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The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope

The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope Research Article The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope 1,2 1 1 1 1,2 1,2 V.C. Padmakumar , Thorsten Libotte , Wenshu Lu , Hafida Zaim , Sabu Abraham , Angelika A. Noegel , 3, 3 1, Josef Gotzmann *, Roland Foisner and Iakowos Karakesisoglou * 1 2 Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria *Authors for correspondence (e-mail: [email protected]; [email protected]) Accepted 6 May 2005 Journal of Cell Science 118, 3419-3430 Published by The Company of Biologists 2005 doi:10.1242/jcs.02471 Summary Nesprins form a novel class of nuclear envelope-anchored does not require functional A-type lamins for its spectrin-repeat proteins. We show that a direct association localisation at the inner nuclear membrane in mammalian of their highly conserved C-terminal luminal domain with cells. Our findings propose a conserved nuclear anchorage the inner nuclear membrane protein Sun1 mediates their mechanism between Caenorhabditis elegans and mammals nuclear envelope localisation. In Nesprin-1 and Nesprin-2 and suggest a model in which Sun1 serves as a ‘structural the conserved C-terminal amino acids PPPX are essential bridge’ connecting the nuclear interior with the actin for the interaction with a C-terminal region in Sun1. In cytoskeleton. fact, Sun1 is required for the proper nuclear envelope localisation of Nesprin-2 as shown using dominant-negative Key words: Emerin, Enaptin, Lamin A/C, NUANCE, SUN domain, mutants and by knockdown of Sun1 expression. Sun1 itself Syne Introduction them in the nuclear envelope (NE), followed by a stretch of amino acids displaying strong homology to the C-terminus of Several lines of evidence suggest the presence of molecular the Drosophila Klarsicht protein (Mosley-Bishop et al., 1999). links, which ‘hard-wire’ plasma membrane receptors, Nesprin genes are huge and complex, having the propensity to cytoskeletal components and nuclear scaffolds together, generate a wide variety of isoforms. These differ in length, allowing the physical integration of the nucleus within a cell domain composition, expression pattern and maybe also in and the generation of nuclear signalling and/or mechanical their functional properties, and consequently a plethora of responses to various intracellular and extracellular cues. names is currently used for these proteins by various groups. Testimony of the existence of such connections are genetic data Nesprin-1 isoforms have been described as CPG2, syne-1, from a wide variety of species, which identify microtubules, myne-1 and Enaptin, whereas Nesprin-2 variants are also microtubule-associated proteins, proteins containing giant known as syne-2 and NUANCE (Apel et al., 2000; Mislow et spectrin repeats and SUN domain-containing molecules al., 2002a; Zhang et al., 2001; Zhang et al., 2002; Zhen et al., involved in nuclear anchorage and nuclear migration (Reinsch 2002; Gough et al., 2003; Cottrell et al., 2004; Padmakumar et and Gönczy, 1998; Starr and Han, 2003; Patterson et al., 2004). al., 2004). Even though terms such as ‘nucleoskeleton’ and the long- Nesprins are widely expressed in a variety of tissues and cell disputed ‘nuclear matrix’ have gained more significance and types (Zhen et al., 2002; Padmakumar et al., 2004). At the popularity, it is still not understood which molecular set-up is subcellular level they were detected at the NE and also in required to determine nuclear architecture and the linkage of the nucleus. In contrast to Nesprin-2, which localises the nucleus to cytoplasmic structures. The recently described ANC-1 of C. elegans (Starr and Han, 2002), MSP-300 of predominantly to the nucleus and the NE, Nesprin-1 has a Drosophila melanogaster (Rosenberg-Hasson, 1996; Zhang et rather heterogeneous subcellular distribution and can also be al., 2002) and the mammalian proteins Nesprin-1/Enaptin and detected at F-actin-rich structures (Zhang et al., 2001; Padmakumar et al., 2004). Consistent with their subcellular Nesprin-2/NUANCE (Zhang et al., 2001; Zhang et al., 2002; localisation, genetic studies involving their orthologues in Zhen et al., 2002; Padmakumar et al., 2004) form a novel lower eukaryotes suggest roles in the attachment of family of proteins containing nuclear spectrin repeats that may intracellular membrane compartments to the actin organise and connect the nuclear membrane to the cytoskeleton cytoskeleton. Mutations in anc-1 of C. elegans disrupt the as well as the nuclear lamina. These proteins are very large positioning of nuclei and mitochondria (Starr and Han, 2002). (>800 kDa) and are composed of an N-terminal α-actinin-type actin binding domain, a long rod containing spectrin repeats MSP-300 in Drosophila is required for embryonic muscle and a highly conserved C-terminal type II transmembrane morphogenesis and the formation of embryonic muscle domain (KASH domain) (Starr and Han, 2002), which anchors attachments (Rosenberg-Hasson et al., 1996). Journal of Cell Science 3420 Journal of Cell Science 118 (15) Genetic studies in C. elegans have recently identified two tmNesprin-1 (amino acids 8611-8749; AAN03486) and GFP- dnNesprin-1 (aa 8369-8749; AAN03486) were cloned into inner nuclear membrane proteins, SUN1 and UNC-84, as EcoRI/SalI-cut EGFPC2 (Clontech) vector. GFP-tmNesprin-2 (aa structural links between the nucleoplasm and the cytoplasm. 6833-6883; AAL33548) was cloned into the EcoRI/BamHI site of SUN1/Matefin (Fridkin et al., 2004) and UNC-84 are EGFPC1 vector (Clontech). The full-length GFP-Sun1 (aa 1-913) was characterised by the presence of the SUN domain, which cloned into the EcoRI/BamHI site of EGFPC2. GFP-Sun1ΔSUN (aa derived its name from proteins containing the Sad1p-UNC-84 1-720), GFP-Sun1N+1TM (aa 1-384) and GFP-Sun1-TM-C (aa 358- homology domain (Hagan et al., 1995; Malone et al., 1999). 913) were cloned into the EcoRI/SalI site of EGFPC2 vector. GFP- SUN1 is essential for early embryonic and germ cell Sun1-N+2TM (aa 1-412), GFP-Sun1-TM-SD1,2 (aa 358-737), GFP- development (Fridkin et al., 2004) and required for the Sun1-TM-CΔSD2SUN (aa 358-632) and GFP-Sun1-CΔCC (358-491, localisation of ZYG-12 to the NE. ZYG-12 functions in 633-913) were cloned in EGFPC2 vector between the EcoRI and centrosomal attachment and the nuclear localisation of dynein BamHI sites. The KIAA0810 clone containing full-length human Sun1 was kindly provided by the KAZUSA research institute (Kikuno (Malone et al., 2003). In contrast to UNC-84 (Lee et al., 2002), et al., 2004). The Gateway -compatible destination vector pTB-RFB- germ-line-specific SUN1 does not require Ce-lamin for its EcoRV was created by insertion of the Gateway-transfer cassette into nuclear localisation (Fridkin et al., 2004). UNC-84 is required the EcoRV-site of pTRACER-EF1-Bsd (Invitrogen). Full-length Sun1 for proper nuclear migration and nuclear anchorage during was amplified from the KIAA-vector by PCR. The amplicon was worm development (Lee et al., 2002) and null alleles or cloned into the pTOPO-D/Entry-vector (Invitrogen) to create a missense mutations in UNC-84 also affect the nuclear envelope Gateway -compatible Sun1 entry vector. The expression vector targeting of ANC-1 (Starr and Han, 2002). In mammals, two expressing human KIAA0810-Sun1 harbouring C-terminal V5, was SUN domain-containing proteins have been described, created by LR-Clonase II-reaction using the Sun1 entry vector, pTB- although at least four unrelated members of the SUN domain RFB-EcoRV and the LR-enzyme mix according to manufacturer’s family are present in the databases (J.G. and R.F., unpublished instructions (Invitrogen). GFP-B1Δ2+ was a generous gift of Chris Hutchison (University of Durham, UK). The luminal domain of data) (Malone et al., 2003). The mammalian Sun1 protein is Nesprin-1 (LDN-1 residues 109-138, BE917568) was amplified from the real UNC-84 orthologue and was identified as an NE embryonic stem cell genomic DNA and cloned between EcoRI and protein in a proteomic analysis of neuronal nuclear membranes SalI sites in pGBKT7 (Clontech) and into pGEX4T-1 (Pharmacia) (Dreger et al., 2001). Sun2 is transcribed from a separate gene vectors. The luminal domain of Nesprin-2 (LDN-2, aa 6854-6883; and shows all properties of an inner nuclear membrane protein, AAL33548) was cloned into EcoRI and SalI sites of pGEX-4T-1. The with the SUN domain extending into the periplasmic space C-terminus of Sun1 (Sun1-C, aa 432-913), Sun1-CΔSUN (aa 432- between the inner and outer nuclear membranes (Hodzic et al., 737), Sun1-SUN (aa 738-913) was cloned into EcoRI/BamHI- 2004). Both Sun1 and Sun2 have also been identified in a digested pGADT7 and pGBKT7 vectors (Clontech). Sun1-SD1 (aa comprehensive proteomic screen of inner nuclear membrane 431-633) and Sun1-SD2 (aa 632-737) were cloned into proteins in rat liver (Schirmer et al., 2003). EcoRI/BamHI-cut pGADT7. In this report, we identify and analyse the direct interaction between the mammalian inner nuclear membrane protein Sun1 Yeast two-hybrid assay and Nesprins, giant NE-cytoskeletal linker proteins. Our Y190 yeast cells were co-transformed with the plasmids and findings provide novel insights into the NE anchorage transformants were selected on SD-Trp-Leu plates. Interaction was mechanism of Nesprins, demonstrating an evolutionarily monitored by growth on plates containing selection media SD-Trp- conserved strategy from worms to mammals. Leu-His and 60 mM 3-amino 1,2,4-triazole. X-Gal assay was also performed to confirm the interaction. The protocols for performing the yeast transformation and X-gal assay are described in detail Materials and Methods elsewhere (Yeast Protocols Handbook PT3024-1; Clontech). Tissue culture and transfection COS7 and C3H/10T1/2 cells were grown as described (Padmakumar Purification of GST fusion proteins and in vitro binding assays et al., 2004). HaCaT, HeLa cells and skin fibroblasts from wild-type and lamin A knockout mice (Sullivan et al., 1999) were routinely Purification of GST fusion proteins and GST pull-down experiments cultivated in DMEM supplemented with 10% fetal calf serum (Gibco were performed as described (Dreuillet et al., 2002). For the pull- Life Technologies) at 37°C in a humidified atmosphere containing 5% down assay, COS7 cells were lysed with 50 mM Tris-HCl, pH 7.5, CO . 150 mM NaCl, 1% Triton X-100 and protease inhibitors (Roche). The All the GFP fusion constructs were transfected in COS7 and 100,000 g supernatant of the lysate was incubated with equal amounts C3H/10T1/2 cell lines by electroporation (200V, 950 μF; Gene Pulser of GST fusions and the solutions were incubated at 4°C overnight with Xcell, Bio-Rad). HaCaT cells were transfected by using the Amaxa GST-sepharose beads on a roller. Samples were centrifuged and the nucleofector technology according to the manufacturer’s instructions pellets (washed three times with PBS) were analysed by SDS-PAGE (Amaxa Biosystems). HeLa cells were transfected using and western blot analysis. Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Antibodies and immunofluorescence microscopy Sun1 polyclonal antibodies were produced against a peptide Cloning strategies containing the first 14 amino acids of human Sun1 All the GFP fusion proteins carry GFP at their N-terminus. The (MDFSRLHMYSPPQC; NP_079430) to create antiserum 281 appropriate DNA fragments were amplified by PCR and cloned in (Eurogentec, Seraing, Belgium). either pGEMTeasy (Promega) or pCR-2.1-TOPO (Invitrogen) vectors Western blotting and immunofluorescence (IF) studies were before cloning them in the GFP, yeast two-hybrid or GST vectors. performed as described (Zhen et al., 2002; Gotzmann et al., 2000). Nesprin-1 constructs were amplified from BC054456 IMAGE clone The following antibodies were used: affinity-purified rabbit and Sun1 was amplified from IMAGE clone AAH48156. GFP- polyclonal anti-Nesprin-1/Enaptin (1:50 for IF), mouse monoclonal Journal of Cell Science Sun1 interacts with Nesprins 3421 anti-Nesprin-2/NUANCE mAb K20-478 (Zhen et al., 2002), transmembrane domain followed by a stretch of 30 amino acids unpurified rabbit polyclonal hSun1 281 (1:50 IF; 1:400 western (luminal domain) extending into the perinuclear space, which is blotting), rabbit polyclonal Nesprin-2 pAbK1 (1:50 IF, 1:1000 highly conserved in this family of nuclear proteins (Fig. 1A). In western blotting), mouse monoclonal anti-lamin A/C (1:50 IF; JoL2, order to identify the sequences necessary for the NE localisation Chemicon), mouse monoclonal anti-V5 (1:500 IF; 1:3000 western of Nesprins and elucidate the mechanism by which they are blotting; Invitrogen), polyclonal serum to LAP2α (1:5000; tethered at the NE, several GFP fusion proteins of their C-termini ImmuQuest) (Vlcek et al., 2002), guinea pig anti-LBR was a kind gift (Fig. 1B) were transiently expressed in various cell lines. of Harald Hermann (1:250 IF) (Dreger et al., 2002), mouse TmNesprin-1, representing the transmembrane domain and monoclonal anti-LAP2β (1:5 IF) (Dechat et al., 1998) goat polyclonal the periplasmic region of the protein localised to the NE in anti-GST antibody (Amersham Biosciences), GFP-specific mAb K3- C3H/10T1/2 cells (Fig. 1C,E, arrow) and exerted a dominant- 184-2 (Noegel et al., 2004). The secondary antibodies used were conjugated with