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First published online 25 January 2005
doi: 10.1242/jcs.01642

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Nesprin-2 is a multi-isomeric protein that binds lamin and emerin at the nuclear envelope and forms a subcellular network in skeletal muscle

¹ Department of Medicine, ACCI, Box 110, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK
² Multi-Imaging Centre, Department of Anatomy, Downing Street, Cambridge, CB2 3DY, UK
³ Division of Medical and Molecular Genetics, GKT Medical School, 8th Floor, Guy's Tower, Guy's Hospital, London, SE1 9RT, UK
⁴ Randall Centre for the Molecular Mechanism of Cell Function, Kings College, New Hunts House, Guy's Campus, London, SE1 1UL, UK

View larger version (19K): [in a new window]   Fig. 1. Domain structure of the major nesprin-2 isoforms identified to date. Isoforms lacking the transmembrane/KLS domain are designated TM. The size of the mRNA in kb is shown as well as the predicted molecular mass in kDa for each isoform. Regions shown in green are predicted to form a continuous rod domain in the presence or absence of amino acids encoded by the small alternatively spliced exons shown in green with an asterisk in Fig. 2.

View larger version (24K): [in a new window]   Fig. 2. Diagrammatic representation, drawn to scale, of the intron/exon splicing events that generate all nesprin-2 isoforms identified to date. Sites of alternate initiation and termination as well as alternatively spliced coding exons along the length of the giant isoform are shown. Coding exons are blue while alternatively spliced coding exons are green and starred. These are not specific to any isoform but utilized in some tissues in a number of isoforms. The 5' and 3' UTRs of each isoform are shown in yellow and red, respectively. Introns are represented by lines, with the majority of introns 1, 2 and 45, omitted because of size constraints.

View larger version (50K): [in a new window]   Fig. 3. (A) Northern blots showing nesprin-2 isoform expression in human tissues. Note that a >20 kb band (asterisks) is present in spleen using 5' UTR (1-333 bp)-, 3' UTR (20,673-21,364 bp)- and CH-domain (161-1787 bp)-specific probes confirming that the giant isoform predicted by RT-PCR is transcribed. Highly expressed short transcripts are present in skeletal muscle, heart and pancreas with numerous transcripts of different sizes expressed at low levels in most tissues. (B) Table showing tissue distributions and levels of expression of specific nesprin-2 domains and isoforms as determined by RT-PCR in various human tissue cDNA samples (for details, see Fig. S1 in supplementary material).

View larger version (77K): [in a new window]   Fig. 4. (A) Western blot of different human tissues using nesprin-2 N2, N3 and CH3 antibodies demonstrating tissue-specific and multiple isoforms in heart (H), kidney (K), skeletal muscle (S) and testis (T). Note the dominant small isoforms and the similar pattern of bands using the N2 antibody in heart and skeletal muscle and the multiple bands in testis; consistent with northern blot results. (B) Multiple isoforms were identified by all antibodies in vascular smooth muscle cells (V) and fibroblasts (F) with these cell types having identical isoform patterns on western blots. All bands were competed by excess peptide confirming antibody specificity. (C) 35S-labelled in vitro translation of nesprin-1 (asterisk), 2 (cross) and 2 ß(diamond) (first panel) and subsequent western blot analysis demonstrates specificity of N2 (middle panel) and N3 (end panel) antibodies. N2 recognised nesprin-2 and -2ß while N3 recognised only nesprin-2ß. Neither antibody recognised the closely related nesprin-1. Luciferase () (D) 35S-labelled in vitro translation of nesprin-1 and nesprin-2 CH domains (left panel) and western blot using the CH3 antibody (right panel) shows that CH3 recognises only the nesprin-2 domain and not the closely related nesprin-1 domain confirming antibody specificity. Multiple unlabelled bands on western blots in C and D represent rabbit nesprin-2 isoforms present in the rabbit reticulocyte lysate. (E) Western blot using nesprin-2 N2, N3 and CH3 antibodies and sub-cellular fractions of human VSMCs to identify nesprin-2 isoforms in cytoplasmic and nuclear domains. Predicted sizes of isoforms identified from cDNA analysis are marked with arrowheads and it is probable that the N2 antibody identifies nesprin-2 and ß isoforms. (F) Control blot shows -SM actin, SF-2 and emerin to confirm cytoplasmic, nuclear and NE fractionation. Cyto, cytoplasmic; NS, nuclear soluble; NE, nuclear envelope.

View larger version (74K): [in a new window]   Fig. 5. (A-C) Immunofluorescent images showing the sub-cellular localization of nesprin-2 isoforms using N2, N3 and CH3 antibodies co-localized with lamin, in human VSMCs. All three antibodies localize to domains in the nucleus with N2 and CH3 showing nuclear rim staining and N2 and N3 abundant around nucleoli (identified by domain-specific antibody; not shown). Cytoplasmic staining was also observed using all three antibodies and this is shown in detail using human skin fibroblasts. (D-F) In the cytoplasm of skin fibroblasts, nesprin-2 is diffusely distributed in a reticular pattern and is localized to lamellipodia (arrow in D), to focal adhesions (arrows in E) and to filopodia (arrow in F; red stain shows ß-tubulin). All stainings were competed completely by excess peptide (not shown).

