had any effect on the distribution of these proteins. GFP-nesprin-3
KASH did not significantly alter the distribution of plectin and was only partially colocalized with plectin in the cytoplasm of the PA-JEB cells (Fig. S3). However, the GFP-nesprin-3
KASH proteins were specifically localized along the keratin-5 IFs and they did not become localized with the actin cytoskeleton (Fig. S3). Because nesprin-3
binds to the ABD of plectin, the plakin repeats are free to make associations with the IF cytoskeleton, which can partially account for the localization of GFP-nesprin-3
KASH along IFs and, similarly, GFP-nesprin-3
KASH will compete with actin for binding to the plectin ABD, which is why it was not present along actin filaments. The observed imaging of GFP-nesprin-3
KASH along IFs in the absence of plectin staining may be a result of other plectin isoforms that are present in PA-JEB cells that are not recognized by the mAb 121 against plectin (i.e., the rod-less variant; Fig. S3). Additionally, the filamentous patterning of GFP-nesprin-3
KASH in the absence of keratin-5 staining is most likely caused by the association of these proteins with other IF networks (Fig. S3). Together, the nesprin-3
proteins lacking the KASH domain do not obviously affect the IF network or the distribution of plectin, but they do provide additional evidence that plectin links nesprin-3
to the IF cytoskeleton.
The integrin 6ß4 and nesprin-3
both associate with plectin in keratinocytes
Our findings suggest the possibility that in basal keratinocytes plectin links the IFs to nesprin-3 at the ONM and to integrin
6ß4 at hemidesmosomes at the plasma membrane. This would allow for a continuous protein scaffolding from the extracellular environment to the nucleus. We decided to take a closer look at PA-JEB/ß4 cells that stably express either GFP-nesprin-3
or GFP-nesprin-3ß. In most PA-JEB/ß4 cells expressing GFP-nesprin-3
, we saw staining of plectin at the NE and the presence of plectin in hemidesmosomes (Fig. 5 C). However, in some PA-JEB/ß4 cells expressing GFP-nesprin-3
, there was almost no staining of plectin at the NE, which coincided with a strong expression of plectin in hemidesmosomes, or alternatively, in some cells there was strong staining of plectin at the NE with almost no hemidesmosomal staining (Fig. 5 E). This effect was never seen in PA-JEB/ß4 cells expressing GFP-nesprin-3ß, which consistently showed hemidesmosomal staining of plectin (Fig. 5 E). These results suggest that ß4 and nesprin-3
compete for binding to the same pool of plectin and, moreover, that plectin may link these two proteins to the same IF cytoskeleton.
Endogenous nesprin-3 and plectin associate in cells
To verify that endogenous nesprin-3 and plectin can associate at the ONM in cells, we used small interfering RNA (siRNA) knock-down of nesprin-3 in the mouse Sertoli cell line TM-4. We selected these cells because it has previously been reported that there is a high concentration of plectin around their nuclear perimeter (Guttman et al., 1999), and therefore any effects on plectin redistribution will be more easily detected. As expected, nesprin-3 is expressed in TM-4 cells (Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200506083/DC1). We then designed a siRNA construct in a region of the mouse nesprin-3 cDNA that was present in both splice variants and tested its efficacy by coexpressing cDNAs of VSV-nesprin-3
and -3ß, along with cDNAs encoding either the nesprin-3 siRNA, a luciferase control siRNA, or an empty vector control plasmid in human-derived 293 human embryonic kidney cells. The results show that the nesprin-3 siRNA specifically knocks down the expression of transiently expressed nesprin-3
and -3ß proteins (Fig. S4 B).