Cy3 (Sigma), Alexa 488 or Alexa 568 (Molecular negative effect on the endogenous Nesprin-1 protein. Probes, Leiden, The Netherlands), Texas Red (Jackson Laboratories, Untransfected fibroblasts displayed strong Nesprin-1 staining West-Grove, PA) and FITC (Sigma). Samples were analysed by wide- at the NE (Fig. 1D,E, arrowhead), which disappeared in field fluorescence microscopy (DMR, Leica, Bensheim, Germany) or transfected cells (Fig. 1D,E, arrows). A similar effect was confocal laser-scanning microscopy (TCS-SP, Leica). observed when COS7 cells expressed the corresponding tmNesprin-2 GFP fusion protein. Like tmNesprin-1, tmNesprin-2 localised to the NE (Fig. 1F, arrow) and displaced Subcellular fractionation the endogenous Nesprin-2 (Fig. 1G,H, arrowheads) from the Subcellular fractionation was done essentially as described (Dechat et NE (Fig. 1G, arrow). As tmNesprin-2 did not contain regions al., 1998; Gotzmann et al., 2000). In brief, cells were broken in upstream of the transmembrane domain as found in tmNesprin- hypotonic buffer using a Dounce homogeniser with a tight-fitting pestle. After addition of 8% sucrose, the soluble cytoplasmic and the 1 (Fig. 1B), we concluded that the transmembrane domain and insoluble nuclear fractions were separated by centrifugation at 2000 the luminal domains are sufficient to mediate NE targeting. The g for 15 minutes at 4°C. The nuclei-containing pellets were extracted high homology between the C-termini of Nesprin-1 and in the same buffer supplemented with 1% Triton X-100, or 200 mM Nesprin-2 further prompted us to investigate the effects of a NaCl or combinations of both. Another extraction was performed dominant-negative Nesprin-1 GFP fragment on endogenous using hypotonic buffer containing 7 M urea. The extracts were Nesprin-2. In these experiments we used the dnNesprin-1 GFP centrifuged at 15,000 g for 10 minutes and supernatants and pellets fusion (~75 kDa), which also harbours the two final spectrin were analysed by western blotting. repeats upstream to the transmembrane domain, thus resembling Nesprin-2α in its domain organisation (Zhang et siRNA knockdown of Sun1 al., 2001). The studies were performed in COS7 cells, which The RNA interference-competent pSHAG-1 vector (containing express Nesprin-2 strongly at the NE (Zhen et al., 2002). a human U6 promoter fragment; –265 to +1), constructed Similar to tmNesprin-1, dnNesprin-1 localised to the NE in using a pTOPO-ENTR/D backbone (Invitrogen, Carlsbad, USA), has COS7 cells (Fig. 1I,K, arrow) and displaced the endogenous been described recently (Paddison et al., 2002; http://katahdin.cshl. Nesprin-2 (Fig. 1J, arrow). Experiments involving shorter org:9331/RNAi_web/scripts/main2.pl). Oligonucleotides A and B, versions of dnNesprin-1, such as tmNesprin-1 fusions, encoding a short hairpin RNA, were designed according to the RNAi- exhibited similar dominant-negative effects on Nesprin-2 (data retriever protocol at http://katahdin.cshl.org:9331/homepage/portal/ not shown). To further study the potential significance of the html/protocols/. For knocking down specifically human Sun1 the highly conserved C-terminal proline residues (Fig. 1A, green following oligonucleotides were used to create vectors pJG173 and bar) for the NE localisation of Nesprin-2, a GFP fusion protein pJG174, respectively: Oligo 173A, 5′-CTCGGACAGCATGCTGC- tmNesprin-2ΔP lacking the PPPT-motif was generated. In AGTTGCTGCAGGAAGCTTGCTGCGGCGACTGCGGCATGTTG- TCCGAGCGCTTTTTT-3′; Oligo 173B, 5′-GATCAAAAAAGCG- contrast to tmNesprin-2 (Fig. 1F, arrow), this protein CTCGGACAACATGCCGCAGTCGCCGCAGCAAGCTTCCTGCA- accumulated in the nuclear interior (Fig. 1L, arrowhead) and GCAACTGCAGCATGCTGTCCGAGCG-3′; Oligo 174A, 5′-AG- in ER-like structures (Fig. 1L, arrows) and did not affect the GACGTGACCTGCCTTGACACGTGGTTGAAGCTTGAGCCGC- location of endogenous Nesprin-2 (Fig. 1M, arrowhead). GTGTTAAGGCAGGTCACGTTCTCTGTTTTTT-3′; Oligo 174B, Taken together, these experiments underline the functional 5′-GATCAAAAAACAGAGAACGTGACCTGCCTTAACACGCG- significance of the conserved proline-rich stretch of Nesprins GCTCAAGCTTCAACCACGTGTCAAGGCAGGTCACGTCCTCG- for NE targeting. Moreover, the C-terminal domains act in a 3′. Annealed oligonucleotides were cloned into pSHAG-1 by insertion dominant-negative manner on the endogenous Nesprin of resulting overhangs into the BseRI/BamHI-sites. The control vector proteins, displacing them from the NE. These findings also pSHAG-FF1 encoding an shRNA targeting Firefly Luciferase was a imply that the associations of the luminal domain of Nesprin- kind gift of Greg Hannon (Cold Spring Harbor Laboratory, NY). The integrity of all vectors was verified by sequencing from both ends. 1 and Nesprin-2 in the perinuclear space involve identical RNAi-vectors targeting hSun1 were always used in combination for binding partners conferring anchorage at the NE. transient knockdown experiments. The cells were fixed 4 days after the transfection and examined by indirect immunofluorescence. Sun1 binds to the luminal domain of both Nesprin-1 and Nesprin-2 Results A genetic interaction between ANC-1 and UNC-84 has been Common mechanisms tether Nesprin-1 and Nesprin-2 reported in C. elegans, although efforts to elucidate the direct at the NE molecular interaction failed (Starr and Han, 2002). A search of Nesprin-1 and Nesprin-2 localise to the NE by virtue of their the mouse EST database for mammalian UNC-84 orthologues conserved C-terminal domain, which includes a type II as potential Nesprin binding partners yielded two SUN Journal of Cell Science 3422 Journal of Cell Science 118 (15) domain-containing proteins, Sun1 (accession number kDa protein composed of 913 amino acids (Fig. 2A). It AAH48156, mouse chromosomal locus 5G.2) and Sun2 contains three putative transmembrane domains (aa 358-383, (AAT90499, residing on chromosome 15). Mouse Sun1 and 386-407 and 413-431) located approximately in the middle of Sun2 display 65% identity and 81% homology in their SUN the protein, a predicted ZnF-C2H2 domain near the N- domain and 47%/39% identity and 63%/59% similarity to terminus, and two predicted coiled-coil domains in the C- the SUN domain of UNC-84, respectively. As Sun1 is more terminus (aa 492-527 and 563-632). The last 175 residues are closely related to UNC-84 than Sun2, Sun1 was chosen for the highly homologous to C. elegans UNC-84 and S. pombe Sad1 current study. In addition, we used human Sun1, the closest forming the evolutionarily conserved SUN domain. The human orthologue to UNC-84, displaying 48% identity and domain structure of human Sun1 is identical to that of mouse, 64% similarity to the SUN domain of UNC-84. Human Sun1 except that it lacks the proposed zinc-finger motif. The region was originally identified as KIAA0810 by the Kazusa between the transmembrane and the SUN domain was divided DNA research institute (Kikuno et al., 2004; into subdomains SD1 and SD2 for functional tests. SD1 http://www.kazusa.or.jp/huge/). Mouse Sun1 encodes a 100 contains the two coiled-coil regions whereas SD2 does not display any known structural features (Fig. 2A). In yeast two-hybrid assays we investigated the possible interaction between the luminal domain of Nesprin- 1 and mouse Sun1. The last 30 amino acids (luminal domain) of mouse Nesprin-1 were fused in-frame to the binding domain of Gal4 and were tested for an interaction with five different C- terminal Sun1 fusion constructs with the activating domain of Gal4. These included Sun1-C (C-terminus of Sun1), Sun1-CΔSUN, which lacks the SUN domain, Sun1-SUN composed solely of the SUN domain, Sun1-SD1 and Sun1- SD2 (Fig. 2B). Co-transformation of the Fig. 1. The C-termini of Nesprins are conserved and sufficient for NE localisation. (A) Alignment (using MultiAlign) of the 30 amino acid luminal domains of various KASH-domain NE proteins. The green bar denotes the highly conserved C- terminal prolines. (B) The tmNesprin-1, tmNesprin- 2, tmNesprin-2ΔP (lacks the last four aa) and dnNesprin-1 GFP fusion constructs used for the experiments shown in C-N. LD, luminal domain; SR, spectrin repeats; TM, transmembrane domain. (C-K) Dominant-negative effect of tmNesprin-1, tmNesprin-2 and dnNesprin-1 GFP fusions on the endogenous Nesprin proteins. Transiently transfected cells were fixed and subjected to immunofluorescence using the monoclonal K20- 478 anti-Nesprin-2 and a rabbit polyclonal Nesprin- 1 antibody. These antibodies did not recognise epitopes on the ectopically expressed polypeptides. Note the nuclear rim staining of endogenous Nesprin proteins in untransfected cells (arrowheads in E,H,K) and the absence of Nesprin staining in GFP-positive cells (arrows in E,H,K). (L-N) Confocal images demonstrate a cytoplasmic (panel L, arrows) and a diffuse nuclear staining pattern (panel L, arrowhead) for GFP-Nesprin-2ΔP, which does not affect endogenous Nesprin-2 at the nuclear envelope (arrowhead in M). The cell lines used are indicated in the lower right-hand corner of the first column of frames (C,F,I). DNA was stained with DAPI. Images were obtained by confocal laser- scanning microscopy. Bars, 10 μm. Journal of Cell Science Sun1 interacts with Nesprins 3423 plasmids into Y190 yeast cells followed by β-galactosidase tmNesprin-2ΔP was unable to displace Nesprin-2 from the NE assays revealed an interaction between Nesprin-1 and Sun1-C, (Fig. 1L-N). whereas controls remained negative. Further experiments identified the SD2 of Sun1 (residues 632-737) as the primary Sun1 is a component of the nuclear envelope and the Nesprin-1 binding site, whereas the SUN domain itself showed inner nuclear membrane only a weak binding to Nesprin-1 in this assay. No interaction was found with SD1 (Fig. 2B). To determine whether mouse and human Sun1 are inner These observations were further supported by biochemical nuclear membrane proteins like UNC-84 in C. elegans, we assays where we used GST fusion proteins containing the performed a series of analyses using the polyclonal Sun1 luminal domain of Nesprin-1 and Nesprin-2 (yielding GST- antiserum 281 generated against a peptide derived from the N- LDN-1 and -2 respectively) to pull down the GFP-Sun1-C terminus of human Sun1. Western blot analysis of HaCaT cell fusion protein, which lacks the transmembrane domains (GFP- lysates using the unpurified Sun1 serum, detected a major 100 Sun1-C, aa 432-913). Both GST fusion proteins were able to kDa band (Fig. 3A). Furthermore, the Sun1 antibody detected precipitate GFP-Sun1-C from COS7 cell lysates (Fig. 2D,E). ectopically expressed full-length human Sun1 protein in cell A Nesprin-2 luminal domain deletion GST construct (GST- lysates, which could be efficiently competed by increasing LDN-2ΔP) lacking the last four highly conserved amino acids amounts of the peptide antigen (data not shown). To examine (PPPT) (Fig. 2C) was generated and used for precipitation whether Sun1 interacts with Nesprin-2 in vivo, we performed assays from COS7 lysates containing GFP-Sun1-C. In contrast immunoprecipitation studies. When the Nesprin-2 to GST-LDN-1 and GST-LDN-2, no interaction with GFP- immunocomplexes (Fig. 3B, lane 4) were resolved by SDS- Sun1-C was observed for the GST-LDN-2ΔP fusion protein PAGE and subjected to silver staining we observed a faint 100 (Fig. 2E), which is consistent with the observation, that kDa band (Fig. 3B, lane 4, arrowhead), which was specifically recognised by the anti-Sun1 antibody (Fig. 3B, lane 4, right panel). These results indicate that Sun1 interacts with Nesprin-2 in vivo. The Sun1 antiserum preferentially stained the NE where it colocalised with Nesprin-2 in HaCaT cells (Fig. 3C-E). In addition, the antibodies produced cytoplasmic background staining resulting most probably because we used Sun1 antiserum, which was not affinity- purified. The background staining also persisted after specific Sun1 knockdown (Fig. 7D,G and J) and therefore does not mirror a natural localisation for the endogenous Sun1 protein. Furthermore stably expressed V5 epitope tagged human Sun1 colocalised with Lamin B receptor (LBR), a well- characterised inner nuclear membrane protein, in a number of cell lines, including HeLa (Fig. 3F-H), human Hek293, COS7 and SW-480 cells, murine NIH-3T3 fibroblasts and canine epithelial MDCK-cells (data not shown). To test the association of human Sun1 with the NE at the biochemical level, Fig. 2. The C-terminus of Sun1 associates directly with the luminal domains of Nesprin-1 nuclei of HeLa cells stably expressing and Nesprin-2. (A) Domain organisation of mouse Sun1. The domain locations as well as Sun1 were extracted in buffers containing their amino acid positions are indicated according to the GenBank entry AAH48156. CC, urea and non-ionic detergents. The coiled-coil domain; ZnF, zinc-finger domain; Tm, transmembrane domain. (B) Sun1 polypeptides corresponding to various Sun1 domains were fused to the Gal4 activating distribution of human Sun1 in soluble (S) domain, whereas the Nesprin-1 luminal domain was fused to the Gal4 DNA-binding and insoluble (P) fractions was analysed domain. The corresponding plasmids were co-transformed into yeast cells and the by immunoblot analysis (Fig. 3I). The interactions were assessed by the filter lift β-galactosidase assay. ++++, strong; ++, weak; distribution of the well-characterised –, no blue colour development. (C) Schematic overview of the fusion proteins (GST-LDN- inner nuclear membrane protein