QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, Q. Articles by Shanahan, C. M. Search for Related Content PubMed PubMed Citation Articles by Zhang, Q. Articles by Shanahan, C. M.

Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues

¹ Department of Medicine, Division of Cardiovascular Medicine, University of Cambridge, Box 110, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, UK
² Department of Anatomy, Multi-Imaging Centre, Tennis Court Rd, Cambridge, CB2 3DY, UK
³ Centre for Veterinary Science, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK
⁴ Randall Centre for the Molecular Mechanism of Cell Function, Kings College, New Hunts House, Guy’s Campus, London, SE1 1UL, UK
⁵ Division of Medical & Molecular Genetics, GKT Medical School, 8th Floor, Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK

View larger version (37K): [in a new window]   Fig. 1. Cloning and expression of rat nesprin-1. (A) Northern blot showing high expression of a 10 kb transcript of rat nesprin-1 (1RA1) in aortic tissue compared with cultured, passage 12, dedifferentiated VSMCs and fibroblasts. (B) Semi-quantitative RT-PCR analysis of rat nesprin-1 (1RA1), showing an increase in expression during developmental differentiation of the aorta. f17, foetal day 17; f19, foetal day 19; n1, neonatal day 1; w2-w8, weeks 2 to 8; ad, adult and c, negative control. The graph shows relative expression of 1RA1 at each stage normalized to a GAPDH control. (C) Schematic representation of isolation of rat nesprin-1 cDNA sequence.

View larger version (69K): [in a new window]   Fig. 2. Human nesprin-1 expression and gene analysis. (A) Northern blot of nesprin-1 expression in human tissues showing transcripts at 10.7 kb and 3.8 kb. The ß-actin loading control is shown. (B) Gene structure of human nesprin-1. The numbered boxes indicate exons, the horizontal lines indicate introns. Nesprin-1 comprises 19 exons, starting at exon 45 and excluding exon 47; nesprin-1ß comprises 61 exons (excluding exons 45, 47 and 55); nesprin-12 comprises18 exons and is initiated in exon 47, which contains an additional 31 N-terminal residues. (C) The deduced amino acid sequence of human nesprin-1 and -1ß. The first methionine of nesprin-1 and -1ß are shown in bold and underlined, the spectrin repeats (SR) are shown by numbered grey boxes. The open box indicates the TM region and the dashed open boxes indicate NLS. The amino acids encoded by exon 55 are shown in bold, whereas the open triangle indicates the insertion site of the 31 residues encoded by exon 47 (not shown). GenBank accession numbers for human nesprin-1 and 1ß are AY061756 and AY061755, respectively.

View larger version (65K): [in a new window]   Fig. 3. Characterization and expression analysis of human nesprin-2. (A) Northern blot analysis of nesprin-2 expression in human tissues. Major transcripts at 11.1 kb, 3.4 kb and 2.7 kb are indicated. The ß-actin loading control is shown. (B) RT-PCR of human nesprin-2 gene expression in aorta indicated that the larger 2 transcript was present in these cells. (C) Gene structure of human nesprin-2. Nesprin 2 comprises 11 exons (55-65), nesprin-2ß is initiated in exon 49, which contains only 5' UTR and comprises 16 exons (excluding exon 56). Nesprin-2 comprises 63 exons (excluding exons 49 and 56). Alternatively spliced exons using cryptic splice sites are shown as open boxes. Alternate splicing at these sites produces transcripts 22 and 2ß2. (D) The deduced amino acid sequence of human nesprin-2, -2ß and -2. The first methionines of nesprin-2, -2ß and -2 are shown in bold and underlined. The 23 residues encoded by exon 56 and specific to nesprin-2 are shown in bold. The open triangle shows the insertion site for 14 residues exclusive to 22 and encoded by exon 61 spliced at a cryptic splice site (hatched box in 3C). The spectrin repeats (SR) are shown by numbered grey boxes, the open box indicates the TM region and the dashed open box indicates NLS. GenBank accession numbers for human nesprin-2, -2ß and -2 are AY061758, AY061757 and AY061759, respectively.

View larger version (54K): [in a new window]   Fig. 4. Predicted protein structure of nesprins-1 and -2. (A) Human nesprin protein domains generated by SMART and ProfileScan programmes. (B) Alignment of the 21 spectrin repeats of the nesprin-1 protein, showing homology domains within the three helices of each repeat. All G (orange) and P (yellow) residues are coloured. Other colouring is by conserved property in >55% of any column: uncoloured residues lack a sufficiently conserved property. Blue, hydrophobic residues; purple, negative residues, green, hydrophilic residues. (C) TMpred analysis showing the predicted hydrophobic TM domain in nesprin-1. (D) Alignment of the 60 C-terminal residues of nesprins showing homology with Drosophila Klarischt and C.elegans (unknown). Note the lack of sequence homology with C-terminal TM domains (shown in bold) found in other NE proteins, including emerin and myoferlin.