We next wanted to verify that the nesprin-3 siRNA can knock-down the expression of the protein in TM-4 cells and to determine the location of plectin in these cells. The nesprin-3 siRNA or luciferase control siRNAcontaining vectors were independently transfected into TM-4 cells, along with a monomeric red fluorescent protein (mRFP) expression plasmid, and the cells were stained for endogenous plectin and nesprin-3 (Fig. 6 A). The mRFP protein was visualized to determine whether the cells express the siRNAs. The results show that the nesprin-3 siRNA can specifically knock-down the expression of the protein at the NE in TM-4 cells. The results also show that the knock-down of nesprin-3 does not obviously affect the distribution of plectin, but do show that in some cells plectin is concentrated around the NE as reported previously (Guttman et al., 1999). Interestingly, in fully spread cells plectin is primarily present in FCs and colocalized with F-actin (Fig. 6 B). Like that from MEF cells, the data obtained from TM-4 cells suggests that nesprin-3 and actin filaments compete for binding to plectin. Therefore, we decided to disrupt the actin network in TM-4 cells using latrunculin B. This should have resulted in an increased amount of plectin available to interact with nesprin-3
at the ONM and, indeed, this is what was observed (Fig. 6 B). To determine if the recruitment of plectin to the nuclear perimeter is dependent on the presence of nesprin-3
, we expressed the nesprin-3 siRNAcontaining vector in TM-4 cells and treated the cells with latrunculin B. The concentration of plectin around the nuclear perimeter in these cells was significantly reduced, with plectin now being distributed in the cytoplasm (Fig. 6 B), which suggests that the recruitment of plectin to the ONM after actin depolymerization is indeed dependent on nesprin-3
. These data also infer that the plectin that is concentrated around the nuclear perimeter in cells that were not fully spread is also dependent on an association with nesprin-3
. Together, the data presented thus far clearly demonstrate that nesprin-3
can bind plectin at the ONM in cells.
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Discussion |
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Nesprin-1 and -2 are gigantic molecules of 976 and 764 kD, respectively. However, several smaller variants of nesprin-1 and -2, which are produced by alternative RNA splicing and/or initiation sites of transcription, have been identified that lack the NH2-terminal ABD. The structure of nesprin-3 closely resembles that of nesprin-1
and -2ß, which suggested to us that nesprin-3
and -3ß are most likely smaller splice variants of a larger nesprin-3 molecule. However, several lines of evidence show that this is not the case. First, we could not identify cDNAs in either the mouse or the human databases containing sequences in exons upstream of the alternatively spliced exon 1, and careful analysis of upstream genomic sequences failed to identify sequences present in a calponin homology motif. Second, 5'-RACE-PCR on mRNA from different cell lines failed to identify any longer transcripts containing exons upstream of those for nesprin-3
, as indicated in Fig. 1. Finally, our nesprin-3 pAbs failed to detect any larger nesprin-3 variants in any of the cell lines or tissues we tested. Based on these data, it appears that nesprin-3
is the largest variant of this new nesprin family member.
The results from our immunofluorescence studies using either full-length nesprin-3 or a nesprin-3
protein lacking the KASH domain provide strong, although circumstantial, evidence that plectin can mediate the linkage of the IF cytoskeleton to nesprin-3
at the ONM. This is especially relevant because the IF interaction site and the ABD are on opposite ends of the plectin molecule (
500 kD), therefore it seems unlikely that the binding of plectin to the IF cytoskeleton would interfere with its binding to nesprin-3
. Thus, it is reasonable to conclude that nesprin-3
can indeed link the nucleus to the IF cytoskeleton through plectin. In nesprin-1/2, the KASH domain is located at the COOH-terminus, whereas the ABD is located at the NH2-terminus at a distance of
150200 nm (Starr and Han, 2003). This suggests that there must be a relatively large space between the ONM and the actin cytoskeleton. It became apparent to us that a similar space may be important between the ONM and the IFs because, like nesprin-1/2, plectin is extremely large and when it is bound to nesprin-3
, a comparable distance is achieved. At present, it is not clear why such a space is required between the ONM and the different cytoskeletal systems. It has also been reported that some isoforms of nesprin-1/2 are localized at the INM (Zhang et al., 2001; Libotte et al., 2005). However, we found no concrete evidence that nesprin-3 is so localized. It therefore appears that the nesprin family of proteins have evolved to link the nucleus to the various cytoskeletal systems, either directly (i.e., nesprin-1/2) or indirectly via members of the plakin family (i.e., nesprin-3). However, we detected only subtle, inconsistent effects on the IF cytoskeleton when nesprin-3
was overexpressed or when nesprin-3 was knocked-down by siRNAs. Moreover, our data with the TM-4 cells suggests that plectin may only be associated with nesprin-3
at the ONM during specific cellular events because we consistently saw that plectin was more concentrated around the nucleus when the TM-4 cells were more contracted or when the actin cytoskeleton was disrupted, whereas there is very little plectin around the nucleus in fully spread cells containing well defined actin stress fibers. It is possible that the function of nesprin-3
is to sequester plectin during such processes as cell division or migration when the cells contract and become more rounded because of cytoskeletal rearrangements.