LAP2β 1, GST-LDN-2 and GST-LDN-2ΔP lacking the last 4 aa) used for the GST pull-down assay served as a control. Sun1 and LAP2β of COS7 cell homogenates expressing GFP-Sun1-C. LDN-1, luminal domain Nesprin-1; displayed similar properties, being LDN-2, luminal domain Nesprin-2. (D,E) COS7 cell lysates expressing the C-terminal half completely resistant to extraction with of Sun1 (Sun1-C) were incubated with the immobilised GST-fusion proteins as indicated high salt, chaotropic agents and detergent and GST for control. Unbound (S) and specifically bound (P) proteins were subjected to at low salt concentration (Fig. 3I). Only SDS-PAGE followed by western blot analysis using GFP-specific mAb K3-184-2. Journal of Cell Science 3424 Journal of Cell Science 118 (15) Fig. 3. Sun1 behaves like an integral inner nuclear membrane protein. (A) Western blotting analysis of HaCaT cell lysates using polyclonal Sun1-specific antibodies detects a major 100 kDa band. (B) Endogenous Sun1 protein co-immunoprecipitates with Nesprin- 2. Immunocomplexes obtained from HaCaT cells with anti-Nesprin- 2 (pAbK1) antibodies were analysed by SDS-PAGE and subjected to silver staining (left panel) or immunoblotting with anti-Nesprin-2 (mAb K20-478) and anti-Sun1 antibody (right panel). The major 800, ~400 and 75 kDa Nesprin-2 isoforms present in HaCaT cells are indicated by arrows (right panel). Lane 1, input lysate; lane 2, control precipitate with Protein A sepharose beads; lane 3, mock-IP control IgG antibody; lane 4, co-immunoprecipitate with anti-Nesprin-2 antibody pAb-K1. The bands observed in lane 4 represent signals obtained after short exposure whereas lanes 1-3 were obtained after prolonged ECL detection (30 minutes). Positions of molecular mass markers in kDa are shown on the left-hand side of the blots. (C-E) HaCaT cells were subjected to immunofluorescence using Sun1 (281) and Nesprin-2 antibodies (mAb K20-478), demonstrating the colocalisation of Sun1 with Nesprin-2 at the NE (E). The inset is a higher magnification of the dotted white box. (F-H) Ectopically expressed full-length human Sun1 (C-terminal V5-tag) is targeted to the nuclear envelope in HeLa cells, displaying strict colocalisation with the Lamin B receptor (LBR). Images were obtained using a confocal microscope. (I) Solubilisation properties of human Sun1 under various extraction conditions. Purified nuclei (Nuc) of HeLa cells, stably expressing V5-tagged human Sun1, were extracted in RIPA buffer containing urea, Triton X-100, salt or combinations thereof, as indicated. Soluble (S) and insoluble (P) fractions were analysed by western blotting. Cytosol (Cyt) served as a purity control. The same lysates were analysed for LAP2β, a known integral inner nuclear membrane protein. Bars, 7 μm. permeabilisation assays suggested that Nesprin-2 is integrated into the outer nuclear membrane (Zhen et al., 2002). To investigate whether the highly homologous Nesprin-1 shares a similar localisation we analysed C3H/10T1/2 fibroblasts treated with Triton X-100 and digitonin. Unlike lamin A, Nesprin-1 staining could still be detected at the NE after treatment with detergent and medium salt concentrations (1% selective digitonin permeabilisation indicating its presence at Triton X-100/200 mM NaCl) efficiently solubilised Sun1 and the outer nuclear membrane (Fig. 4J-L). However our findings LAP2β. In accordance with these data KIAA0810 has been do not exclude the possibility that Nesprin-1 also localises to identified as a component of both the detergent- and chaotrope- the inner nuclear membrane. Collectively our findings suggest resistant fractions in a proteomics screen (Dreger et al., 2001). an asymmetric distribution at the nuclear membrane of the More recently, Sun1 was exclusively detected in the salt- and interaction partners Sun1 and Nesprins. sodium hydroxide-resistant fractions of a novel protocol to isolate unknown NE constituents (Schirmer et al., 2003). The N- and C-termini of Sun1 localise independently to In order to define the topology of Sun1 at the NE we the NE performed digitonin permeabilisation of cells, which selectively disrupts the plasma membrane leaving the NE The current sorting mechanism, which defines the localisation membranes intact (Adam et al., 1990), whereas Triton X-100 of NE transmembrane proteins during interphase is the permeabilises all membranes. Antibodies to both Sun1 and ‘diffusion-retention’ model (Worman and Courvalin, 2000). lamin A/C stained the NE in Triton X-100-permeabilised According to this model NE proteins are co-translationally COS7 cells (Fig. 4A-C, arrows). In addition, the antiserum integrated into the ER followed by a lateral diffusion from the strongly stained the nucleoplasm (Fig. 4A), whereas in HaCaT ER to the outer and inner nuclear membranes, interconnected cells the staining was preferentially found at the NE (Fig. 3C). by the nuclear pore complex. Proteins are then retained at the In digitonin-treated COS7 cells Sun1 and lamin A/C remain inner nuclear membrane owing to the presence of nuclear undetectable using these antibodies at the NE. Only the retention sequences allowing binding to nuclear proteins, cytoplasmic staining of Sun1 antiserum 281 was still observed chromatin or both. (Fig. 4D and F, arrowheads). Identical results were obtained To investigate the subcellular localisation and to determine the for ectopically expressed human as well as mouse Sun1 (data NE retention domains of mouse Sun1 we transiently transfected not shown). Altogether, these data strongly suggest that Sun1 various Sun1-GFP fusion proteins (Fig. 5A) into COS7 cells. is an integral inner nuclear membrane protein. The expression and the appropriate molecular masses of the Earlier studies on Nesprin-2 using digitonin fusion proteins were confirmed by western blotting (data not Journal of Cell Science Sun1 interacts with Nesprins 3425 fusion protein remained in the ER (Fig. 5F, arrowheads). Although more than 75% of the Sun1- N+1TM and Sun1-N+2TM fusions localised to the NE, only 58% of Sun1-TM-C displayed NE localisation. The accumulation of the proteins in the ER may reflect either the absence of important domains or result from the overexpression of improperly folded proteins. In summary, both the N- and C-termini of Sun1 localise independently to the NE, which is most likely facilitated by binding to different proteins in the nucleoplasm and the perinuclear space. Sun1 associates with itself in vivo and the two coiled-coil domains are sufficient to target the C-terminus of Sun1 to the NE In order to study the C-terminal Sun1 nuclear targeting sequences in more detail, three additional GFP constructs (Sun1-TM-SD1,2; Sun1-TM- CΔSD2SUN; Sun1-TM-CΔCC; Fig. 5A) were expressed in COS7 cells (Fig. 5G-I). The GFP fusion protein Sun1-TM-SD1,2 comprising SD1, SD2 and the three transmembrane domains displayed a NE localisation in 64% of transfected cells (Fig. 5G, arrow and Fig. 5J). As this polypeptide lacks the SUN domain, the SUN domain seems dispensable and not required to confer the NE localisation, as shown above for the GFP-ΔSUN mutant. To investigate whether the Nesprin binding domain SD2 in Sun1 is involved in the NE targeting of Sun1, we constructed a GFP fusion construct of the three transmembrane domains followed by SD1, composed of the two coiled-coil domains, but lacking SD2 (Sun1-TM- CΔSD2SUN). In 73% of transiently transfected Fig. 4. Asymmetric distribution of Sun1 and Nesprin-1 at the nuclear membrane. COS7 cells we observed a clear NE association of (A-C) Triton X-100 treated COS7 cells subjected to immunofluorescence with Sun1 and lamin A/C antibodies, indicate the nuclear localisation of Sun1 the fusion protein (Fig. 5H, arrow and Fig. 5J), (arrows). Non-specific staining of antibody 281 was observed in the cytoplasm suggesting therefore the presence of a nuclear (arrowheads; see also Fig. 3C). (D-F) In digitonin-treated COS7 cells only the retention signal within SD1 of Sun1. Coiled-coils non-specific staining remains (arrowheads) suggesting a localisation of Sun1 at have traditionally been recognised as an the inner nuclear membrane. The integrity of the nuclear membrane is oligomerisation unit in a large number of proteins documented by the absence of lamin A/C staining (E). (G-I) In Triton X-100- (Burkhard et al., 2001). As SD1 is composed of two permeabilised fibroblasts Nesprin-1 antibodies strongly stain the nucleus. coiled-coil regions (CC1 and CC2, see Fig. 2A), it (J-L) Nesprin-1 staining at the NE persists after digitonin treatment suggesting is possible that oligomerisation of the GFP-fusion the presence of Nesprin-1 at the outer nuclear membrane. Note the absence of protein with the endogenous Sun1 leads to retention lamin A/C staining (K). DAPI was used to counterstain the nucleus. Confocal at the nuclear envelope. This is supported by data images are shown. Bars, 5 μm. from yeast two-hybrid experiments, which indicated an interaction of SD1 with itself (data not shown). To test the hypothesis that the NE localisation of N-terminally shown). Similar to its human orthologue (Fig. 3F) the full-length truncated Sun1 polypeptides is mediated by the coiled-coil mouse Sun1 protein also localised to the NE (Fig. 5B). Deletion region, we generated GFP-Sun-TM-CΔCC removing the two of the SUN domain (GFP-ΔSUN) did not affect targeting to the coiled-coil regions between amino acids 491-633, leaving the NE (Fig. 5C, arrow) showing that the SUN domain is not SUN as well as the transmembrane domains intact. Only 33% required for retention at the NE. Accumulation at the NE was of transfected cells (Fig. 5J) displayed a NE localisation (Fig. also obtained with GFP fusion proteins containing the entire 5I, arrows). In the majority of cases we observed substantial Sun1 N-terminus with a single (Sun1-N+1TM) or two accumulation of the chimeric protein in the ER (arrowhead). transmembrane domains (Sun1-N+2TM) (Fig. 5D,E, arrows). Our data suggest that the coiled-coil region of Sun1 is important Surprisingly, the C-terminus containing the three transmembrane to mediate NE association, however the ability of the Sun1-TM- domains (Sun1-TM-C) also localised to the NE in COS7 cells CΔCC fusion protein to localise to the NE is indicative of the (Fig. 5F, arrows; Fig. 6A, arrow). Thus, the C-terminus of Sun1 existence of additional nuclear retention signal(s) in the C- is sufficient to confer NE targeting, however, much of the GFP Journal of Cell Science 3426 Journal of Cell Science 118 (15) Fig. 5. Sun1 contains multiple, independent nuclear targeting signals. (A) Schematic representation of Sun1 GFP fusion constructs. Domain labelling is as in Fig. 2A. (B-I) Subcellular localisation of GFP Sun1 fusion proteins in COS7 cells observed by direct fluorescence confocal microscopy. Arrows indicate NE localisation whereas arrowheads indicate ER localisation. DAPI was used to counter-stain the nuclei. (J) Histogram representing a statistical evaluation (percentage of transfected cells) of the localisation profiles of the various SUN1-GFP fusions to the ER and the NE. Bars, 10 μm. Sun1 antiserum in COS7 cells transiently expressing Sun1-TM-C (which is not detected by the antiserum) revealed a displacement of endogenous Sun1 from the NE (Fig. 6B-C, arrow). Untransfected cells, however, displayed a proper NE localisation of Sun1 (Fig. 6B-C, arrowhead). Out of 200 transfected cells 82% showed significantly reduced NE staining of Sun1 (Fig. 6G). As Nesprin-2 associates with Sun1 we tested its localisation in Sun1-TM-C transfected cells as well (Fig. 6D-F). Similar to the endogenous Sun1, Nesprin-2 was displaced from the NE (Fig. 6E-F, arrow) in transfected cells, whereas in untransfected cells Nesprin-2 was properly localised at the NE (Fig. 6D-F, arrowhead). In a statistical analysis of 200 transfected cells Nesprin-2 was no longer found at the NE in 97% of cells (Fig. 6G). To further support the requirement of Sun1 for the localisation of Nesprin-2, we performed knockdown studies employing a mixture of two independently expressed siRNAs targeting the N-terminus of Sun1. Transient transfections of the RNAi- competent plasmids pJG173 and pJG174 in HeLa cells, which stably express V5-tagged hSun1 verified the efficacy of the Sun1 knockdown (Fig. 7A-C). Many transfected cells displayed a reduced anti-V5 hSun1 staining (asterisks), whereas the lamin A/C pattern appeared unaffected (Fig. 7B). Similar observations were made in transiently transfected Sun1 knockdown HaCAT cells (Fig. 7D-F). In keratinocytes where the nuclear Sun1 staining was absent (Fig. 7D; asterisks), lamin A/C staining was still observed at the nuclear envelope (Fig. terminus of Sun1. Furthermore we cannot exclude the 7E; asterisks), suggesting therefore that Sun1 is not essential possibility that the Sun1 transmembrane domains themselves for lamin A/C localisation. In sharp contrast, however, in Sun1 contain sorting signals and determine the NE localisation in a knockdown cells (Fig. 7G-L) the Nesprin-2 staining pattern similar fashion to the lamin B receptor (Wozniak and Blobel, was either very faint or absent using both N-terminally (Fig. 1992; Smith and Blobel, 1993). The presence of multiple and 7H; asterisks) as well as C-terminally (Fig. 7K; asterisks) independent nuclear retention signals across Sun1 is further directed antibodies. In summary our data indicate that the supported by the fact