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First published online 9 October 2007
doi: 10.1242/jcs.008219

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Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery

¹ Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
² Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
³ Cell Dynamics Program, University of Massachusetts Medical School, Worcester, MA 01605, USA

View larger version (39K): [in this window] [in a new window]   Fig. 1. Overexpressed supervillin decreases the rate of cell spreading. (A) Quantification of spreading and spread cells. As compared with the diameter of rounded cells (a), `spreading cells' (b) are smaller than twice the diameter of the average rounded COS7 cell, which is 23.0±0.7 µm (means±s.e.m., n=17), and `spread cells' (c) are larger than twice the diameter of rounded cells. Bar, 46 µm. (B) Percentage of spread COS7 cells after plating onto 10 µg/ml fibronectin for 15, 30, 45, 60 or 90 minutes. Results for control nontransfected cells or cells expressing EGFP alone or EGFP-SV were obtained. Means±s.e.m. of 100 cells counted per experiment, n=6. Sample images are shown in supplementary material Fig. S1. (C) Histogram of the percentages of spread COS7 cells at 30 minutes after plating on 10 µg/ml fibronectin. Means±s.e.m.; n=6; ***P<0.001 for EGFP-SV versus both control and EGFP-expressing cells.

View larger version (59K): [in this window] [in a new window]   Fig. 2. EGFP-supervillin (EGFP-SV) and myosin heavy chain (MHC) IIB co-localize at the cell periphery during spreading. (A,B) COS7 cells expressing EGFP-SV (upper panels) or EGFP only (lower panels) after 30 (A) or 60 (B) minutes of spreading were counterstained with anti-MHC IIB antibodies. EGFP signals (a,d; green in c,f) and MHC IIB signals visualized in the red channel (b,e; red in c,f) were merged (c,f); signal overlaps are shown in yellow. Arrows indicate peripheral co-localizations. Bar, 20 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Co-localization of endogenous proteins during cell spreading. After 30 minutes of spreading on 10 µg/ml fibronectin, A549 cells were stained with antibodies against MHC IIA (a), tubulin (c), MHC IIB (d), vinculin (f), mono-phosphorylated serine-19 in MLC (pS19-MLC; g,j,m), di-phosphorylated threonine-18, serine-19 in MLC (pT18pS19-MLC; h), or supervillin (k,n) and with Alexa-Fluor-350-conjugated phalloidin for visualizing F-actin (b,e,i,l,o). Alexa-Fluor-488-conjugated (a,d,h,k) and Alexa-Fluor-594-conjugated (c,f,g,j) secondary antibodies were used. (m-o) Enlargements of the boxed areas in j-l. (p-r) Cross-sections of signal intensities along the vertical lines in m-o, respectively. Bars, 10 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Supervillin binds to the S2 domains of both of the nonmuscle myosins – myosin IIA and myosin IIB. GST-tagged SV1-174 (left) or GST only (right) were pre-bound to glutathione-agarose beads and incubated with the hexa-histidine (6xHis)-tagged recombinant S2 domain from the heavy chain of nonmuscle myosin IIA (S2-MHC IIA), nonmuscle myosin IIB (S2-MHC II) or skeletal muscle myosin II (S2-Sk-MHC II). Samples (15 µl) of void volumes (V, lanes 1), washes with binding buffer (W, lanes 2), washes with the indicated concentrations of NaCl in binding buffer (lanes 3-6) and eluates with 5 mM glutathione (E, lanes 7) were analyzed on immunoblots stained with anti-GST antibodies (top panels) or anti-6xHis antibodies (lower panels).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Supervillin knockdown increases the rate of cell spreading. (A) Immunoblots of endogenous SV, MHC IIA, MHC IIB, long and short isoforms of MLCK (L-MLCK and S-MLCK), and ERK1/2, in lysates from cells treated with transfection reagent alone (lanes 1; Mock), dsRNAs 1680 (hSV1) or 2026 (hSV2) that target SV (lanes 2, 3), or control dsRNAs (lanes 4, Con). (B) Histogram showing the rates of area change for populations of A549 cells treated with control (white) or hSV1 (black) dsRNAs spreading on 10 µg/ml fibronectin. Spreading velocities in the bimodal distributions typical of unsynchronized cells (Dubin-Thaler et al., 2004) appear to be displaced, but the difference between the means (36.2±5.7 µm2/minute for controls; 51.7±11.3 µm2/minute for hSV1) is not statistically significant for the numbers of living cells (n=16; n=17) assayed. Movies 1 and 2 in the supplementary material show cell morphologies during linear spreading for average control- and hSV1-treated cells. (C) Percentage of spread A549 cells after plating onto 10 µg/ml fibronectin at 15, 30, 45, 60 or 90 minutes. Spread cells were defined as in Fig. 1; the average diameter of a rounded A549 cell was taken as 18.0±0.8 µm (means±s.e.m., n=18). Cells were mock-transfected, or were treated with hSV1, hSV2 or control dsRNA. Means±s.e.m. of 200 cells counted per experiment, n=5. (D) Percentage of spread A549 cells plated on 10 µg/ml fibronectin for 30 minutes. Means±s.e.m.; n=5; *P<0.05 for each SV dsRNA versus both mock and control dsRNA treatments.