We have also identified a splice variant of nesprin-3 that is unable to associate with plectin. The original nesprin-3ß RIKEN clone 4831426I19 is derived from head tissue of a day 0 neonatal mouse. Consistent with the origin of the RIKEN clone, we observed a faint protein band corresponding to the molecular mass of nesprin-3ß in a lysate derived from brain tissue. However, nesprin-3ß is not ubiquitously or strongly expressed in cells because we did not detect it by Western blotting or RT-PCR analysis in several different cell lines and tissues. The existence of this splice variant suggests that nesprin-3 must have functions other than connecting the nucleus to the IFs or sequestering plectin.
The results of our Y2H and pull-down assays show that nesprin-3 and the ß4 subunit have the same binding specificity for the different ABDs. We found this surprising because the plectin ABD binding region on the first pair of FNIII domains of ß4 (i.e., ß sheets; de Pereda et al., 1999) is structurally very different from the SRs in nesprin-3
(i.e.,
-helices; Yan et al., 1993). In fact, our original Y2H screen with the plectin-1C ABD also identified a novel interaction with an SR-rich region of ß2-spectrin, suggesting that the binding of an ABD to SRs is not unique for nesprin-3 (unpublished data). It has been shown previously that the binding of the plectin ABD to the ß4 FNIII domains and to actin is mutually exclusive (Geerts et al., 1999). Because of the similarities in binding specificity, we believe that this is also true for the binding of the plectin ABD to nesprin-3
and actin, although competition studies will have to prove this. If confirmed, this would suggest that in a cell the recruitment of plectin to actin filaments and to nesprin-3
is competitive, which could partially explain why we saw minimal staining of plectin at the nuclear perimeter in cells with well-defined actin stress fibers, but saw recruitment of plectin to the ONM when nesprin-3
was overexpressed or when the actin cytoskeleton was disrupted. Our results also show that nesprin-3
can bind the ABDs of MACF and BPAG1-n. These two proteins would allow nesprin-3
to also link the nucleus to MTs. However, we could not confirm an interaction between full-length MACF and nesprin-3
in PA-JEB or EBS-MD keratinocytes, suggesting that the endogenous proteins do not associate or that the binding between the MACF ABD and nesprin-3
is regulated differently in these cells.
Our finding that in keratinocytes nesprin-3 can compete with the integrin ß4 subunit for binding to plectin suggests that these two molecules are connected to the same IF system. Indeed, immunofluorescence studies showed that the keratin-14 IF system is colocalized with both plectin at the NE and plectin in hemidesmosomes (Geerts et al., 1999). This implies that plectin can be the link in a continuous protein scaffold from the extracellular matrix to the nucleus via the IF cytoskeleton (Fig. 7), analogous to the manner in which nesprin-1/2 can link the nucleus to FCs at the plasma membrane via actin filaments (Fig. 7). This notion is consistent with the cellular tensegrity model, which describes the cell as a prestressed structure whereby forces can be transferred from the extracellular environment through the different cytoskeletons into the nucleus (Ingber, 2003a,b). This model integrates and explains many observations in cell biology, such as nuclear architecture, but importantly it can explain how external forces may affect events in the nucleus such as chromatin organization (Maniotis et al., 1997). The nesprins are the prime candidates for mediating these processes because they have been shown to interact with INM proteins associated with the nuclear lamins (Gruenbaum et al., 2005). The lamins are known to bind chromatin and have even been suggested to play a role in regulating transcription by RNA polymerase II and DNA replication (Spann et al., 1997, 2002). If force can indeed directly alter gene expression, the need for cytoplasmic signaling events would be circumvented, allowing for a more instantaneous regulation of transcriptional events during cell migration or situations of applied force. Although these ideas are intriguing, clearly more studies are necessary to determine the extent to which the nesprins can mediate the transfer of force into the nucleus.