that none of the fusion proteins localised proper localisation of Nesprin-2 at the NE requires Sun1. as efficiently to the NE as the full-length Sun1 (Fig. 5J). However, its interaction with Nesprins is apparently not Targeting of Sun1 to the NE is independent of a involved in retaining Sun 1 at the NE. functional lamin A/C network In C. elegans UNC-84 localises to the NE in a lamin-dependent Sun1 affects the NE localisation of Nesprin-2 manner (Lee et al., 2002). To explore whether the UNC-84 Immunofluorescence analyses of endogenous Sun1 using the orthologue Sun1 also depends on the lamin network we Journal of Cell Science Sun1 interacts with Nesprins 3427 Fig. 6. GFP-Sun1-TM-C acts in a dominant-negative manner on endogenous Sun1 and Nesprin-2. COS7 cells expressing GFP-Sun1- TM-C were stained using specific antibodies to Sun1 (B,C) and Nesprin-2 (E,F). (A-F) Confocal images illustrating that GFP-Sun1- TM-C (transfected cells are indicated by arrows) interferes with the localisation of endogenous Sun1 (B and C, arrows) and Nesprin-2 (E and F, arrows). Note the differences in the Sun1 and Nesprin-2 Fig. 7. Nesprin-2 localisation is affected in cells where Sun1 staining pattern in transfected (arrows) versus untransfected expression has been silenced by siRNA. (A-C) HeLa cells expressing (arrowheads) cells. (G) Histogram illustrating the displacement stably V5-tagged hSun1 and HaCaT cells (D-L) were transiently effects of the GFP-Sun1-TM-C fusion on the endogenous Sun1 and transfected with a combination of plasmids (pJG173/174) encoding Nesprin-2 proteins. Data were obtained by evaluating 200 transfected siRNAs targeting hSun1. The distribution of Sun1 (panel A, anti-V5; cells. Bars, 7 μm. panels D,G,J, anti-Sun1 281 serum), lamin A/C (panels B and E) and Nesprin-2 (panel H, mAb K20-478; panel K, mAb K49-260) was investigated by indirect immunofluorescence in knockdown cells transfected lamin A/C knockout fibroblasts (Sullivan et al., (indicated by asterisks). In Sun1 knockdown cells, the lamin A/C 1999) with the mouse Sun1 and Sun1-N+2TM GFP fusion localisation remained unaltered (B and E), whereas Nesprin-2 staining was either absent or reduced. DNA was stained by DAPI. proteins (Fig. 8A-C). In both wild-type and knockout The images shown were taken by confocal microscopy and merged fibroblasts the fusion proteins localised properly to the NE. to visualise colocalisation (panels C,F,I,L). Bars, 10 μm. Identical results were obtained with human V5 epitope-tagged Sun1 in lamin A/C knockout fibroblasts (Fig. 8D). To further substantiate our findings, we transiently transfected HeLa cells stably expressing human Sun1 with the Xenopus mutant GFP- (Burke et al., 2001; Gotzmann and Foisner, 2004). B1Δ2+ (Fig. 8G-I). This fusion protein accumulates in Understanding the pathology of all these diseases requires the intranuclear aggregates (Fig. 8H) and recruits endogenous identification and functional characterisation of all nuclear lamins A/C, thus disturbing the functional organisation of the envelope constituents as a first step, as well as knowledge of lamin A/C network (Dechat et al., 2000; Vaughan et al., 2001). the networking interactions that take place at the NE. Towards Importantly, the expression of GFP-B1Δ2+ and loss of a this end, using both biochemical as well as cell biological data functional A-type lamin network did not affect the NE we unravel the first link between an inner nuclear membrane localisation of Sun1 (Fig. 8G-I). Altogether, lamin A/C is not protein (Sun1) and constituents of the outer and inner nuclear essential for the localisation of Sun1 at the NE. Based on membranes (Nesprins). UNC84 data, one could assume that B-type lamins may be required for retention of Sun1 at the inner nuclear membrane. Nesprins are targeted to the NE by binding to Sun1 through their conserved C-terminus Discussion In an effort to study the nuclear localisation mechanism of the Recent focus on NE composition and function has been giant actin binding Nesprin-1 and Nesprin-2 proteins, we primarily powered by the unexpected involvement of several demonstrated that the evolutionarily conserved C-terminus of NE components and associated proteins in human diseases Nesprins is sufficient to target the proteins to the NE. Journal of Cell Science 3428 Journal of Cell Science 118 (15) perinuclear space. In addition, the SUN domain may have different functions in C. elegans compared to higher eukaryotes. In fact, in C. elegans the SUN domain of UNC-84 is directly involved in the NE recruitment of UNC-83 a nuclear transmembrane protein, which is essential for proper nuclear migration (Starr et al., 2001; Lee et al., 2002). To date no orthologue of UNC-83 is known in higher eukaryotes. Irrespective of SUN domain function in various organisms, its conservation in evolution implies that Sun1 exhibits additional functions besides the tethering of Nesprins to the NE. Sun1 is an inner nuclear membrane protein By performing digitonin experiments we demonstrated the presence of Sun1 in the inner membrane of the NE. Our results are in accordance with a nuclear envelope proteomics approach, which identified Sun1 as an integral membrane protein of the inner nuclear membrane (Dreger et al., 2001). Similar findings were also obtained for its paralogue Sun2, also a type II transmembrane domain protein (Hodzic et al., 2004). Moreover, experiments involving proteinase K protection assays and digitonin experiments with Sun2 suggested the presence of the C-terminus in the perinuclear space whereas Fig. 8. Lamin A/C does not influence the NE localisation of Sun1. the N-terminus points to the nucleoplasm thus allowing an (A-C) Wild type (A) and lamin A/C knockout (B and C) mouse interaction with the nuclear lamina (Hodzic et al., 2004). dermal fibroblasts were transfected with the mouse GFP-Sun1 and Because of the overall similarity between Sun1 and Sun2 we GFP-Sun1-N+2TM fusion proteins. Transiently transfected cells assume a similar topology for the Sun1 full-length protein. were processed for direct fluorescence microscopy. Note that both This arrangement is required to allow the interaction of the GFP fusion proteins localise to the nuclear envelope in the absence –/– of lamin A/C (B and C). (D-F) Lamin A/C fibroblasts were SD2 domain with the luminal domain of Nesprins. transfected with plasmid encoding V5-tagged human Sun1 and processed for immunofluorescence using antibodies to V5 and the LAP2α. (G-I) HeLa cells stably expressing human Sun1 (V5-tagged) The NE localisation of Sun1 at the NE does not depend were transiently transfected with a plasmid coding for the dominant on lamin A/C negative GFP-lamin B1Δ2+ protein and stained for the V5 epitope. The proper localisation of full-length or the N-terminal half of Images were obtained by confocal microscopy. Bars, 6 μm. Sun1 in lamin A/C knockout fibroblasts demonstrated that lamin A/C is not required for the NE localisation of Sun1. Furthermore, we showed that the overexpression of C-terminal Moreover, disruption of a functional lamin A/C system had no Nesprin peptides caused a dominant-negative effect on the detectable effects on the proper localisation of Sun1. The C. distribution of endogenous Nesprins provoking their elegans UNC-84 requires the B-type Ce-lamin for its envelope displacement from the NE. These results not only suggest that localisation (Lee et al., 2002). Thus localisation of mammalian their NE retention mechanism may be the same, but in addition Sun1 at the NE may also depend on B-type lamins. In contrast, C-terminal isoforms may have regulatory functions. both Nesprin-1 and Nesprin-2 localisation at the NE depend on Temporary and spatially controlled expression of Nesprins-1α, lamin A/C, and NE-targeting of human Sun2 also requires a β and Nesprin-2α-γ may allow a modulation of Nesprin-based functional lamin A/C network (Libotte et al., 2005) (our contacts to the actin cytoskeleton, as their presence would unpublished data). Therefore, those findings suggest that result in the reduction or absence of the large ABD-containing additional proteins are implicated in the tethering of Nesprins isoforms from the NE. at the NE. Whether additional SUN domain-containing Based on data from C. elegans demonstrating a genetic proteins in addition to Sun1 are involved in those associations interaction between C. elegans ANC-1 and UNC-84 (Starr and warrants further investigation. Han, 2002), we studied the NE anchorage of Nesprins by the UNC-84 orthologue in higher eukaryotes. Yeast two-hybrid Sun1 connects through the Nesprin proteins the nucleus and GST pull-down experiments demonstrated that a region to the cytoskeleton termed SD2, composed of amino acids 632-737 of Sun1 does indeed interact in vivo and in vitro with both Nesprin-1 and Our studies support a model whereby Nesprin-1 and Nesprin- Nesprin-2. In C. elegans, missense mutations in or near the 2 are anchored at the nuclear envelope through a Sun1- SUN domain of UNC-84 probably disrupt the capacity of SD2 mediated interaction (Fig. 9). Although the N-terminus of Sun1 to associate with ANC-1. Our yeast two-hybrid data suggest, may provide a link to the nuclear lamina, its C-terminal however, that the SUN domain is not the main Nesprin subdomains are implicated in intramolecular (SD1) and interaction domain. It may well be that these particular SUN intermolecular (SD2 and SUN) perinuclear space interactions. domain mutations affect the proper folding of the protein Whereas SD2 was identified as the Nesprin binding domain we resulting in non-functional C-terminal domains in the failed to assign a specific function to the evolutionarily Journal of Cell Science Sun1 interacts with Nesprins 3429 proximity of the two NE membranes. Recent evidence implicates TorsinA in connecting the NE to the cytoskeleton. Torsin A an AAA+ ATPase of the ER localises to the NE when mutated in the neurological human disorder early-onset torsion dystonia (Goodchild and Dauer, 2004; Naismith et al., 2004; Gerace, 2004). TorsinA mutants affect several aspects of NE structure, including its morphology, perinuclear spacing and nuclear pore distribution (Naismith et al., 2004). Whether TorsinA modulates the Sun1/Nesprin interactions needs to be seen. In any case, molecular interactions governing and regulating the connections between the nucleoskeleton and cytoskeleton seem to be highly complex and we may have encountered only the tip of the iceberg. Nuclear envelope proteomic approaches suggest the presence of 67 novel or uncharacterised nuclear membrane proteins (Schirmer et al., 2003), which might potentially be involved in these linker complexes. The human KIAA0810 was obtained from the Kazusa DNA Research Institute, Japan. We acknowledge the gift of GFP-B1Δ2+ vector by Chris Hutchison, University of Durham, UK. Wild-type and lamin-A knockout mice skin fibroblasts were kindly provided by Colin Stewart (NCI, Frederick). The pSHAG-FF1 plasmid was a generous gift of Greg Hannon, Cold Spring Harbor Laboratory, USA. Fig. 9. Model illustrating the interactions of Sun1 with Nesprins at Anti-LBR antibodies were a kind gift of Harald Hermann, DKFZ, the nuclear envelope. Unknown nuclear envelope proteins and Heidelberg, Germany. This work was supported by grants from the interactions are indicated by X and ?, respectively. To reduce Austrian Science Research Fund (FWF, P15312) to R.F.; by grants complexity a homotypic dimerisation of Sun1 via the coiled-coil from the CMMC and the DFG (SFB 589) to A.A.N. and by a grant regions is postulated, although other coiled-coil-containing proteins from the Maria-Pesch-Foundation to I.K. might form heterotypic complexes with Sun1. INM, inner nuclear membrane; LD, luminal domain; N, N-terminal domain; ONM, outer nuclear membrane; PNS, perinuclear space. References Adam, S. A., Marr, R. S. and Gerace, L. (1990). 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The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope

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0021-9533
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10.1242/jcs.02471
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Abstract