View larger version (58K): [in this window] [in a new window]   Fig. 6. Supervillin knockdown decreases the number of peripheral bundles of MHC IIB and disorganizes the peripheral localization of phosphorylated MLC. (A) Localization of F-actin (a,d; green in c,f) and MHC IIB (b,e; red in c,f) in A549 cells treated with hSV1 (a-c) or control (d-f) dsRNAs after spreading on fibronectin for 30 minutes. (B) Localization of activated MLC using antibodies against mono-phosphorylated serine-19 (pS19-MLC; a,d), di-phosphorylated threonine-18, serine-19 (pT18pS19-MLC; b,e) and F-actin visualized with Alexa-Fluor-350-phalloidin (c,f). Cells were treated with hSV2 dsRNA (a-c) or were mock-treated (d-f) before spreading on fibronectin for 30 minutes. Signals were visualized in the red (a,d) and green (b,e) channels. Arrows, peripheral bundles. Bars, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 7. Inhibition of myosin ATPase activity directly or via MLCK, but not via Rho kinase, increases the rate of A549 cell spreading. Histogram showing the percentage of spread A549 cells plated for 30 minutes on fibronectin in the absence (Control) and presence of specific inhibitors of: myosin II ATPase (blebbistatin, 20 µM), MLCK (ML-7, 1 µM), the MLCK activator MEK (U0126, 2.5 µM) or Rho kinase (Y-27632, 20 µM). Means±s.e.m.; 200 cells/experiment, n=6; **P<0.01 versus control.

View larger version (109K): [in this window] [in a new window]   Fig. 8. Inhibition of MLCK mimics the effect of supervillin knockdown on the formation of peripheral bundles of activated myosin during spreading of A549 cells. A549 cells treated with 1 µM ML-7 to specifically inhibit MLCK (a-i) lack the peripheral bundles of activated myosin filaments found in control cells (j-l) after 30 minutes of spreading. Primary antibody staining for MHC IIA (a), vinculin (c,f), MHC IIB (d), pSer19-MLC (g,j) and pThr18pSer19-MLC (h,k) was visualized in the green (a,d,h,j) and red (c,f,g,k) channels. F-actin was visualized with Alexa-Fluor-350–phalloidin (b,e,i,l). Bar, 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 9. At low concentrations, specific inhibitors of myosin II activity abolish supervillin-mediated decreases in spreading of COS7 cells. Percentages of spread COS7 cells expressing either EGFP-SV or EGFP at 30 minutes after plating on fibronectin in the presence of inhibitors of myosin function: (A) MHC ATPase inhibitor, blebbistatin, **P=0.003 (20 µM versus 0 µM for EGFP-SV); (B) MLCK inhibitor, ML-7, **P=0.002 (1.0 µM versus 0 µM for EGFP-SV); (C) MEK inhibitor, U0126, *P=0.014 (2.5 µM versus 0 µM for EGFP-SV); (D) Rho kinase inhibitor, Y-27632, no significant effects. Means±s.e.m.; 100 cells per data point per experiment, n=4.