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Materials and methods |
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Cell lines, antibodies, and other reagents
COS7 and MEF cells were grown in DME (Life Technologies) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin. COS7 cells were transiently transfected with cDNA constructs using the DEAE-dextran method (Seed and Aruffo, 1987), and MEF cells were transfected with Effectene (QIAGEN). The PA-JEB and EBS-MD keratinocyte cell lines were maintained in keratinocyte serum-free medium (Life Technologies) supplemented with 50 µg/ml bovine pituitary extract, 5 ng/ml EGF, 100 U/ml penicillin, and 100 U/ml streptomycin. The mouse Sertoli cell line TM-4 was maintained in DME containing 2.5% fetal calf serum, 5% horse serum, 15 mM Hepes, pH 7.4, 100 U/ml penicillin, and 100 U/ml streptomycin. The following mouse-derived cell lines were also used: Tam2D2-lymphoma; N115-neuroblastoma; GE11-epithelial; C2C12-myoblast; J774-macrophage; NMK-1-keratinocyte; mTSC-tumor stem cell; mBCC-1-basal cell carcinoma; and RAC-11P and mSCC-1, -2, -3, -4-squamous cell carcinoma. Stable integration of the ß4 integrin and GFP-nesprin-3/ß were performed as described previously (Sterk et al., 2000). Murine nesprin-3 rabbit pAbs were raised against the seventh SR (Fig. S1 A) common to both 3
and 3ß and fused to GST (see cDNA constructs for specifics). This antigen sequence shows 93% (rat), 67% (dog), 64% (human), 63% (chimpanzee), and 43% (chicken) homology to the same region of nesprin-3 in other species. This sequence also shows 34% homology to nesprin-1 (mouse) and 23% homology to spectrin-ß2 (mouse), and is therefore not likely to cross react. The lamin A mAb (133A2) was a gift from F. Ramaekers (University of Maastricht, Maastricht, Netherlands). The mouse mAb 121 against plectin/HD1 was provided by K. Owaribe (University of Nagoya, Nagoya, Japan; Hieda et al., 1992). The monoclonal ß4 antibody (450-11A) was purchased from BD Biosciences. The monoclonal tubulin antibody was obtained from Sigma-Aldrich. The polyclonal MACF antibody CU119 was a gift from R. Liem (Columbia University, New York, NY) and has been described previously (Lin et al., 2005). The polyclonal GFP antibody was made at Sanquin Blood Supply Foundation and has been described previously (van Ham et al., 1997). The affinity columnpurified plectin ABD rabbit pAb (D16) was made as described previously (Geerts et al., 1999). Keratin-5, -6 (PRB-169P), and -14 (AF64) rabbit pAbs were purchased from Covance Research Products, Inc. The HA mAb (12CA5) and polyclonal HA antibody (Y-11) were purchased from Santa Cruz Biotechnology, Inc.; the donkey antirabbit and goat antimouse horseradish peroxidaseconjugated antibodies were purchased from GE Healthcare; the goat antimouse FITC-conjugated antiserum was obtained from Rockland Immunochemicals, Inc.; and the goat antirabbit Texas redconjugated antibodies were purchased from Invitrogen. Phalloidin-Texas red and latrunculin B were purchased from Sigma-Aldrich and TOP-RO was obtained from Invitrogen.
cDNA constructs
pcDNA3-VSV was generated by inserting a 100-bp fragment, containing two VSV tags (YTDIEMNRLGK) flanked by KpnI, ClaI, and NcoI sites upstream and a BamHI site downstream, into the KpnIBamHI sites of pcDNA3 (Invitrogen).
Nesprin-3.
