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First published online 23 April 2003
doi: 10.1242/jcs.00422

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Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton

Sang W. Oh^*, Robert K. Pope, Kelly P. Smith, Jessica L. Crowley, Thomas Nebl, Jeanne B. Lawrence and Elizabeth J. Luna^¶

Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA
^* Present address: Central Research Institute of BodiTech, Inc. Chuncheon, Kangwon-Do 200-160, South Korea
Present address: Department of Biological Sciences, Indiana University South Bend, South Bend, IN 46634, USA
Present address: Department of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, VIC 3050, Australia

View larger version (31K): [in a new window]   Fig. 1. Muscle contains a 250 kDa F-actin binding protein that is related to supervillin. (A) Two antibodies (-H340, -pepA) against human supervillin (asterisk) recognize a larger protein in muscle cells (line). Immunoblots of human cervical carcinoma (HeLa S3) cells (lane 1), human 50MB-1 myoblasts (lane 2), hamster skeletal muscle (lane 3) and rabbit skeletal muscle (lane 4) were probed with affinity-purified rabbit polyclonal -H340 (lanes 1-3) or -pepA (lane 4) antibodies. Each lane of the 5% polyacrylamide SDS-gel was loaded with 100 µg total protein. Supervillin is a 205 kDa polypeptide in non-muscle cells (lane 1, asterisk). (B) Specificity of the -H340 antibody on immunoblots. Affinity-purified -H340 antibody (0.5 µg/ml) was pre-incubated for 1 hour at 0°C without (–) or with (+) bacterially expressed H340 protein (60 µg/ml) before incubation with blot strips containing rabbit skeletal muscle (100 µg/lane) and visualization by ECL. Staining of both the major 250 kDa polypeptide (line) and a less-prominent 95 kDa band are competed by immunogen. Progressive loss of the larger band with time suggests proteolytic degradation. (C) Specificity of the -H340 antibody in immunofluorescence. Phase images (a,c) and indirect immunofluorescence micrographs (b,d) of proliferating 50MB-1 cells stained with affinity-purified -H340 and secondary antibodies. To show specificity, the -H340 antibody used for C and D was pre-incubated with 20 µg/ml of the H340 immunogen for 1 hour before use. Bar, 5 µm. (D) Direct binding of 32P-labeled F-actin to the 250 kDa supervillin-like protein from mouse muscle. Immunoprecipitation with rabbit IgG as a negative control (lane 1) and with -H340 (lane 2). The polypeptide specifically immunoprecipitated by -H340 IgG binds both 32P-labeled F-actin (top panel) and -H340 antibody (lower panel). (E) Co-fractionation of the 250 kDa supervillin-like protein with dystrophin and caveolin-3 in the crude plasma membrane fraction from rabbit skeletal muscle (Ohlendieck et al., 1991). Immunoblots with antibodies against H340, dystrophin and caveolin-3 in a higher-density membrane fraction enriched in T-tubules and sarcoplasmic reticulum (lane 1, Dense) and in the low-density membrane fraction enriched in sarcolemmal membranes (lane 2, Light) are shown.

View larger version (60K): [in a new window]   Fig. 2. Cloning of muscle supervillin (archvillin) and northern analyses. (A) Diagram showing the coding regions (boxes), the 5'- and 3'-UTRs, and the sequences used as an archvillin-specific probe (probe 1) and as a probe for both archvillin and supervillin messages (probe 2). Lines represent partial sequences derived from gene-specific primers (no arrows) and 5'- (left arrows) and 3'- (right arrows) RACE products from skeletal muscle cDNAs. Archvillin-specific sequences in the 5'-halves of the coding regions (gold), predicted nuclear targeting signals (blue), and coding sequences for the highly conserved villin/gelsolin homology regions at the 3'-ends of the coding regions (pink) are indicated. Asterisks on clones M03 and M08 denote the location of an additional 23 bp of sequence. Consensus sequences of these human and murine cDNAs are available (accession nos AF109135, AF317422), as is the potential alternatively spliced murine cDNA sequence (AF317423). (B) Multiple tissue northern blot (Clontech) containing 2 µg per lane of poly(A)+ RNA from heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7) and pancreas (lane 8) after hybridization with 32P-labeled probe 2. The blot was exposed to XAR/MS film at –80°C with an intensifying screen for 24 hours. (C) Lanes from the same northern blot containing heart (lane 1) and skeletal muscle RNA (lane 6) after exposure to film at –80°C for only 3 hours. (D) The same northern blot after stripping and re-probing with archvillin-specific probe 1. (E) Hybridization with ß-actin probe to show equal RNA loading. Migration positions of archvillin message are shown (AV, asterisks), as are the positions of supervillin mRNA (SV, arrowheads).