Research Article The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope 1,2 1 1 1 1,2 1,2 V.C. Padmakumar , Thorsten Libotte , Wenshu Lu , Hafida Zaim , Sabu Abraham , Angelika A. Noegel , 3, 3 1, Josef Gotzmann *, Roland Foisner and Iakowos Karakesisoglou * 1 2 Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria *Authors for correspondence (e-mail: [email protected]; [email protected]) Accepted 6 May 2005 Journal of Cell Science 118, 3419-3430 Published by The Company of Biologists 2005 doi:10.1242/jcs.02471 Summary Nesprins form a novel class of nuclear envelope-anchored does not require functional A-type lamins for its spectrin-repeat proteins. We show that a direct association localisation at the inner nuclear membrane in mammalian of their highly conserved C-terminal luminal domain with cells. Our findings propose a conserved nuclear anchorage the inner nuclear membrane protein Sun1 mediates their mechanism between Caenorhabditis elegans and mammals nuclear envelope localisation. In Nesprin-1 and Nesprin-2 and suggest a model in which Sun1 serves as a ‘structural the conserved C-terminal amino acids PPPX are essential bridge’ connecting the nuclear interior with the actin for the interaction with a C-terminal region in Sun1. In cytoskeleton. fact, Sun1 is required for the proper nuclear envelope localisation of Nesprin-2 as shown using dominant-negative Key words: Emerin, Enaptin, Lamin A/C, NUANCE, SUN domain, mutants and by knockdown of Sun1 expression. Sun1 itself Syne Introduction them in the nuclear envelope (NE), followed by a stretch of amino acids displaying strong homology to the C-terminus of Several lines of evidence suggest the presence of molecular the Drosophila Klarsicht protein (Mosley-Bishop et al., 1999). links, which ‘hard-wire’ plasma membrane receptors, Nesprin genes are huge and complex, having the propensity to cytoskeletal components and nuclear scaffolds together, generate a wide variety of isoforms. These differ in length, allowing the physical integration of the nucleus within a cell domain composition, expression pattern and maybe also in and the generation of nuclear signalling and/or mechanical their functional properties, and consequently a plethora of responses to various intracellular and extracellular cues. names is currently used for these proteins by various groups. Testimony of the existence of such connections are genetic data Nesprin-1 isoforms have been described as CPG2, syne-1, from a wide variety of species, which identify microtubules, myne-1 and Enaptin, whereas Nesprin-2 variants are also microtubule-associated proteins, proteins containing giant known as syne-2 and NUANCE (Apel et al., 2000; Mislow et spectrin repeats and SUN domain-containing molecules al., 2002a; Zhang et al., 2001; Zhang et al., 2002; Zhen et al., involved in nuclear anchorage and nuclear migration (Reinsch 2002; Gough et al., 2003; Cottrell et al., 2004; Padmakumar et and Gönczy, 1998; Starr and Han, 2003; Patterson et al., 2004). al., 2004). Even though terms such as ‘nucleoskeleton’ and the long- Nesprins are widely expressed in a variety of tissues and cell disputed ‘nuclear matrix’ have gained more significance and types (Zhen et al., 2002; Padmakumar et al., 2004). At the popularity, it is still not understood which molecular set-up is subcellular level they were detected at the NE and also in required to determine nuclear architecture and the linkage of the nucleus. In contrast to Nesprin-2, which localises the nucleus to cytoplasmic structures. The recently described ANC-1 of C. elegans (Starr and Han, 2002), MSP-300 of predominantly to the nucleus and the NE, Nesprin-1 has a Drosophila melanogaster (Rosenberg-Hasson, 1996; Zhang et rather heterogeneous subcellular distribution and can also be al., 2002) and the mammalian proteins Nesprin-1/Enaptin and detected at F-actin-rich structures (Zhang et al., 2001; Padmakumar et al., 2004). Consistent with their subcellular Nesprin-2/NUANCE (Zhang et al., 2001; Zhang et al., 2002; localisation, genetic studies involving their orthologues in Zhen et al., 2002; Padmakumar et al., 2004) form a novel lower eukaryotes suggest roles in the attachment of family of proteins containing nuclear spectrin repeats that may intracellular membrane compartments to the actin organise and connect the nuclear membrane to the cytoskeleton cytoskeleton. Mutations in anc-1 of C. elegans disrupt the as well as the nuclear lamina. These proteins are very large positioning of nuclei and mitochondria (Starr and Han, 2002). (>800 kDa) and are composed of an N-terminal α-actinin-type actin binding domain, a long rod containing spectrin repeats MSP-300 in Drosophila is required for embryonic muscle and a highly conserved C-terminal type II transmembrane morphogenesis and the formation of embryonic muscle domain (KASH domain) (Starr and Han, 2002), which anchors attachments (Rosenberg-Hasson et al., 1996). Journal of Cell Science 3420 Journal of Cell Science 118 (15) Genetic studies in C. elegans have recently identified two tmNesprin-1 (amino acids 8611-8749; AAN03486) and GFP- dnNesprin-1 (aa 8369-8749; AAN03486) were cloned into inner nuclear membrane proteins, SUN1 and UNC-84, as EcoRI/SalI-cut EGFPC2 (Clontech) vector. GFP-tmNesprin-2 (aa structural links between the nucleoplasm and the cytoplasm. 6833-6883; AAL33548) was cloned into the EcoRI/BamHI site of SUN1/Matefin (Fridkin et al., 2004) and UNC-84 are EGFPC1 vector (Clontech). The full-length GFP-Sun1 (aa 1-913) was characterised by the presence of the SUN domain, which cloned into the EcoRI/BamHI site of EGFPC2. GFP-Sun1ΔSUN (aa derived its name from proteins containing the Sad1p-UNC-84 1-720), GFP-Sun1N+1TM (aa 1-384) and GFP-Sun1-TM-C (aa 358- homology domain (Hagan et al., 1995; Malone et al., 1999). 913) were cloned into the EcoRI/SalI site of EGFPC2 vector. GFP- SUN1 is essential for early embryonic and germ cell Sun1-N+2TM (aa 1-412), GFP-Sun1-TM-SD1,2 (aa 358-737), GFP- development (Fridkin et al., 2004) and required for the Sun1-TM-CΔSD2SUN (aa 358-632) and GFP-Sun1-CΔCC (358-491, localisation of ZYG-12 to the NE. ZYG-12 functions in 633-913) were cloned in EGFPC2 vector between the EcoRI and centrosomal attachment and the nuclear localisation of dynein BamHI sites. The KIAA0810 clone containing full-length human Sun1 was kindly provided by the KAZUSA research institute (Kikuno (Malone et al., 2003). In contrast to UNC-84 (Lee et al., 2002), et al., 2004). The Gateway -compatible destination vector pTB-RFB- germ-line-specific SUN1 does not require Ce-lamin for its EcoRV was created by insertion of the Gateway-transfer cassette into nuclear localisation (Fridkin et al., 2004). UNC-84 is required the EcoRV-site of pTRACER-EF1-Bsd (Invitrogen). Full-length Sun1 for proper nuclear migration and nuclear anchorage during was amplified from the KIAA-vector by PCR. The amplicon was worm development (Lee et al., 2002) and null alleles or cloned into the pTOPO-D/Entry-vector (Invitrogen) to create a missense mutations in UNC-84 also affect the nuclear envelope Gateway -compatible Sun1 entry vector. The expression vector targeting of ANC-1 (Starr and Han, 2002). In mammals, two expressing human KIAA0810-Sun1 harbouring C-terminal V5, was SUN domain-containing proteins have been described, created by LR-Clonase II-reaction using the Sun1 entry vector, pTB- although at least four unrelated members of the SUN domain RFB-EcoRV and the LR-enzyme mix according to manufacturer’s family are present in the databases (J.G. and R.F., unpublished instructions (Invitrogen). GFP-B1Δ2+ was a generous gift of Chris Hutchison (University of Durham, UK). The luminal domain of data) (Malone et al., 2003). The mammalian Sun1 protein is Nesprin-1 (LDN-1 residues 109-138, BE917568) was amplified from the real UNC-84 orthologue and was identified as an NE embryonic stem cell genomic DNA and cloned between EcoRI and protein in a proteomic analysis of neuronal nuclear membranes SalI sites in pGBKT7 (Clontech) and into pGEX4T-1 (Pharmacia) (Dreger et al., 2001). Sun2 is transcribed from a separate gene vectors. The luminal domain of Nesprin-2 (LDN-2, aa 6854-6883; and shows all properties of an inner nuclear membrane protein, AAL33548) was cloned into EcoRI and SalI sites of pGEX-4T-1. The with the SUN domain extending into the periplasmic space C-terminus of Sun1 (Sun1-C, aa 432-913), Sun1-CΔSUN (aa 432- between the inner and outer nuclear membranes (Hodzic et al., 737), Sun1-SUN (aa 738-913) was cloned into EcoRI/BamHI- 2004). Both Sun1 and Sun2 have also been identified in a digested pGADT7 and pGBKT7 vectors (Clontech). Sun1-SD1 (aa comprehensive proteomic screen of inner nuclear membrane 431-633) and Sun1-SD2 (aa 632-737) were cloned into proteins in rat liver (Schirmer et al., 2003). EcoRI/BamHI-cut pGADT7. In this report, we identify and analyse the direct interaction between the mammalian inner nuclear membrane protein Sun1 Yeast two-hybrid assay and Nesprins, giant NE-cytoskeletal linker proteins. Our Y190 yeast cells were co-transformed with the plasmids and findings provide novel insights into the NE anchorage transformants were selected on SD-Trp-Leu plates. Interaction was mechanism of Nesprins, demonstrating an evolutionarily monitored by growth on plates containing selection media SD-Trp- conserved strategy from worms to mammals. Leu-His and 60 mM 3-amino 1,2,4-triazole. X-Gal assay was also performed to confirm the interaction. The protocols for performing the yeast transformation and X-gal assay are described in detail Materials and Methods elsewhere (Yeast Protocols Handbook PT3024-1; Clontech). Tissue culture and transfection COS7 and C3H/10T1/2 cells were grown as described (Padmakumar Purification of GST fusion proteins and in vitro binding assays et al., 2004). HaCaT, HeLa cells and skin fibroblasts from wild-type and lamin A knockout mice (Sullivan et al., 1999) were routinely Purification of GST fusion proteins and GST pull-down experiments cultivated in DMEM supplemented with 10% fetal calf serum (Gibco were performed as described (Dreuillet et al., 2002). For the pull- Life Technologies) at 37°C in a humidified atmosphere containing 5% down assay, COS7 cells were lysed with 50 mM Tris-HCl, pH 7.5, CO . 150 mM NaCl, 1% Triton X-100 and protease inhibitors (Roche). The All the GFP fusion constructs were transfected in COS7 and 100,000 g supernatant of the lysate was incubated with equal amounts C3H/10T1/2 cell lines by electroporation (200V, 950 μF; Gene Pulser of GST fusions and the solutions were incubated at 4°C overnight with Xcell, Bio-Rad). HaCaT cells were transfected by using the Amaxa GST-sepharose beads on a roller. Samples were centrifuged and the nucleofector technology according to the manufacturer’s instructions pellets (washed three times with PBS) were analysed by SDS-PAGE (Amaxa Biosystems). HeLa cells were transfected using and western blot analysis. Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Antibodies and immunofluorescence microscopy Sun1 polyclonal antibodies were produced against a peptide Cloning strategies containing the first 14 amino acids of human Sun1 All the GFP fusion proteins carry GFP at their N-terminus. The (MDFSRLHMYSPPQC; NP_079430) to create antiserum 281 appropriate DNA fragments were amplified by PCR and cloned in (Eurogentec, Seraing, Belgium). either pGEMTeasy (Promega) or pCR-2.1-TOPO (Invitrogen) vectors Western blotting and immunofluorescence (IF) studies were before cloning them in the GFP, yeast two-hybrid or GST vectors. performed as described (Zhen et al., 2002; Gotzmann et al., 2000). Nesprin-1 constructs were amplified from BC054456 IMAGE clone The following antibodies were used: affinity-purified rabbit and Sun1 was amplified from IMAGE clone AAH48156. GFP- polyclonal anti-Nesprin-1/Enaptin (1:50 for IF), mouse monoclonal Journal of Cell Science Sun1 interacts with Nesprins 3421 anti-Nesprin-2/NUANCE mAb K20-478 (Zhen et al., 2002), transmembrane domain followed by a stretch of 30 amino acids unpurified rabbit polyclonal hSun1 281 (1:50 IF; 1:400 western (luminal domain) extending into the perinuclear space, which is blotting), rabbit polyclonal Nesprin-2 pAbK1 (1:50 IF, 1:1000 highly conserved in this family of nuclear proteins (Fig. 1A). In western blotting), mouse monoclonal anti-lamin A/C (1:50 IF; JoL2, order to identify the sequences necessary for the NE localisation Chemicon), mouse monoclonal anti-V5 (1:500 IF; 1:3000 western of Nesprins and elucidate the mechanism by which they are blotting; Invitrogen), polyclonal serum to LAP2α (1:5000; tethered at the NE, several GFP fusion proteins of their C-termini ImmuQuest) (Vlcek et al., 2002), guinea pig anti-LBR was a kind gift (Fig. 1B) were transiently expressed in various cell lines. of Harald Hermann (1:250 IF) (Dreger et al., 2002), mouse TmNesprin-1, representing the transmembrane domain and monoclonal anti-LAP2β (1:5 IF) (Dechat et al., 1998) goat polyclonal the periplasmic region of the protein localised to the NE in anti-GST antibody (Amersham Biosciences), GFP-specific mAb K3- C3H/10T1/2 cells (Fig. 1C,E, arrow) and exerted a dominant- 184-2 (Noegel et al., 2004). The secondary antibodies used were conjugated with Cy3 (Sigma), Alexa 488 or Alexa 568 (Molecular negative effect on the endogenous Nesprin-1 protein. Probes, Leiden, The Netherlands), Texas Red (Jackson Laboratories, Untransfected fibroblasts displayed strong Nesprin-1 staining West-Grove, PA) and FITC (Sigma). Samples were analysed by wide- at the NE (Fig. 1D,E, arrowhead), which disappeared in field fluorescence microscopy (DMR, Leica, Bensheim, Germany) or transfected cells (Fig. 1D,E, arrows). A similar effect was confocal laser-scanning microscopy (TCS-SP, Leica). observed when COS7 cells expressed the corresponding tmNesprin-2 GFP fusion protein. Like tmNesprin-1, tmNesprin-2 localised to the NE (Fig. 1F, arrow) and displaced Subcellular fractionation the endogenous Nesprin-2 (Fig. 1G,H, arrowheads) from the Subcellular fractionation was done essentially as described (Dechat et NE (Fig. 1G, arrow). As tmNesprin-2 did not contain regions al., 1998; Gotzmann et al., 2000). In brief, cells were broken in upstream of the transmembrane domain as found in tmNesprin- hypotonic buffer using a Dounce homogeniser with a tight-fitting pestle. After addition of 8% sucrose, the soluble cytoplasmic and the 1 (Fig. 1B), we concluded that the transmembrane domain and insoluble nuclear fractions were separated by centrifugation at 2000 the luminal domains are sufficient to mediate NE targeting. The g for 15 minutes at 4°C. The nuclei-containing