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First published online 21 September 2004
doi: 10.1242/jcs.01378

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Smooth muscle archvillin: a novel regulator of signaling and contractility in vascular smooth muscle

¹ Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA
² Cell Dynamics Group, Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue, Worcester, MA 01605, USA
³ Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA

View larger version (26K): [in a new window]   Fig. 8. Quantification of subcellular distribution of CaP and SmAV in differentiated smooth-muscle cells. Fluorescence intensities were quantified from confocal micrographs. In all cases, between 14 and 53 cells were analysed. (Inset) Method used to produce line scans for quantification of ratios. (A) Comparison of the average (with standard error) of surface:core ratios for the distribution of SmAV in the absence and presence of phenylephrine (PE). (B) Comparison of the average surface:core ratios for the distribution of CaP in the absence and presence of PE. (C) Average ratios (+ s.e.m.) of SmAV surface-membrane to cytosol fluorescence intensities in single smooth-muscle cells that were unstimulated or stimulated with PE for 2 minutes, 4 minutes or 10 minutes. ****P<0.0001 compared with unstimulated values.

View larger version (43K): [in a new window]   Fig. 1. Identification of SmAV as a CaP interacting protein. (A) Interactions between SmAV and CaP in reciprocal yeast two-hybrid assays. The growth of yeast host (YRG2) with plasmids or constructs in Trp-, Leu- and His-depleted selective medium. (1) Yeast Host-YRG2. (2) pBD-GAL4 Cam. (3) pBD-GAL4 Cam-CaP. (4) pBD-GAL4 Cam-CaP and pAD-GAL4-2.1-c-SmAV. (5) pBD-GAL4 Cam-c-SmAV. (6) pBD-GAL4 Cam-c-SmAV and pAD-GAL4-2.1-CaP. (7) pBD-GAL4 Cam-c-SmAV and pAD-GAL4-2.1-Cap136. (8) pBDGAL4 Cam-c-SmAV and pAD-GAL4-2.1-CaP163. (B) CaP domains indicating the amino acid residue numbers (ferret sequence) and the domains (Morgan and Gangopadhyay, 2001). Double-headed arrows indicate the deletion constructs used. (C) Expression of LacZ as detected by blue color development with X-gal. Colonies of yeast cells (either host only or transfected with plasmid/constructs) are numbered as in (A).

View larger version (68K): [in a new window]   Fig. 2. Sequence comparisons of SmAV with human supervillin and archvillin. (A, top) The SmAV sequence. The muscle-specific sequence (insert 1) is in yellow (Oh et al., 2003) and the nuclear targeting sequences is in blue. The CaP-binding domain is represented by the red box. (A, bottom) The alignment of N-terminal amino acid sequences of ferret SmAV with those of human supervillin and human archvillin. Identical residues are in black; the second muscle-specific insert in skeletal-muscle archvillin is in green. The three sequences in skeletal-muscle but not smooth-muscle archvillin are boxed and numbered. Residue numbers are given on the right. The SmAV sequence data are available from GenBank (accession number AY380816). (B) Domain analysis of SmAV, based on homology with domains in supervillin (Chen et al., 2003) and motif searching programs (http://scansite.mit.edu/, http://www.ncbi.nlm.nih.gov/). Domains are marked with residue numbers. Sequences corresponding to the predicted binding sites for myosin II (lavender) and F-actin (brown) are shown. The CaP-binding domain is represented by the red box.

View larger version (49K): [in a new window]   Fig. 3. Overlay analysis of binding interactions between CaP and supervillin/archvillin fragments. (A, left) Naphthol blue black (NBB) stain of total protein on blot before incubation with overlay solution. (A, right) Immunostain with anti-CaP antibody after processing with overlay buffer containing 1.25 µg ml-1 CaP. Purified proteins and their corresponding overlay signals in lanes are indicated with arrowheads. All three proteins were loaded at 0.2 µg per lane. (B) Quantitative analysis of results obtained in (A). The chart shows the ratio of densitometry obtained from the overlay assay to that from the NBB-stained blot. The results are from n=7 (GST-c-BSV), n=6 (GST-c-SmAV) and n=3 (GST) samples in each case. (C, left) NBB stain of total protein on blot before incubation with overlay solution. (C, right) Immunostain with anti-GST antibody after processing with overlay buffer containing 1.25 µg ml-1 GST-c-SmAV or GST only as indicated. Purified proteins and their corresponding overlay signals in lanes are indicated with arrowheads. Both recombinant CaP and recombinant calmodulin were at 3 µg per lane. (D) Quantitative analysis of results obtained in (C). The chart shows the ratio of densitometry obtained from the overlay assay to that from the NBB-stained blot. The results are from n=5 (CaP) and n=3 (CaM) samples for the GST-c-SmAV overlay, and n=3 (CaP) and n=3 (CaM) samples for the GST overlay. (E) Overlay assay of GST-c-SmAV with chymotrypsin-digested fragments of CaP. (left) NBB stain of blot before incubation with overlay solution. (right) Immunostain with anti-GST antibody after processing with overlay buffer containing 1.25 µg ml-1 GST-c-SmAV. Undigested (full-length) and fragments of CaP are indicated with arrowheads. (F) Quantitative analysis of results obtained in (E). The chart shows the ratio of densitometry obtained from the overlay assay to that from the NBB-stained blot. The results are from n=5 samples.