PCR fragments of nesprin-3ß (clone 4831426I19; RIKEN) were cloned into the BamHIEcoRV sites of the pcDNA3-VSV vector in two steps. First, the COOH-terminal part containing the sequence starting from the internal BamHI site (exon 10) until the end was cloned, and, second, the NH2-terminal part until the internal BamHI site was inserted. The published sequence for the clone contains a single cytosine nucleotide insertion just after position 2,652. The original nesprin-3 construct was made by inserting the NH2-terminal fragment (containing the upstream, noncoding sequences present in the Y2H clone [Fig. S1 A]) until the internal BamHI site into a pcDNA3-VSV vector that already contained the COOH-terminal part of nesprin-3ß. A nesprin-3
construct was also made that had the upstream, noncoding sequences removed. This was done by inserting a PCR fragment that was generated from a primer annealing to the internal BamHI site and a primer containing an upstream BamHI site fused to sequences annealing to nucleotides that encode for the first six amino acids of nesprin-3
(Fig. S1 A) into the original nesprin-3
pcDNA3-VSV vector using BamHIBamHI sites. Nesprin-3
was cloned into pEGFP-C3 (CLONTECH Laboratories, Inc.) using HindIIIEcoRI sites. Subsequently, nesprin-3
in pLZRS-IRES-zeo was obtained by first inserting GFP into the BamHIEcoRI sites of this vector with subsequent insertion of the EcoRI digest of nesprin-3
from pEGFP-C3. Nesprin-3ß was cloned into pEGFP-C3 using HindIIISalI sites. The NheISalI sites of this construct were used to obtain the GFP-nesprin-3ß fragment for cloning into the SwaIXhoI sites of pLZRS-IRES-zeo. The NheI site was blunt ended using T4 polymerase. The nesprin-3
lacking the KASH domain (GFP-nesprin-3
KASH) construct was made by amplifying the region of the nesprin-3
sequence identified in the original Y2H plasmid (Fig. S1 A) with primers containing BamHI and XbaI restriction sites, and this PCR fragment was then subsequently cloned into the pcDNA3-VSV vector. The insert was then cut out of pcDNA3-VSV with HindIII and XbaI and the XbaI site was then blunt ended. The pEGFP-C3 vector (CLONTECH Laboratories, Inc.) was cut with HindIII and EcoRI and the EcoRI site was blunt ended. The insert was then cloned into this vector.
HA-tagged ABDs.
The HA-tagged plectin constructs have been described previously (Litjens et al., 2003). The sequence for the BPAG1-n (dystonin-1) ABD (amino acids 1388) in pcDNA3-HA was generated by PCR using BamHINotI restriction sites. The MACF ABD cDNA sequence (amino acids 1327) in pcDNA3-HA was generated by digestion out of a construct provided by R. Liem using EcoRIBamHI restriction sites. The -actinin-1 ABD cDNA (amino acids 1337) in pcDNA3-HA was generated by PCR using EcoRINotI restriction sites. Both the
-actinin-1 and BPAG1-n ABD cDNAs in pAS2-1 on which the PCRs were performed have been described previously (Litjens et al., 2003). The GST-nesprin-3-SR7 antigen was made by cloning the nesprin-3 SR7derived PCR product (using primers 5'-cgcggggaattcccaccttcagtcacaggctg-3' and 5'-ggccgcgcggccgctctcatgagactcagcat-3') into pGEX-4T-2 (GE Healthcare) digested with EcoRI and NotI. GST preparations were done as previously described (Wilhelmsen et al., 2002) and fusion proteins were eluted from glutathione-Sepharose beads using 15 mM of reduced glutathione. All PCR fragments were generated using the proofreading Pwo DNA polymerase (Roche). All plasmids were verified by sequencing, and protein expression and size were confirmed by Western blotting.
Coimmunoprecipitation experiments
Cells were grown to confluency on 10-cm tissue culture dishes, rinsed twice with cold TBS, and lysed in 1 ml of 10 mM Na-phosphate, pH 7.0, 150 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 2 mM EDTA, 50 mM NaF, 100 µM sodium vanadate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (radioimmunoprecipitation [RIPA]-lysis buffer) per 10-cm tissue culture dish. Lysates were cleared by centrifugation at 10,000 rpm in a microfuge at 4°C for 45 min. Nesprin-3 proteins were immunoprecipitated by incubation of cell lysates with 100 µl of a 10% slurry of polyclonal antiNesprin-3-SR7 antiserum coupled to protein ASepharose beads with dimethyl pimelimidate dihydrochloride (Sigma-Aldrich) with rocking for 1 h at 4°C. Immunoprecipitates were washed four times with 1 ml RIPA-lysis buffer, boiled for 5 min in 62.5 mM Tris/Cl, pH 6.8, 10% glycerol, 5% ß-mercaptoethanol, 5 mM DTT, 2.3% SDS, and 0.025% Bromophenol blue (SDSsample buffer) and resolved by SDS-PAGE. For anti-HA immunoprecipitates, cell lysates were incubated by rocking for 1 h at 4°C with 100 µl of a 10% slurry of anti-HA mAb prebound to -bindSepharose (GE Healthcare) in the presence of 1% BSA.