pellets were extracted high homology between the C-termini of Nesprin-1 and in the same buffer supplemented with 1% Triton X-100, or 200 mM Nesprin-2 further prompted us to investigate the effects of a NaCl or combinations of both. Another extraction was performed dominant-negative Nesprin-1 GFP fragment on endogenous using hypotonic buffer containing 7 M urea. The extracts were Nesprin-2. In these experiments we used the dnNesprin-1 GFP centrifuged at 15,000 g for 10 minutes and supernatants and pellets fusion (~75 kDa), which also harbours the two final spectrin were analysed by western blotting. repeats upstream to the transmembrane domain, thus resembling Nesprin-2α in its domain organisation (Zhang et siRNA knockdown of Sun1 al., 2001). The studies were performed in COS7 cells, which The RNA interference-competent pSHAG-1 vector (containing express Nesprin-2 strongly at the NE (Zhen et al., 2002). a human U6 promoter fragment; –265 to +1), constructed Similar to tmNesprin-1, dnNesprin-1 localised to the NE in using a pTOPO-ENTR/D backbone (Invitrogen, Carlsbad, USA), has COS7 cells (Fig. 1I,K, arrow) and displaced the endogenous been described recently (Paddison et al., 2002; http://katahdin.cshl. Nesprin-2 (Fig. 1J, arrow). Experiments involving shorter org:9331/RNAi_web/scripts/main2.pl). Oligonucleotides A and B, versions of dnNesprin-1, such as tmNesprin-1 fusions, encoding a short hairpin RNA, were designed according to the RNAi- exhibited similar dominant-negative effects on Nesprin-2 (data retriever protocol at http://katahdin.cshl.org:9331/homepage/portal/ not shown). To further study the potential significance of the html/protocols/. For knocking down specifically human Sun1 the highly conserved C-terminal proline residues (Fig. 1A, green following oligonucleotides were used to create vectors pJG173 and bar) for the NE localisation of Nesprin-2, a GFP fusion protein pJG174, respectively: Oligo 173A, 5′-CTCGGACAGCATGCTGC- tmNesprin-2ΔP lacking the PPPT-motif was generated. In AGTTGCTGCAGGAAGCTTGCTGCGGCGACTGCGGCATGTTG- TCCGAGCGCTTTTTT-3′; Oligo 173B, 5′-GATCAAAAAAGCG- contrast to tmNesprin-2 (Fig. 1F, arrow), this protein CTCGGACAACATGCCGCAGTCGCCGCAGCAAGCTTCCTGCA- accumulated in the nuclear interior (Fig. 1L, arrowhead) and GCAACTGCAGCATGCTGTCCGAGCG-3′; Oligo 174A, 5′-AG- in ER-like structures (Fig. 1L, arrows) and did not affect the GACGTGACCTGCCTTGACACGTGGTTGAAGCTTGAGCCGC- location of endogenous Nesprin-2 (Fig. 1M, arrowhead). GTGTTAAGGCAGGTCACGTTCTCTGTTTTTT-3′; Oligo 174B, Taken together, these experiments underline the functional 5′-GATCAAAAAACAGAGAACGTGACCTGCCTTAACACGCG- significance of the conserved proline-rich stretch of Nesprins GCTCAAGCTTCAACCACGTGTCAAGGCAGGTCACGTCCTCG- for NE targeting. Moreover, the C-terminal domains act in a 3′. Annealed oligonucleotides were cloned into pSHAG-1 by insertion dominant-negative manner on the endogenous Nesprin of resulting overhangs into the BseRI/BamHI-sites. The control vector proteins, displacing them from the NE. These findings also pSHAG-FF1 encoding an shRNA targeting Firefly Luciferase was a imply that the associations of the luminal domain of Nesprin- kind gift of Greg Hannon (Cold Spring Harbor Laboratory, NY). The integrity of all vectors was verified by sequencing from both ends. 1 and Nesprin-2 in the perinuclear space involve identical RNAi-vectors targeting hSun1 were always used in combination for binding partners conferring anchorage at the NE. transient knockdown experiments. The cells were fixed 4 days after the transfection and examined by indirect immunofluorescence. Sun1 binds to the luminal domain of both Nesprin-1 and Nesprin-2 Results A genetic interaction between ANC-1 and UNC-84 has been Common mechanisms tether Nesprin-1 and Nesprin-2 reported in C. elegans, although efforts to elucidate the direct at the NE molecular interaction failed (Starr and Han, 2002). A search of Nesprin-1 and Nesprin-2 localise to the NE by virtue of their the mouse EST database for mammalian UNC-84 orthologues conserved C-terminal domain, which includes a type II as potential Nesprin binding partners yielded two SUN Journal of Cell Science 3422 Journal of Cell Science 118 (15) domain-containing proteins, Sun1 (accession number kDa protein composed of 913 amino acids (Fig. 2A). It AAH48156, mouse chromosomal locus 5G.2) and Sun2 contains three putative transmembrane domains (aa 358-383, (AAT90499, residing on chromosome 15). Mouse Sun1 and 386-407 and 413-431) located approximately in the middle of Sun2 display 65% identity and 81% homology in their SUN the protein, a predicted ZnF-C2H2 domain near the N- domain and 47%/39% identity and 63%/59% similarity to terminus, and two predicted coiled-coil domains in the C- the SUN domain of UNC-84, respectively. As Sun1 is more terminus (aa 492-527 and 563-632). The last 175 residues are closely related to UNC-84 than Sun2, Sun1 was chosen for the highly homologous to C. elegans UNC-84 and S. pombe Sad1 current study. In addition, we used human Sun1, the closest forming the evolutionarily conserved SUN domain. The human orthologue to UNC-84, displaying 48% identity and domain structure of human Sun1 is identical to that of mouse, 64% similarity to the SUN domain of UNC-84. Human Sun1 except that it lacks the proposed zinc-finger motif. The region was originally identified as KIAA0810 by the Kazusa between the transmembrane and the SUN domain was divided DNA research institute (Kikuno et al., 2004; into subdomains SD1 and SD2 for functional tests. SD1 http://www.kazusa.or.jp/huge/). Mouse Sun1 encodes a 100 contains the two coiled-coil regions whereas SD2 does not display any known structural features (Fig. 2A). In yeast two-hybrid assays we investigated the possible interaction between the luminal domain of Nesprin- 1 and mouse Sun1. The last 30 amino acids (luminal domain) of mouse Nesprin-1 were fused in-frame to the binding domain of Gal4 and were tested for an interaction with five different C- terminal Sun1 fusion constructs with the activating domain of Gal4. These included Sun1-C (C-terminus of Sun1), Sun1-CΔSUN, which lacks the SUN domain, Sun1-SUN composed solely of the SUN domain, Sun1-SD1 and Sun1- SD2 (Fig. 2B). Co-transformation of the Fig. 1. The C-termini of Nesprins are conserved and sufficient for NE localisation. (A) Alignment (using MultiAlign) of the 30 amino acid luminal domains of various KASH-domain NE proteins. The green bar denotes the highly conserved C- terminal prolines. (B) The tmNesprin-1, tmNesprin- 2, tmNesprin-2ΔP (lacks the last four aa) and dnNesprin-1 GFP fusion constructs used for the experiments shown in C-N. LD, luminal domain; SR, spectrin repeats; TM, transmembrane domain. (C-K) Dominant-negative effect of tmNesprin-1, tmNesprin-2 and dnNesprin-1 GFP fusions on the endogenous Nesprin proteins. Transiently transfected cells were fixed and subjected to immunofluorescence using the monoclonal K20- 478 anti-Nesprin-2 and a rabbit polyclonal Nesprin- 1 antibody. These antibodies did not recognise epitopes on the ectopically expressed polypeptides. Note the nuclear rim staining of endogenous Nesprin proteins in untransfected cells (arrowheads in E,H,K) and the absence of Nesprin staining in GFP-positive cells (arrows in E,H,K). (L-N) Confocal images demonstrate a cytoplasmic (panel L, arrows) and a diffuse nuclear staining pattern (panel L, arrowhead) for GFP-Nesprin-2ΔP, which does not affect endogenous Nesprin-2 at the nuclear envelope (arrowhead in M). The cell lines used are indicated in the lower right-hand corner of the first column of frames (C,F,I). DNA was stained with DAPI. Images were obtained by confocal laser- scanning microscopy. Bars, 10 μm. Journal of Cell Science Sun1 interacts with Nesprins 3423 plasmids into Y190 yeast cells followed by β-galactosidase tmNesprin-2ΔP was unable to displace Nesprin-2 from the NE assays revealed an interaction between Nesprin-1 and Sun1-C, (Fig. 1L-N). whereas controls remained negative. Further experiments identified the SD2 of Sun1 (residues 632-737) as the primary Sun1 is a component of the nuclear envelope and the Nesprin-1 binding site, whereas the SUN domain itself showed inner nuclear membrane only a weak binding to Nesprin-1 in this assay. No interaction was found with SD1 (Fig. 2B). To determine whether mouse and human Sun1 are inner These observations were further supported by biochemical nuclear membrane proteins like UNC-84 in C. elegans, we assays where we used GST fusion proteins containing the performed a series of analyses using the polyclonal Sun1 luminal domain of Nesprin-1 and Nesprin-2 (yielding GST- antiserum 281 generated against a peptide derived from the N- LDN-1 and -2 respectively) to pull down the GFP-Sun1-C terminus of human Sun1. Western blot analysis of HaCaT cell fusion protein, which lacks the transmembrane domains (GFP- lysates using the unpurified Sun1 serum, detected a major 100 Sun1-C, aa 432-913). Both GST fusion proteins were able to kDa band (Fig. 3A). Furthermore, the Sun1 antibody detected precipitate GFP-Sun1-C from COS7 cell lysates (Fig. 2D,E). ectopically expressed full-length human Sun1 protein in cell A Nesprin-2 luminal domain deletion GST construct (GST- lysates, which could be efficiently competed by increasing LDN-2ΔP) lacking the last four highly conserved amino acids amounts of the peptide antigen (data not shown). To examine (PPPT) (Fig. 2C) was generated and used for precipitation whether Sun1 interacts with Nesprin-2 in vivo, we performed assays from COS7 lysates containing GFP-Sun1-C. In contrast immunoprecipitation studies. When the Nesprin-2 to GST-LDN-1 and GST-LDN-2, no interaction with GFP- immunocomplexes (Fig. 3B, lane 4) were resolved by SDS- Sun1-C was observed for the GST-LDN-2ΔP fusion protein PAGE and subjected to silver staining we observed a faint 100 (Fig. 2E), which is consistent with the observation, that kDa band (Fig. 3B, lane 4, arrowhead), which was specifically recognised by the anti-Sun1 antibody (Fig. 3B, lane 4, right panel). These results indicate that Sun1 interacts with Nesprin-2 in vivo. The Sun1 antiserum preferentially stained the NE where it colocalised with Nesprin-2 in HaCaT cells (Fig. 3C-E). In addition, the antibodies produced cytoplasmic background staining resulting most probably because we used Sun1 antiserum, which was not affinity- purified. The background staining also persisted after specific Sun1 knockdown (Fig. 7D,G and J) and therefore does not mirror a natural localisation for the endogenous Sun1 protein. Furthermore stably expressed V5 epitope tagged human Sun1 colocalised with Lamin B receptor (LBR), a well- characterised inner nuclear membrane protein, in a number of cell lines, including HeLa (Fig. 3F-H), human Hek293, COS7 and SW-480 cells, murine NIH-3T3 fibroblasts and canine epithelial MDCK-cells (data not shown). To test the association of human Sun1 with the NE at the biochemical level, Fig. 2. The C-terminus of Sun1 associates directly with the luminal domains of Nesprin-1 nuclei of HeLa cells stably expressing and Nesprin-2. (A) Domain organisation of mouse Sun1. The domain locations as well as Sun1 were extracted in buffers containing their amino acid positions are indicated according to the GenBank entry AAH48156. CC, urea and non-ionic detergents. The coiled-coil domain; ZnF, zinc-finger domain; Tm, transmembrane domain. (B) Sun1 polypeptides corresponding to various Sun1 domains were fused to the Gal4 activating distribution of human Sun1 in soluble (S) domain, whereas the Nesprin-1 luminal domain was fused to the Gal4 DNA-binding and insoluble (P) fractions was analysed domain. The corresponding plasmids were co-transformed into yeast cells and the by immunoblot analysis (Fig. 3I). The interactions were assessed by the filter lift β-galactosidase assay. ++++, strong; ++, weak; distribution of the well-characterised –, no blue colour development. (C) Schematic overview of the fusion proteins (GST-LDN- inner nuclear membrane protein LAP2β 1, GST-LDN-2 and GST-LDN-2ΔP lacking the last 4 aa) used for the GST pull-down assay served as a control. Sun1 and LAP2β of COS7 cell homogenates expressing GFP-Sun1-C. LDN-1, luminal domain Nesprin-1; displayed similar properties, being LDN-2, luminal domain Nesprin-2. (D,E) COS7 cell lysates expressing the C-terminal half completely resistant to extraction with of Sun1 (Sun1-C) were incubated with the immobilised GST-fusion proteins as indicated high salt, chaotropic agents and detergent and GST for control. Unbound (S) and specifically bound (P) proteins were subjected to at low salt concentration (Fig. 3I). Only SDS-PAGE followed by western blot analysis using GFP-specific mAb K3-184-2. Journal of Cell Science 3424 Journal of Cell Science 118 (15) Fig. 3. Sun1 behaves like an integral inner nuclear membrane protein. (A) Western blotting analysis of HaCaT cell lysates using polyclonal Sun1-specific antibodies detects a major 100 kDa band. (B) Endogenous Sun1 protein co-immunoprecipitates with Nesprin- 2. Immunocomplexes obtained from HaCaT cells with anti-Nesprin- 2 (pAbK1) antibodies were analysed by SDS-PAGE and subjected to silver staining (left panel) or immunoblotting with anti-Nesprin-2 (mAb K20-478) and anti-Sun1 antibody (right panel). The major 800, ~400 and 75 kDa Nesprin-2 isoforms present in HaCaT cells are indicated by arrows (right panel). Lane 1, input