View larger version (56K): [in a new window]   Fig. 3. Muscle-specific sequences in human and murine archvillin. (A) Schematic representation of archvillin cDNA and protein domain structure showing the amino acid similarity of each domain in the predicted proteins (percentages), the amino acid sequences of the two muscle-specific coding inserts, the five predicted nuclear targeting sequences (blue bars), the predicted coiled-coil domain (green patterned box) and the location of a 23 bp insert sequence found in clones M03 and M08 (asterisk). Gold shading denotes the relative extents of the two N-terminal muscle-specific inserts and the highly conserved C-terminal villin/gelsolin homology regions. If stable protein can be produced from clones M03 and M08, the C-terminal sequence after Q-1948 in mouse archvillin would be altered to ALFSFLWKILEVLTSRPACSSSAAPPETSLRQSLCTPHKRPLPSAPCLSCRRTCTARRSQLSSLLTTITRCTSGKAGGPLKTR, resulting in a smaller, more basic protein (227 kDa, pI=8.16) without the villin-like headpiece. (B) Fluorescence localization in C2C12 myotubes expressing a chimera of EGFP and murine archvillin insert 1 sequence (MAV 257-629, left) or EGFP alone (right) is consistent with the prediction that muscle insert 1 contains functional nuclear targeting sequences. Bar, 5 µm. (C) The muscle-specific upstream exon M-3 contains regions of high sequence similarity. Regions of 254 nt and 34 nt in exon M-3 that exhibit 88% and 94% identity, respectively, between human and mouse archvillin cDNAs are shown (arrows). Intron locations are denoted schematically by thick vertical bars, and intron sizes are shown. Exons –2 and –1 are present in both muscle and non-muscle cDNAs. Exon M-3 is consistently observed in muscle-specific cDNAs and in ESTs from muscle-rich tissues. Exon nomenclature was based on the location of the initiator AUG because the large size of the first muscle-specific intron and the growing number of ESTs with homology to other upstream genomic sequences suggest the potential for many other exons encoding supervillin/archvillin 5'-UTR sequences. Upstream open reading frames (uORFs), two of which are conserved across species (thick bars) are present in human (HAV) and murine (MAV) archvillin 5'-UTRs.

View larger version (98K): [in a new window]   Fig. 4. Endogenous archvillin mRNA is clustered at the ends of myotubes, but no clustering is observed for supervillin mRNA in polarized human fibroblasts or for archvillin mRNA in myoblasts. (A) WI-38 fibroblasts, (B) 50MB-1 myoblasts and (C,D) 50MB-1 myotubes were fixed, probed with digoxigenin-labeled sequences conserved in human supervillin and archvillin mRNAs, and visualized with rhodamine-labeled anti-digoxigenin. Note the strong granular staining at the tips of myotubes (arrows); the most proximal of the centrally localized nuclei is indicated (nucleus). Bars, 5 µm.

View larger version (114K): [in a new window]   Fig. 5. Archvillin mRNA localizes to the tips of myotubes, in contrast to the more central distribution observed for mRNA encoding the ß-cardiac myosin heavy chain. Double labeling of a human 50MB-1 myotube with probes for (A) archvillin and (B) myosin heavy chain mRNAs. (C) The composite image shows archvillin (red), myosin heavy chain (green) and nuclear DNA (blue). Archvillin mRNA was hybridized using a cDNA probe labeled with digoxigenin-11-dUTP and detected with rhodamine-conjugated anti-digoxigenin; mRNA encoding the ß-cardiac myosin heavy chain was hybridized with a nick-translated probe containing biotin-16-dUTP and detected with FITC-conjugated avidin. DNA was labeled with 4',6-diamidino-2-phenylindole (DAPI). The most proximal of the centrally localized nuclei is at the left of the image. Bar, 5 µm.

