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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02471

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

¹ Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany
² Center for Molecular Medicine Cologne, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany
³ 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

View larger version (72K): [in a new window]   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-2P (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-2P, 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.

View larger version (29K): [in a new window]   Fig. 2. The C-terminus of Sun1 associates directly with the luminal domains of Nesprin-1 and Nesprin-2. (A) Domain organisation of mouse Sun1. The domain locations as well as their amino acid positions are indicated according to the GenBank entry AAH48156 CC, coiled-coil domain; ZnF, zinc-finger domain; Tm, transmembrane domain. (B) Sun1 polypeptides corresponding to various Sun1 domains were fused to the Gal4 activating domain, whereas the Nesprin-1 luminal domain was fused to the Gal4 DNA-binding domain. The corresponding plasmids were co-transformed into yeast cells and the interactions were assessed by the filter lift ß-galactosidase assay. ++++, strong; ++, weak; –, no blue colour development. (C) Schematic overview of the fusion proteins (GST-LDN-1, GST-LDN-2 and GST-LDN-2P lacking the last 4 aa) used for the GST pull-down assay of COS7 cell homogenates expressing GFP-Sun1-C. LDN-1, luminal domain Nesprin-1; LDN-2, luminal domain Nesprin-2. (D,E) COS7 cell lysates expressing the C-terminal half of Sun1 (Sun1-C) were incubated with the immobilised GST-fusion proteins as indicated and GST for control. Unbound (S) and specifically bound (P) proteins were subjected to SDS-PAGE followed by western blot analysis using GFP-specific mAb K3-184-2.

View larger version (60K): [in a new window]   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 LAP2ß, a known integral inner nuclear membrane protein. Bars, 7 µm.

View larger version (82K): [in a new window]   Fig. 4. Asymmetric distribution of Sun1 and Nesprin-1 at the nuclear membrane. (A-C) Triton X-100 treated COS7 cells subjected to immunofluorescence with Sun1 and lamin A/C antibodies, indicate the nuclear localisation of Sun1 (arrows). Non-specific staining of antibody 281 was observed in the cytoplasm (arrowheads; see also Fig. 3C). (D-F) In digitonin-treated COS7 cells only the non-specific staining remains (arrowheads) suggesting a localisation of Sun1 at the inner nuclear membrane. The integrity of the nuclear membrane is documented by the absence of lamin A/C staining (E). (G-I) In Triton X-100-permeabilised fibroblasts Nesprin-1 antibodies strongly stain the nucleus. (J-L) Nesprin-1 staining at the NE persists after digitonin treatment suggesting the presence of Nesprin-1 at the outer nuclear membrane. Note the absence of lamin A/C staining (K). DAPI was used to counterstain the nucleus. Confocal images are shown. Bars, 5 µm.

View larger version (52K): [in a new window]   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.

View larger version (88K): [in a new window]   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 staining pattern in transfected (arrows) versus untransfected (arrowheads) cells. (G) Histogram illustrating the displacement effects of the GFP-Sun1-TM-C fusion on the endogenous Sun1 and Nesprin-2 proteins. Data were obtained by evaluating 200 transfected cells. Bars, 7 µm.

View larger version (99K): [in a new window]   Fig. 7. Nesprin-2 localisation is affected in cells where Sun1 expression has been silenced by siRNA. (A-C) HeLa cells expressing stably V5-tagged hSun1 and HaCaT cells (D-L) were transiently transfected with a combination of plasmids (pJG173/174) encoding siRNAs targeting hSun1. The distribution of Sun1 (panel A, anti-V5; 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 (indicated by asterisks). In Sun1 knockdown cells, the lamin A/C localisation remained unaltered (B and E), whereas Nesprin-2 staining was either absent or reduced. DNA was stained by DAPI. The images shown were taken by confocal microscopy and merged to visualise colocalisation (panels C,F,I,L). Bars, 10 µm.

View larger version (95K): [in a new window]   Fig. 8. Lamin A/C does not influence the NE localisation of Sun1. (A-C) Wild type (A) and lamin A/C knockout (B and C) mouse dermal fibroblasts were transfected with the mouse GFP-Sun1 and GFP-Sun1-N+2TM fusion proteins. Transiently transfected cells were processed for direct fluorescence microscopy. Note that both 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 transfected with plasmid encoding V5-tagged human Sun1 and processed for immunofluorescence using antibodies to V5 and the LAP2. (G-I) HeLa cells stably expressing human Sun1 (V5-tagged) were transiently transfected with a plasmid coding for the dominant negative GFP-lamin B12+ protein and stained for the V5 epitope. Images were obtained by confocal microscopy. Bars, 6 µm.

View larger version (39K): [in a new window]   Fig. 9. Model illustrating the interactions of Sun1 with Nesprins at the nuclear envelope. Unknown nuclear envelope proteins and interactions are indicated by X and?, respectively. To reduce complexity a homotypic dimerisation of Sun1 via the coiled-coil regions is postulated, although other coiled-coil-containing proteins might form heterotypic complexes with Sun1. INM, inner nuclear membrane; LD, luminal domain; N, N-terminal domain; ONM, outer nuclear membrane; PNS, perinuclear space.

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