View larger version (75K): [in a new window]   Fig. 4. CaP and the supervillin C-terminus colocalize in COS-7 cells. Confocal images of immunostained transfected COS-7 cells. (A, left) Single expression of the EGFP-tagged C-terminal 783 residues of bovine supervillin (EGFP-c-SV). (A, right) Single expression of h1-CaP. (B) CaP (left) and EGFP-c-SV (middle) co-expressed in COS-7 cells. Merged image is shown to the right; regions of signal overlap appear in yellow. (C) Co-expression of CaP and EGFP control. CaP was visualized by staining with an anti-CaP antibody and EGFP-c-SV was visualized with an anti-GFP antibody. Scale bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 5. Co-transfection with CaP prevents extraction of EGFP-c-SV by Triton X-100 from COS-7 cells. (A, left) Unextracted COS-7 cell singly transfected with EGFP-c-SV. (A, right) COS-7 cell singly transfected with EGFP-c-SV and extracted with 0.5% Triton X-100 before fixation. (B, left) Unextracted COS-7 cell singly transfected with CaP. (B, right) COS-7 cell singly transfected with CaP and extracted with 0.5% Triton X-100 before fixation. (C, left) Unextracted COS-7 cell co-transfected with EGFP-c-SV (top) and CaP (middle). (C, right) COS-7 cell co-transfected with EGFP-c-SV (top) and CaP (middle) and extracted with 0.5% Triton X-100 before fixation. Merged images shown at the bottom, with regions of overlap in yellow. Scale bar, 10 µm.

View larger version (44K): [in a new window]   Fig. 6. Tissue fractionation of CaP and SmAV. Immunodetection of SmAV (top) and CaP (bottom) in cytosolic, Triton-X-100-extractable (`membrane') and Triton-X-100-resistant (`cytoskeletal') fractions from unstimulated tissue or from tissue stimulated for 10 minutes with 10-5 M phenylephrine.

View larger version (85K): [in a new window]   Fig. 7. CaP and SmAV colocalize in an agonist-dependent manner in differentiated aortic smooth-muscle cells. (A) Western blot of aorta tissue homogenate probed with anti-H340 antibody. Molecular weight standards are indicated on the left. (B) Confocal images of a freshly isolated ferret aorta cell fixed under control conditions and labeled for endogenous levels of CaP (left, green) and SmAV (middle, red). Merged images are shown on the right. (C) Confocal images of a freshly isolated ferret aorta cell fixed in the presence of 10-5 M phenylephrine for 10 minutes and labeled for CaP and SmAV. Merged images are on the right, with signal overlap in yellow. Scale bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 9. Antisense knockdown of SmAV decreases contractility and signaling in smooth-muscle tissue. (A) Densitometric analysis of SmAV protein levels from immunoblots of antisense- and sham-treated muscles expressed as a precentage of random loaded tissue. **P0.01. (Inset) A typical western blot for SmAV. (B) Magnitude of steady-state contraction in response to a depolarizing physiological saline solution containing 51 mM KCl. Forces are normalized to the amplitude of contraction of each muscle in response to 51 mM KCl physiological saline solution on day 1. (C) Contraction of muscle strips in response to the phorbol ester DPBA (3 µM). Forces are normalized to the amplitude of contraction of each muscle in response to 51 mM KCl on day 1. **P0.01 for antisense compared with sham; ++P0.001 for antisense compared with random. (D) Contraction of muscle strips in response to 10 µM phenylephrine in the absence of extracellular calcium. Forces are normalized to the amplitude of contraction of each muscle in response to 51 mM KCl physiological saline on day 1. *P0.05 for antisense compared with sham; +P0.05 for antisense compared with random. (E) Densitometric analysis of phospho-ERK1/2 on immunoblots for antisense- or sham-treated muscles exposed to DPBA, normalized to that for muscles treated with a random sequence. (Inset) A typical western blot for phospho-ERK1/2. **P0.05. All values are from between three and five separate experiments.

View larger version (105K): [in a new window]   Fig. 10. Localization of endogenous SmAV, F-actin, nonmuscle myosin II, CaP and ERK1/2 before and after PDBu treatment of A7r5 smooth-muscle cells. (A) In untreated A7r5 cells, endogenous SmAV (a) appears to be membranous, whereas F-actin (b,e,h), nonmuscle myosin II (d) and CaP (g,k) are primarily associated with microfilaments, and ERK1/2 appears to be cytosolic (j). (B) After PKC activation with PDBu, endogenous SmAV (a,g), F-actin (b,e), nonmuscle myosin II (d), CaP (h,k) and ERK1/2 (j) all localize to podosome-like structures (arrowheads). SmAV, F-actin, myosin IIB and CaP also concentrate at or near membrane ruffles (arrows). Merged images (c,f,i,l) show the overlap of signal from panels on the left (green) with the signal from the middle panels (red); the overlap appears yellow. Scale bar, 25 µm.

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