lysate; lane 2, control precipitate with Protein A sepharose beads; lane 3, mock-IP control IgG antibody; lane 4, co-immunoprecipitate with anti-Nesprin-2 antibody pAb-K1. The bands observed in lane 4 represent signals obtained after short exposure whereas lanes 1-3 were obtained after prolonged ECL detection (30 minutes). Positions of molecular mass markers in kDa are shown on the left-hand side of the blots. (C-E) HaCaT cells were subjected to immunofluorescence using Sun1 (281) and Nesprin-2 antibodies (mAb K20-478), demonstrating the colocalisation of Sun1 with Nesprin-2 at the NE (E). The inset is a higher magnification of the dotted white box. (F-H) Ectopically expressed full-length human Sun1 (C-terminal V5-tag) is targeted to the nuclear envelope in HeLa cells, displaying strict colocalisation with the Lamin B receptor (LBR). Images were obtained using a confocal microscope. (I) Solubilisation properties of human Sun1 under various extraction conditions. Purified nuclei (Nuc) of HeLa cells, stably expressing V5-tagged human Sun1, were extracted in RIPA buffer containing urea, Triton X-100, salt or combinations thereof, as indicated. Soluble (S) and insoluble (P) fractions were analysed by western blotting. Cytosol (Cyt) served as a purity control. The same lysates were analysed for LAP2β, a known integral inner nuclear membrane protein. Bars, 7 μm. permeabilisation assays suggested that Nesprin-2 is integrated into the outer nuclear membrane (Zhen et al., 2002). To investigate whether the highly homologous Nesprin-1 shares a similar localisation we analysed C3H/10T1/2 fibroblasts treated with Triton X-100 and digitonin. Unlike lamin A, Nesprin-1 staining could still be detected at the NE after treatment with detergent and medium salt concentrations (1% selective digitonin permeabilisation indicating its presence at Triton X-100/200 mM NaCl) efficiently solubilised Sun1 and the outer nuclear membrane (Fig. 4J-L). However our findings LAP2β. In accordance with these data KIAA0810 has been do not exclude the possibility that Nesprin-1 also localises to identified as a component of both the detergent- and chaotrope- the inner nuclear membrane. Collectively our findings suggest resistant fractions in a proteomics screen (Dreger et al., 2001). an asymmetric distribution at the nuclear membrane of the More recently, Sun1 was exclusively detected in the salt- and interaction partners Sun1 and Nesprins. sodium hydroxide-resistant fractions of a novel protocol to isolate unknown NE constituents (Schirmer et al., 2003). The N- and C-termini of Sun1 localise independently to In order to define the topology of Sun1 at the NE we the NE performed digitonin permeabilisation of cells, which selectively disrupts the plasma membrane leaving the NE The current sorting mechanism, which defines the localisation membranes intact (Adam et al., 1990), whereas Triton X-100 of NE transmembrane proteins during interphase is the permeabilises all membranes. Antibodies to both Sun1 and ‘diffusion-retention’ model (Worman and Courvalin, 2000). lamin A/C stained the NE in Triton X-100-permeabilised According to this model NE proteins are co-translationally COS7 cells (Fig. 4A-C, arrows). In addition, the antiserum integrated into the ER followed by a lateral diffusion from the strongly stained the nucleoplasm (Fig. 4A), whereas in HaCaT ER to the outer and inner nuclear membranes, interconnected cells the staining was preferentially found at the NE (Fig. 3C). by the nuclear pore complex. Proteins are then retained at the In digitonin-treated COS7 cells Sun1 and lamin A/C remain inner nuclear membrane owing to the presence of nuclear undetectable using these antibodies at the NE. Only the retention sequences allowing binding to nuclear proteins, cytoplasmic staining of Sun1 antiserum 281 was still observed chromatin or both. (Fig. 4D and F, arrowheads). Identical results were obtained To investigate the subcellular localisation and to determine the for ectopically expressed human as well as mouse Sun1 (data NE retention domains of mouse Sun1 we transiently transfected not shown). Altogether, these data strongly suggest that Sun1 various Sun1-GFP fusion proteins (Fig. 5A) into COS7 cells. is an integral inner nuclear membrane protein. The expression and the appropriate molecular masses of the Earlier studies on Nesprin-2 using digitonin fusion proteins were confirmed by western blotting (data not Journal of Cell Science Sun1 interacts with Nesprins 3425 fusion protein remained in the ER (Fig. 5F, arrowheads). Although more than 75% of the Sun1- N+1TM and Sun1-N+2TM fusions localised to the NE, only 58% of Sun1-TM-C displayed NE localisation. The accumulation of the proteins in the ER may reflect either the absence of important domains or result from the overexpression of improperly folded proteins. In summary, both the N- and C-termini of Sun1 localise independently to the NE, which is most likely facilitated by binding to different proteins in the nucleoplasm and the perinuclear space. Sun1 associates with itself in vivo and the two coiled-coil domains are sufficient to target the C-terminus of Sun1 to the NE In order to study the C-terminal Sun1 nuclear targeting sequences in more detail, three additional GFP constructs (Sun1-TM-SD1,2; Sun1-TM- CΔSD2SUN; Sun1-TM-CΔCC; Fig. 5A) were expressed in COS7 cells (Fig. 5G-I). The GFP fusion protein Sun1-TM-SD1,2 comprising SD1, SD2 and the three transmembrane domains displayed a NE localisation in 64% of transfected cells (Fig. 5G, arrow and Fig. 5J). As this polypeptide lacks the SUN domain, the SUN domain seems dispensable and not required to confer the NE localisation, as shown above for the GFP-ΔSUN mutant. To investigate whether the Nesprin binding domain SD2 in Sun1 is involved in the NE targeting of Sun1, we constructed a GFP fusion construct of the three transmembrane domains followed by SD1, composed of the two coiled-coil domains, but lacking SD2 (Sun1-TM- CΔSD2SUN). In 73% of transiently transfected Fig. 4. Asymmetric distribution of Sun1 and Nesprin-1 at the nuclear membrane. COS7 cells we observed a clear NE association of (A-C) Triton X-100 treated COS7 cells subjected to immunofluorescence with Sun1 and lamin A/C antibodies, indicate the nuclear localisation of Sun1 the fusion protein (Fig. 5H, arrow and Fig. 5J), (arrows). Non-specific staining of antibody 281 was observed in the cytoplasm suggesting therefore the presence of a nuclear (arrowheads; see also Fig. 3C). (D-F) In digitonin-treated COS7 cells only the retention signal within SD1 of Sun1. Coiled-coils non-specific staining remains (arrowheads) suggesting a localisation of Sun1 at have traditionally been recognised as an the inner nuclear membrane. The integrity of the nuclear membrane is oligomerisation unit in a large number of proteins documented by the absence of lamin A/C staining (E). (G-I) In Triton X-100- (Burkhard et al., 2001). As SD1 is composed of two permeabilised fibroblasts Nesprin-1 antibodies strongly stain the nucleus. coiled-coil regions (CC1 and CC2, see Fig. 2A), it (J-L) Nesprin-1 staining at the NE persists after digitonin treatment suggesting is possible that oligomerisation of the GFP-fusion the presence of Nesprin-1 at the outer nuclear membrane. Note the absence of protein with the endogenous Sun1 leads to retention lamin A/C staining (K). DAPI was used to counterstain the nucleus. Confocal at the nuclear envelope. This is supported by data images are shown. Bars, 5 μm. from yeast two-hybrid experiments, which indicated an interaction of SD1 with itself (data not shown). To test the hypothesis that the NE localisation of N-terminally shown). Similar to its human orthologue (Fig. 3F) the full-length truncated Sun1 polypeptides is mediated by the coiled-coil mouse Sun1 protein also localised to the NE (Fig. 5B). Deletion region, we generated GFP-Sun-TM-CΔCC removing the two of the SUN domain (GFP-ΔSUN) did not affect targeting to the coiled-coil regions between amino acids 491-633, leaving the NE (Fig. 5C, arrow) showing that the SUN domain is not SUN as well as the transmembrane domains intact. Only 33% required for retention at the NE. Accumulation at the NE was of transfected cells (Fig. 5J) displayed a NE localisation (Fig. also obtained with GFP fusion proteins containing the entire 5I, arrows). In the majority of cases we observed substantial Sun1 N-terminus with a single (Sun1-N+1TM) or two accumulation of the chimeric protein in the ER (arrowhead). transmembrane domains (Sun1-N+2TM) (Fig. 5D,E, arrows). Our data suggest that the coiled-coil region of Sun1 is important Surprisingly, the C-terminus containing the three transmembrane to mediate NE association, however the ability of the Sun1-TM- domains (Sun1-TM-C) also localised to the NE in COS7 cells CΔCC fusion protein to localise to the NE is indicative of the (Fig. 5F, arrows; Fig. 6A, arrow). Thus, the C-terminus of Sun1 existence of additional nuclear retention signal(s) in the C- is sufficient to confer NE targeting, however, much of the GFP Journal of Cell Science 3426 Journal of Cell Science 118 (15) Fig. 5. Sun1 contains multiple, independent nuclear targeting signals. (A) Schematic representation of Sun1 GFP fusion constructs. Domain labelling is as in Fig. 2A. (B-I) Subcellular localisation of GFP Sun1 fusion proteins in COS7 cells observed by direct fluorescence confocal microscopy. Arrows indicate NE localisation whereas arrowheads indicate ER localisation. DAPI was used to counter-stain the nuclei. (J) Histogram representing a statistical evaluation (percentage of transfected cells) of the localisation profiles of the various SUN1-GFP fusions to the ER and the NE. Bars, 10 μm. Sun1 antiserum in COS7 cells transiently expressing Sun1-TM-C (which is not detected by the antiserum) revealed a displacement of endogenous Sun1 from the NE (Fig. 6B-C, arrow). Untransfected cells, however, displayed a proper NE localisation of Sun1 (Fig. 6B-C, arrowhead). Out of 200 transfected cells 82% showed significantly reduced NE staining of Sun1 (Fig. 6G). As Nesprin-2 associates with Sun1 we tested its localisation in Sun1-TM-C transfected cells as well (Fig. 6D-F). Similar to the endogenous Sun1, Nesprin-2 was displaced from the NE (Fig. 6E-F, arrow) in transfected cells, whereas in untransfected cells Nesprin-2 was properly localised at the NE (Fig. 6D-F, arrowhead). In a statistical analysis of 200 transfected cells Nesprin-2 was no longer found at the NE in 97% of cells (Fig. 6G). To further support the requirement of Sun1 for the localisation of Nesprin-2, we performed knockdown studies employing a mixture of two independently expressed siRNAs targeting the N-terminus of Sun1. Transient transfections of the RNAi- competent plasmids pJG173 and pJG174 in HeLa cells, which stably express V5-tagged hSun1 verified the efficacy of the Sun1 knockdown (Fig. 7A-C). Many transfected cells displayed a reduced anti-V5 hSun1 staining (asterisks), whereas the lamin A/C pattern appeared unaffected (Fig. 7B). Similar observations were made in transiently transfected Sun1 knockdown HaCAT cells (Fig. 7D-F). In keratinocytes where the nuclear Sun1 staining was absent (Fig. 7D; asterisks), lamin A/C staining was still observed at the nuclear envelope (Fig. terminus of Sun1. Furthermore we cannot exclude the 7E; asterisks), suggesting therefore that Sun1 is not essential possibility that the Sun1 transmembrane domains themselves for lamin A/C localisation. In sharp contrast, however, in Sun1 contain sorting signals and determine the NE localisation in a knockdown cells (Fig. 7G-L) the Nesprin-2 staining pattern similar fashion to the lamin B receptor (Wozniak and Blobel, was either very faint or absent using both N-terminally (Fig. 1992; Smith and Blobel, 1993). The presence of multiple and 7H; asterisks) as well as C-terminally (Fig. 7K; asterisks) independent nuclear retention signals across Sun1 is further directed antibodies. In summary our data indicate that the supported by the fact that none of the fusion proteins localised proper localisation of Nesprin-2 at the NE requires Sun1. as efficiently to the NE as the full-length Sun1 (Fig. 5J). However, its interaction with Nesprins is apparently not Targeting of Sun1 to the NE is independent of a involved in retaining Sun 1 at the NE. functional lamin A/C network In C. elegans UNC-84 localises to the NE in a lamin-dependent Sun1 affects the NE localisation of Nesprin-2 manner (Lee et al., 2002). To explore whether the UNC-84 Immunofluorescence analyses of endogenous Sun1 using the orthologue Sun1 also depends on the lamin network we Journal of Cell Science Sun1 interacts with Nesprins 3427 Fig. 6. GFP-Sun1-TM-C acts in a dominant-negative manner on endogenous Sun1 and Nesprin-2. COS7 cells expressing GFP-Sun1- TM-C were stained using specific antibodies to Sun1 (B,C) and Nesprin-2 (E,F). (A-F) Confocal images illustrating that GFP-Sun1- TM-C (transfected cells are indicated by arrows) interferes with the localisation of endogenous Sun1 (B and C, arrows) and Nesprin-2 (E and F, arrows). Note the differences in the Sun1 and Nesprin-2 Fig. 7. Nesprin-2 localisation is affected in cells where Sun1 staining pattern in transfected (arrows) versus untransfected expression has been silenced by siRNA. (A-C) HeLa cells expressing (arrowheads) cells. (G) Histogram illustrating the displacement stably V5-tagged hSun1 and