Immunofluorescence
PA-JEB, EBS-MD, and MEF cells were grown on glass coverslips, fixed with either 1 or 3% paraformaldehyde in PBS for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. The TM-4 cells were fixed and permeabilized with 1% paraformaldehyde containing 0.5% Tween 20 in PBS for 10 min. After washing in PBS and blocking with 2% BSA in PBS for 60 min at room temperature, the cells were incubated with primary antibodies for 45 min at room temperature and then washed three times with PBS. Cells were then incubated with either FITC, Cy5, or Texas redlabeled secondary antibodies for 45 min at room temperature. Coverslips were washed three times in PBS, mounted in Mowiol/DAPCO, and viewed under a confocal scanning laser microscope (model SP-2 AOBS; Leica) at room temperature. Images were visualized with either a PL APO 40x, 1.25 NA, or a PL APO (CS) 63x 1.4 NA objective lens (Leica) using LCS software and captured with a photomultiplier.
EM slides
EM was done as previously described (Sterk et al., 2000), with the following modifications. Ultrathin sections of cells were fixed in 2% paraformaldehyde and were either labeled with rabbit pAbs against the plectin-ABD (D16), followed by incubation with 10-nm gold-conjugated protein A, or labeled with rabbit pAbs against GFP, followed by incubation with 15-nm gold-conjugated protein A.
Nesprin-3 siRNA
The mouse nesprin-3 sequence (5'-gctacgtagaatcatcaca-3') and luciferase sequence used for siRNA knock-down studies was cloned into the pSUPER vector (Brummelkamp et al., 2002). The vectors were transfected into TM-4 cells using the effectene protocol (QIAGEN) and 293 human embryonic kidney cells using the lipofectamine protocol (Invitrogen). The cells were lysed with RIPA buffer or fixed 72 h after transfection and assayed for nesprin-3 expression.
Exon detection primers
In Fig. 3 C, the primers annealing to either side of the nesprin-3specific exon 3 were 5'-gagcggccctggaggcaaggcttc-3' (primer 1) and 5'-caggagcacagcctgattgtccac-3' (primer 2), and the primers amplifying exons 1518 of nesprin-3 were 5'-gaacagtggctgcaggcag-3' (primer 3) and 5'-ggtgggcggggggccattgtaccg-3' (primer 4). We also used primers annealing in exon 4 (5'-cgcatcgagtgggtgtggctgcac-3') and exon 10 (5'-gtccttgtgctgaacaagcaggct-3') to determine if other potential downstream splice variants exist.
RACE-PCR
5'-RACE-PCR to determine the 5'-sequences of nesprin-3 was performed using the second generation 5'/3' RACE-PCR (Roche) according to the manufacturer's specifications. Antisense primers annealing to the 5' end of the known nesprin-3 sequence used in the RACE reactions were 5'-caggagcacagcctgattgtccac-3', 5'-cacgttgtgcagcagcacctg-3', and 5'-atcctccacgctcctctc-3'.
Database mining (websites)
Information pertaining to this study is available at the following websites: ELM (www.elm.eu.org), ClustalW (www.ebi.ac.uk/clustalw), SMART (smart.embl-heidelberg.de), basic local alignment search tool (www.ncbi.nih.gov/BLAST), mouse genome research (www.ncbi.nih.gov/genome/guide/mouse), and human genome research (www.ncbi.nlm.nih.gov/genome/guide/human).
Online supplemental material
Fig. S1 A shows the organization of the nesprin-3 cDNA, whereas Fig. S1 B lists all the alternative exon 1 sequences that were detected for nesprin-3 by 5'-RACE-PCR. In Fig. S2, the specificity of the nesprin-3 pAb that was generated against the seventh SR is demonstrated. Fig. S3 is a confocal microscopy image showing that the nesprin-3 proteins lacking the KASH domain are localized along keratin-5 IFs and not along F-actin. Fig. S4 A biochemically confirms that TM-4 cells express nesprin-3 and Fig. S4 B demonstrates the knock-down specificity of the siRNAs that were designed against nesprin-3. Finally, the data in Fig. S5 B indicates that nesprin-3
cannot associate with full-length MACF in keratinocytes. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200506083/DC1.
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Acknowledgments |
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This work was supported by grants from the Dutch Cancer Society and the Netherlands Science Organization (Netherlands Organisation of Scientific Research/Earth and Life).
Submitted: 14 June 2005
Accepted: 28 October 2005
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References |
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