View larger version (26K): [in a new window]   Fig. 5. (A) Nesprin-EGFP fusion constructs and (B) their subcellular localization in transfected COS-7 cells as indicated. (IX) EGFP vector alone.

View larger version (34K): [in a new window]   Fig. 6. In vitro transcription/translation of human nesprin-1 cDNA resulted in a 112 kDa product (lane 1). Translation reactions were as follows: nesprin-1 cDNA (lanes 1-9) and controls S. cerevisiae -factor (10-11) and ß-lactamase cDNA (12-13); either in the absence (1-2, 10,12) or in the presence of canine pancreatic microsome (3-9, 11,13); added either co-translationally (3-5, 11,13) or post-translationally (6-9). Reactions were digested with Proteinase-K alone (2, 4 and 7) or with 1% Triton X-100 (5). Translation products were subjected to sedimentation and separated into pellet (lane 8) and supernatant (lane 9) fractions. In these experiments, two controls were used to confirm microsome functionality. S. cerevisiae -factor exhibits an increase in molecular weight due to being N-glycosylated, and pro-ß-lactamase undergoes signal peptide cleavage to produce the lower molecular weight mature form.

View larger version (36K): [in a new window]   Fig. 7. (A) Schematic representation of the positions of the five peptides used for generating polyclonal antibodies against nesprin-1. The peptides N1 and N2 were specific to nesprin-1ß but did not produce functional antibodies. The small black boxes indicate peptides. (B) In vitro transcription and translation of human nesprin-1 cDNA. The expression vector pcDNA3.1 containing the full-length cDNA of human nesprin-1 (112 kDa), the luciferase (61 kDa) T7 control cDNA as a positive control, the TNT lysate reaction without DNA as negative control. (C) Immunoprecipitation of human nesprin-1 generated from radioactive in vitro transcription/translation using antibodies C1 that recognised a 112kDa protein and N1, a non-specific negative control. (D) Western blot of human nesprin-1-EGFP fusion protein in transfected COS-7 cells. Control; untransfected COS-7 cells, GFP: pEGFP-C1 vector alone and nesprin 1-pEGFP-C1. (E) Western blot of human VSMC (I) and peripheral blood leukocyte (II) cell lysates using antibodies N3 and C1. The 112 kDa and 380 kDa bands are indicated by arrows. (F) Immunoprecipitation of C2C12 cells (I) using antibodies N3 and C1. Rabbit IgG as negative control. Western blot of C2C12 whole cell lysates (II) indicating that many of the IP products are also identified on western blots. (G) Immunofluorescent staining of nesprin-1 in mouse C2C12 myoblasts and human VSMCs. Endogenous nesprin-1 (green) was detected by nesprin-1 antibodies (N3, C1 and C2) and visualised by confocal microscope. DAPI is shown as false colour red in composite.

View larger version (78K): [in a new window]   Fig. 8. Subcellular colocalization of endogenous nesprin-1 in C2C12 myoblasts (A,C) and human VMSCs (B,D,E). Cytoskeletal -actin (A), ER protein calnexin (B), and NE proteins, emerin (C), LAP1 (D), lamin A/C (E). Nesprins are shown in green, other markers in red (as indicated) and the nucleus in blue. Yellow in merged images indicates regions of colocalization.

View larger version (127K): [in a new window]   Fig. 9. Immunogold localization of nesprin-1 in C2C12 myoblasts. Gold particles (shown as black) localized along the NE are arrowed. Gold particles are also present (arrows) in heterochromatic regions of the nucleus (N) and are absent from euchromatic zones. In some cells, prominent localization to the nucleolus (Nu) was observed (B), as indicated by arrowheads.

View larger version (64K): [in a new window]   Fig. 10. Subcellular colocalization of Nesprin-1 in human tissues and in C2C12 differentiation. (A) Immunofluorescence of nesprin-1 in human tissues; aorta (I-III), heart (VI-VII), skeletal muscle (VIII-IX), spleen (X-XI) and leukocytes from peripheral blood (XII-XIII). Nesprins are shown in green, emerin and myosin in red, the nucleus in blue and rabbit IgG is a negative control (IV-V). (B) Immunolocalization of nesprin-1 in mouse C2C12 myoblasts (I-III) and myotubes (V-VII). Nesprin-1 is shown in green, emerin (IV and VIII), -actin and myosin are shown in red.