HaCaT cells (D-L) were transiently effects of the GFP-Sun1-TM-C fusion on the endogenous Sun1 and transfected with a combination of plasmids (pJG173/174) encoding Nesprin-2 proteins. Data were obtained by evaluating 200 transfected siRNAs targeting hSun1. The distribution of Sun1 (panel A, anti-V5; cells. Bars, 7 μm. panels D,G,J, anti-Sun1 281 serum), lamin A/C (panels B and E) and Nesprin-2 (panel H, mAb K20-478; panel K, mAb K49-260) was investigated by indirect immunofluorescence in knockdown cells transfected lamin A/C knockout fibroblasts (Sullivan et al., (indicated by asterisks). In Sun1 knockdown cells, the lamin A/C 1999) with the mouse Sun1 and Sun1-N+2TM GFP fusion localisation remained unaltered (B and E), whereas Nesprin-2 staining was either absent or reduced. DNA was stained by DAPI. proteins (Fig. 8A-C). In both wild-type and knockout The images shown were taken by confocal microscopy and merged fibroblasts the fusion proteins localised properly to the NE. to visualise colocalisation (panels C,F,I,L). Bars, 10 μm. Identical results were obtained with human V5 epitope-tagged Sun1 in lamin A/C knockout fibroblasts (Fig. 8D). To further substantiate our findings, we transiently transfected HeLa cells stably expressing human Sun1 with the Xenopus mutant GFP- (Burke et al., 2001; Gotzmann and Foisner, 2004). B1Δ2+ (Fig. 8G-I). This fusion protein accumulates in Understanding the pathology of all these diseases requires the intranuclear aggregates (Fig. 8H) and recruits endogenous identification and functional characterisation of all nuclear lamins A/C, thus disturbing the functional organisation of the envelope constituents as a first step, as well as knowledge of lamin A/C network (Dechat et al., 2000; Vaughan et al., 2001). the networking interactions that take place at the NE. Towards Importantly, the expression of GFP-B1Δ2+ and loss of a this end, using both biochemical as well as cell biological data functional A-type lamin network did not affect the NE we unravel the first link between an inner nuclear membrane localisation of Sun1 (Fig. 8G-I). Altogether, lamin A/C is not protein (Sun1) and constituents of the outer and inner nuclear essential for the localisation of Sun1 at the NE. Based on membranes (Nesprins). UNC84 data, one could assume that B-type lamins may be required for retention of Sun1 at the inner nuclear membrane. Nesprins are targeted to the NE by binding to Sun1 through their conserved C-terminus Discussion In an effort to study the nuclear localisation mechanism of the Recent focus on NE composition and function has been giant actin binding Nesprin-1 and Nesprin-2 proteins, we primarily powered by the unexpected involvement of several demonstrated that the evolutionarily conserved C-terminus of NE components and associated proteins in human diseases Nesprins is sufficient to target the proteins to the NE. Journal of Cell Science 3428 Journal of Cell Science 118 (15) perinuclear space. In addition, the SUN domain may have different functions in C. elegans compared to higher eukaryotes. In fact, in C. elegans the SUN domain of UNC-84 is directly involved in the NE recruitment of UNC-83 a nuclear transmembrane protein, which is essential for proper nuclear migration (Starr et al., 2001; Lee et al., 2002). To date no orthologue of UNC-83 is known in higher eukaryotes. Irrespective of SUN domain function in various organisms, its conservation in evolution implies that Sun1 exhibits additional functions besides the tethering of Nesprins to the NE. Sun1 is an inner nuclear membrane protein By performing digitonin experiments we demonstrated the presence of Sun1 in the inner membrane of the NE. Our results are in accordance with a nuclear envelope proteomics approach, which identified Sun1 as an integral membrane protein of the inner nuclear membrane (Dreger et al., 2001). Similar findings were also obtained for its paralogue Sun2, also a type II transmembrane domain protein (Hodzic et al., 2004). Moreover, experiments involving proteinase K protection assays and digitonin experiments with Sun2 suggested the presence of the C-terminus in the perinuclear space whereas Fig. 8. Lamin A/C does not influence the NE localisation of Sun1. the N-terminus points to the nucleoplasm thus allowing an (A-C) Wild type (A) and lamin A/C knockout (B and C) mouse interaction with the nuclear lamina (Hodzic et al., 2004). dermal fibroblasts were transfected with the mouse GFP-Sun1 and Because of the overall similarity between Sun1 and Sun2 we GFP-Sun1-N+2TM fusion proteins. Transiently transfected cells assume a similar topology for the Sun1 full-length protein. were processed for direct fluorescence microscopy. Note that both This arrangement is required to allow the interaction of the GFP fusion proteins localise to the nuclear envelope in the absence –/– of lamin A/C (B and C). (D-F) Lamin A/C fibroblasts were SD2 domain with the luminal domain of Nesprins. transfected with plasmid encoding V5-tagged human Sun1 and processed for immunofluorescence using antibodies to V5 and the LAP2α. (G-I) HeLa cells stably expressing human Sun1 (V5-tagged) The NE localisation of Sun1 at the NE does not depend were transiently transfected with a plasmid coding for the dominant on lamin A/C negative GFP-lamin B1Δ2+ protein and stained for the V5 epitope. The proper localisation of full-length or the N-terminal half of Images were obtained by confocal microscopy. Bars, 6 μm. Sun1 in lamin A/C knockout fibroblasts demonstrated that lamin A/C is not required for the NE localisation of Sun1. Furthermore, we showed that the overexpression of C-terminal Moreover, disruption of a functional lamin A/C system had no Nesprin peptides caused a dominant-negative effect on the detectable effects on the proper localisation of Sun1. The C. distribution of endogenous Nesprins provoking their elegans UNC-84 requires the B-type Ce-lamin for its envelope displacement from the NE. These results not only suggest that localisation (Lee et al., 2002). Thus localisation of mammalian their NE retention mechanism may be the same, but in addition Sun1 at the NE may also depend on B-type lamins. In contrast, C-terminal isoforms may have regulatory functions. both Nesprin-1 and Nesprin-2 localisation at the NE depend on Temporary and spatially controlled expression of Nesprins-1α, lamin A/C, and NE-targeting of human Sun2 also requires a β and Nesprin-2α-γ may allow a modulation of Nesprin-based functional lamin A/C network (Libotte et al., 2005) (our contacts to the actin cytoskeleton, as their presence would unpublished data). Therefore, those findings suggest that result in the reduction or absence of the large ABD-containing additional proteins are implicated in the tethering of Nesprins isoforms from the NE. at the NE. Whether additional SUN domain-containing Based on data from C. elegans demonstrating a genetic proteins in addition to Sun1 are involved in those associations interaction between C. elegans ANC-1 and UNC-84 (Starr and warrants further investigation. Han, 2002), we studied the NE anchorage of Nesprins by the UNC-84 orthologue in higher eukaryotes. Yeast two-hybrid Sun1 connects through the Nesprin proteins the nucleus and GST pull-down experiments demonstrated that a region to the cytoskeleton termed SD2, composed of amino acids 632-737 of Sun1 does indeed interact in vivo and in vitro with both Nesprin-1 and Our studies support a model whereby Nesprin-1 and Nesprin- Nesprin-2. In C. elegans, missense mutations in or near the 2 are anchored at the nuclear envelope through a Sun1- SUN domain of UNC-84 probably disrupt the capacity of SD2 mediated interaction (Fig. 9). Although the N-terminus of Sun1 to associate with ANC-1. Our yeast two-hybrid data suggest, may provide a link to the nuclear lamina, its C-terminal however, that the SUN domain is not the main Nesprin subdomains are implicated in intramolecular (SD1) and interaction domain. It may well be that these particular SUN intermolecular (SD2 and SUN) perinuclear space interactions. domain mutations affect the proper folding of the protein Whereas SD2 was identified as the Nesprin binding domain we resulting in non-functional C-terminal domains in the failed to assign a specific function to the evolutionarily Journal of Cell Science Sun1 interacts with Nesprins 3429 proximity of the two NE membranes. Recent evidence implicates TorsinA in connecting the NE to the cytoskeleton. Torsin A an AAA+ ATPase of the ER localises to the NE when mutated in the neurological human disorder early-onset torsion dystonia (Goodchild and Dauer, 2004; Naismith et al., 2004; Gerace, 2004). TorsinA mutants affect several aspects of NE structure, including its morphology, perinuclear spacing and nuclear pore distribution (Naismith et al., 2004). Whether TorsinA modulates the Sun1/Nesprin interactions needs to be seen. In any case, molecular interactions governing and regulating the connections between the nucleoskeleton and cytoskeleton seem to be highly complex and we may have encountered only the tip of the iceberg. Nuclear envelope proteomic approaches suggest the presence of 67 novel or uncharacterised nuclear membrane proteins (Schirmer et al., 2003), which might potentially be involved in these linker complexes. The human KIAA0810 was obtained from the Kazusa DNA Research Institute, Japan. We acknowledge the gift of GFP-B1Δ2+ vector by Chris Hutchison, University of Durham, UK. Wild-type and lamin-A knockout mice skin fibroblasts were kindly provided by Colin Stewart (NCI, Frederick). The pSHAG-FF1 plasmid was a generous gift of Greg Hannon, Cold Spring Harbor Laboratory, USA. Fig. 9. Model illustrating the interactions of Sun1 with Nesprins at Anti-LBR antibodies were a kind gift of Harald Hermann, DKFZ, the nuclear envelope. Unknown nuclear envelope proteins and Heidelberg, Germany. This work was supported by grants from the interactions are indicated by X and ?, respectively. To reduce Austrian Science Research Fund (FWF, P15312) to R.F.; by grants complexity a homotypic dimerisation of Sun1 via the coiled-coil from the CMMC and the DFG (SFB 589) to A.A.N. and by a grant regions is postulated, although other coiled-coil-containing proteins from the Maria-Pesch-Foundation to I.K. might form heterotypic complexes with Sun1. INM, inner nuclear membrane; LD, luminal domain; N, N-terminal domain; ONM, outer nuclear membrane; PNS, perinuclear space. References Adam, S. A., Marr, R. S. and Gerace, L. (1990). Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell conserved SUN domain. Additional studies will be needed to Biol. 111, 807-816. Apel, E. D., Lewis, R. M., Grady, R. M. and Sanes, J. R. (2000). Syne-1, a elucidate its biological significance. Unlike many other NE dystrophin- and Klarsicht-related protein associated with synaptic nuclei at proteins such as lamins, emerin and Sun1, Nesprins are present the neuromuscular junction. J. Biol. Chem. 275, 31986-31995. on both sides of the NE. Their presence at the inner nuclear Burke, B., Mounkes, L. C. and Stewart, C. L. (2001). The nuclear envelope membrane, is substantiated in particular by electron in muscular dystrophy and cardiovascular diseases. Traffic 2, 675-683. microscopy studies and by the physical association of Nesprins Burkhard, P., Stetefeld, J. and Strelkov, S. V. (2001). Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82-88. with the inner nuclear membrane proteins lamin A/C and Cottrell, J. R., Borok, E., Horvath, T. L. and Nedivi, E. (2004). CPG2: a emerin (Mislow et al., 2002b; Libotte et al., 2005; Zhang et al., brain- and synapse-specific protein that regulates the endocytosis of 2005). The absence therefore of Nesprin-2 staining in Sun1- glutamate receptors. Neuron 44, 677-690. silenced cells strongly suggests that Sun1 recruits and Dechat, T., Gotzmann, J., Stockinger, A., Harris, C. A., Talle, M. A., Siekierka, J. J. and Foisner, R. (1998). Detergent-salt resistance of integrates both outer as well as inner nuclear membrane LAP2alpha in interphase nuclei and phosphorylation-dependent association Nesprin-2 pools through their luminal domains (Fig. 9). Such with chromosomes early in nuclear assembly implies functions in nuclear a scenario is substantiated by the ability of Sun1 to oligomerise structure dynamics. EMBO J. 17, 4887-4902. and by the fact that Nesprin-2 appears as clusters along both Dechat, T., Korbei, B., Vaughan, O. A., Vlcek, S., Hutchison, C. J. and sides of the nuclear membrane in HaCaT cells (Libotte et al., Foisner, R. (2000). Lamina-associated polypeptide 2α binds intranuclear A- type lamins. J. Cell Sci. 113, 3473-3484. 2005). At the moment however, it is not clear how such a Dreger, C. K., Konig, A. R., Spring, H., Lichter, P. and Herrmann, H. structural crossbridging is established at the molecular level. (2002). Investigation of nuclear architecture with a domain-presenting Are identical domains such as SD2 in Sun1 implicated in those expression system. J. Struct. Biol. 140, 100-115. associations? If so, is the SD2 domain flexible enough to allow Dreger, M., Bengtsson, L., Schoneberg, T., Otto, H. and Hucho, F. (2001). Nuclear envelope proteomics: novel integral membrane proteins of the inner such interactions? Or, do different Sun1 domains recruit nuclear membrane. 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Published: Aug 1, 2005

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