View larger version (113K): [in a new window]   Fig. 6. Archvillin in hamster skeletal muscle is primarily sarcolemmal, localizing at costameres and within peripheral nuclei. The -H340 signal localizes predominantly as `arches' in optical cross-sections (B,D, double arrowheads) that resolve as alternating circumferential thick and thin bands with occasional longitudinal striations in confocal sections within the plane of the sarcolemma (C, inset, arrow). Archvillin colocalizes with costameric dystrophin in both transverse (D-F) and oblique (G-I) muscle sections. Colocalization with ethidium homodimer-1 in peripheral nuclei also is seen (J-L, arrows), as is a small amount of staining within the muscle cells (M-O, asterisks). Phase (A), epifluorescence (B) and confocal fluorescence sections (C-O) show the colocalization of archvillin (B,C,D,G,J,M) with anti-dystrophin (E,H) and ethidium homodimer-1 (K,N). Composite images (F,I,L,O) were generated by superimposition of the -H340 signals in green and dystrophin (E,H) and ethidium homodimer-1 (K,N) signals in red; areas of overlap appear yellow or orange. Bars, 5 µm (C), 10 µm (A-B,G-I) and 20 µm (D-F,J-O).

View larger version (55K): [in a new window]   Fig. 7. Archvillin localizations in 50MB-1 myoblasts. Confocal sections of human 50MB-1 myoblasts showing -H340 signals within nuclei (A-C), in association with actin filament bundles near the basal plasma membrane (D-I) and as punctae on basal membrane surfaces (J-L). Double-label immunofluorescence of archvillin (A,D,G,J) with anti-lamin A/C (B), AlexaTM 594-labeled phalloidin (E,H) and anti-vinculin (K). Composite images (C,F,I,L) were generated by superimposition of the -H340 signals in green and lamin (C), F-actin (F,I) and vinculin (L) signals in red; areas of overlap appear yellow. Arrows denote localizations at the ends of actin filaments (G-I) and occasional sites of colocalization with vinculin at the basal membrane (J-L). Bar, 20 µm.

View larger version (85K): [in a new window]   Fig. 8. EGFP-tagged full-length murine archvillin (MAV-FL) colocalizes with endogenous archvillin, F-actin and non-muscle myosin II. Confocal fluorescence images showing C2C12 myoblasts visualized for (A) endogenous archvillin localization with -H340 in nuclei (arrowhead) and at the plasma membrane (arrow); (B,C) colocalization of EGFP and -H340 signals in cells expressing low (arrows) and moderate (arrowhead) levels of EGFP-tagged archvillin; and colocalization of (D,G) EGFP-tagged full-length archvillin with (E) AlexaTM-594 phalloidin-stained F-actin or (H) antibodies against non-muscle myosin II heavy chain. Composite images (F, I) were generated by superimposition of the -H340 signals in green and phalloidin (E) or nonmuscle myosin II (H) signals in red; areas of overlap appear yellow. Myoblasts in panels G-I were pre-extracted with 0.2% Triton X-100 (5 minutes, 0°C) to better visualize cytoskeletal structures. Bar, 5 µm.

View larger version (77K): [in a new window]   Fig. 9. Increased amounts and polarization of endogenous and EGFP-tagged murine archvillin in myotubes. Confocal images showing increased amounts of archvillin staining in untransfected myotubes (A,B), as compared with adjacent myocytes (asterisks). In addition to the overall increase in staining, large clusters of fluorescence are observed for endogenous archvillin (B) and for EGFP (C,E) and -H340 signals (D,F) at the tips of myotubes expressing low levels of EGFP-tagged murine archvillin (EGFP-MAV). In both small (C,D) and large (E,F) myotubes, EGFP and -H340 archvillin signals colocalize at myotube tips (arrows) and as punctae along the membrane and within the cell. Bars, 20 µm.

View larger version (13K): [in a new window]   Fig. 10. Inhibition of myotube formation by EGFP-tagged murine archvillin sequences. EGFP-tagged proteins encoding the designated murine archvillin amino acids were transfected into C2C12 myoblasts, and the percentages of expressing cells that were multinucleate were scored after 6 days in differentiation medium. Column heights and error bars represent the means and standard deviations, respectively, of four separate experiments. Statistical significance was assessed with the Tukey-Kramer multiple comparisons test and by Student's t-tests. Both analyses indicated that C2C12 cells transfected with EGFP-tagged murine archvillin sequences 1-1353, 1-739 and 257-739 were significantly less likely to be present in myotubes than were cells similarly transfected with either the EGFP tag alone or with EGFP-tagged full-length murine archvillin (*P<0.001).