View larger version (64K): [in a new window]   Fig. 6. In vitro co-immunoprecipitation (IP) assays demonstrating (A) nesprin-2 and -2ß binding to lamin A and (B) emerin. IP of nesprin-2ß with lamin A and emerin could be demonstrated with both N2 and N3 antibodies (N2 only shown). (C) Binding of nesprin-2 or -2ß to lamin C could not be demonstrated. (D) Although both nesprin-1 and nesprin-2 bind lamin A, nesprin-2 does not bind to nesprin-1 (D).

View larger version (31K): [in a new window]   Fig. 7. In vivo co-immunoprecipitation assays demonstrating nesprin-2 binding to lamin-A/C (A) and emerin (B) (arrow) using the N3 antibody and VSMC lysates.

View larger version (89K): [in a new window]   Fig. 8. Mislocalization of nesprin-2 isoforms in SW13 cells, and relocalization in SW13/20 cells. Nesprin-2 isoforms recognised by both the N2 (A, upper panel) and CH3 (B, upper panel) antibodies were mislocalized to the ER where they colocalized with emerin in lamin-A-negative SW13 cells. However, in lamin-A-GFP SW13/20 cells nesprin-2 isoforms recognised by these antibodies were correctly re-localized to the NE (A and B, lower panels) with emerin (C). With the N2 antibody there was also some mislocalization of nuclear nesprin-2 isoforms to punctate spots that were more evenly redistributed in lamin-A GFP SW13/20 cells (A).

View larger version (67K): [in a new window]   Fig. 9. The dominant sub-cellular localization of nesprin-2 isoforms changes during differentiation of C2C12 myoblasts to myotubes. (A) In myoblasts (top panel) the predominant N2 staining was nuclear, in a punctate pattern at the NE with only very weak cytoplasmic staining. In myotubes (lower panel) N2 recognised cytoplasmic structures of the muscle sarcomere organised in a striated pattern (arrows). Bottom inset: myosin staining to show differentiation and myofibre formation (small arrow) with N2 located between myosin positive bands. Top inset: the change in nuclear distribution of N2 in myotubes into a less punctate pattern (arrow). (B) Western blot analysis using the nesprin-2 N2 antibody and sub-cellular fractions of C2C12 myoblasts and myotubes demonstrated a shift in nesprin-2 isoform patterns in the cytoplasm and nucleus. The larger cytoplasmic isoforms were more highly expressed in myotubes with a reduction in smaller cytoplasmic isoforms and there was higher expression of two small nuclear isoforms in myotubes than in myoblasts.

View larger version (162K): [in a new window]   Fig. 10. EM immunogold localization of nesprin-2 epitopes in C2C12 myoblasts (A) and myotubes (B-F). In myoblasts (not shown) and myotubes, CH3 was present at the NE (black arrows in B) and in punctate nuclear (white arrow in B) localizations. In myoblasts CH3 localized to punctate zones along the plasma membrane (arrows in A) while in myotubes CH3 also decorated cytoplasmic structures, including isolated filaments associated with the plasma membrane (C). N3 was present in cytoplasmic domains in myotubes and decorated the rim of mitochondria (asterisks in D). In myoblasts (not shown) and myotubes, N2 was present at the NE (arrows in E) and in the cytoplasm at the Z-band (long arrows in F) and in a repeating pattern along the length of the myofibrils of the sarcomere (short arrows in F). Bar, 100 nm.

View larger version (138K): [in a new window]   Fig. 11. Nesprin-2 epitopes are abundant in skeletal muscle. N3 and CH3 epitopes were localised to the sarcomere in a striated pattern and both colocalized in a punctate manner with titin T12 at the Z-line (A-D). N3 was also distributed around the nucleus (inset in C) and was present at the A/I junction where it colocalized with titin-A/I (E-G). The localization pattern for CH3 was essentially the same as that observed for N3 but this epitope was particularly abundant over the A/I junction where it colocalized with titin A/I (H). Nesprin-2 antibodies did not colocalize with myomesin at the M-line (N3 shown, I-K) confirming the Z-line/A/I junction distribution. The N2 antibody recognised the NE and nucleus, and was localized in a punctate pattern throughout the sarcomere in a pattern consistent with N3 and CH3, only weaker (arrow in L). Bar, 5 µm.

View larger version (157K): [in a new window]   Fig. 12. Nesprin-2 epitopes are present in the sarcoplasmic reticulum in human skeletal muscle. Punctate Nesprin-2 N3 epitopes co-localize in a punctate fashion with RyR, a junctional SR marker (A-C). Larger areas of co-localization are apparent with both N3 and CH3 (not shown) and SERCA2, a free SR marker, in longitudinal sections (D-F) and confirmed in cross sections (G-I). Bar, 4 µm.

View larger version (44K): [in a new window]   Fig. 13. Schematic of a muscle cell showing potential localizations/interactions of nesprin-2 isoforms. Isoforms potentially form a network linking the nucleus and organelles to the sarcomere and plasma membrane via the actin cytoskeleton. Continuity of interactions across the NE may occur if SUN domain proteins form a bridge across the nuclear membranes and bind to nesprin-2 isoforms on both the inner and outer faces (not shown).