View larger version (45K): [in this window] [in a new window]   Fig. 10. MLCK isoforms, especially L-MLCK, co-localize with supervillin, and mimic and potentiate supervillin-mediated decreases in the rates of cell spreading. (A) Fluorescence micrographs of co-transfected COS7 cells after spreading on fibronectin for 30 minutes, showing Flag-tagged MLCK proteins, as indicated (a,e,i,m; red in merged images), EGFP-SV (b,f,j,n; green in merges) and merged images (c,g,k,o; overlap in yellow). MLCK proteins were visualized with anti-Flag antibodies and Alexa-Fluor-594-conjugated anti-rabbit IgG antibodies; F-actin (d,h,l,p) was visualized with Alexa-Fluor-350–phalloidin. Bar, 20 µm. (B) Percentages of spread cells 30 minutes after plating on fibronectin for cells expressing: (1) EGFP only (n=14); (2) EGFP-SV only (n=14); (3) EGFP plus wild-type (Wt) L-MLCK (n=6); (4) EGFP-SV plus wild-type L-MLCK (n=9); (5) EGFP plus L-MLCK KD (n=6); (6) EGFP-SV plus L-MLCK KD (n=9); (7) EGFP plus wild-type S-MLCK (n=6); (8) EGFP-SV plus wild-type S-MLCK (n=9); (9) EGFP-SV plus S-MLCK KD (n=6); or (10) EGFP-SV plus S-MLCK KD (n=9). Means±s.e.m.; 100 cells per experiment; ***P<0.001, **P<0.01. Statistical differences were evaluated using Student-Newman-Keuls multiple comparisons test. Notice that the x axis starts at 20%.

View larger version (38K): [in this window] [in a new window]   Fig. 11. L-MLCK N-terminal sequences reduce the rate of cell spreading and bind the SV N-terminus. (A) Diagram of L-MLCK, S-MLCK and mutants. Catalytic core (dark gray); immunoglobulin-like domains (Ig, stippled); Asp-X-Lysine sequences (DXR, black); and location (*) of the kinase-inactivating (KD) point mutation are shown. (B) EGFP-tagged fusion proteins containing N-terminal L-MLCK sequences (Poperechnaya et al., 2000) reduce the rate of cell spreading. N-terminal sequences unique to L-MLCK without (Ig) or with (Ig+5DXRs) all five of the DXRXXL actin-binding motifs reduce the rate of spreading by 50%; the 5DXR motifs alone slow cell spreading by 30%. Histogram of the percentages of spread COS7 cells transfected for 16 hours with EGFP as a control or EGFP-tagged avian L-MLCK sequences, as indicated. Means±s.e.m.; 100 cells/experiment, n=6; *P<0.05; ***P<0.001 versus EGFP-only control. (C) The L-MLCK Ig domains, but not the 5DXRs, bind directly to SV1-174. GST-tagged SV1-174 (lanes 1-6) or GST only (lanes 7-12) were pre-bound to glutathione-agarose beads and incubated with purified recombinant 6xHis-tagged Ig domains (upper panel) or 6xHis-tagged 5DXRs (lower panel). Void volumes (V, lanes 1, 12), washes with the indicated concentrations of NaCl (lanes 2-5, 8-11) and glutathione eluates (E, lanes 6,7) were analyzed on immunoblots stained with anti-6xHis antibodies.

View larger version (79K): [in this window] [in a new window]   Fig. 12. Both F-actin-bundling and myosin II-binding sequences in supervillin slow cell spreading; SV1-171 co-localizes with mono- and diphosphorylated MLC and L-MLCK, but not with S-MLCK or with the MYPT1 subunit of myosin phosphatase. (A) Percentages of spread COS7 cells transfected with EGFP (Control) or EGFP-SV: full length (SV-FL), SV1-171, SV343-571, SV570-830, SV343-830, SV171-1792. Means±s.e.m.; n=6; ***P<0.001 versus EGFP control; **P<0.01 versus EGFP control. (B) COS7 cells transfected with EGFP-SV1-171 (SV1-171, a,g; green in c,i) or EGFP only (Control, d; green in f) and counterstained with anti-phosphoSer19-MLC antibodies (pS19-MLC, b,e; red in c,f) or antibody against pThr18pSer19-MLC (h; red in i). Overlap in merged images (c,f,i) appears yellow. Bar, 20 µm. (C) Co-transfected COS7 cells expressing EGFP-SV1-171 (SV1-171, a,g) or EGFP only (Control, d) and Flag-tagged L-MLCK (b,e) or S-MLCK (h) and counterstained with Alexa-Fluor-350–phalloidin (c,f,i). Cells expressing EGFP-SV1-171 alone (j,m) were stained with antibodies against the MYPT1 subunit of myosin phosphatase (k,n) and phosphoS19-MLC (l,o). (m-o) Enlargements of the boxed areas in j-l. Arrows show punctae of SV1-171 with pS19-MLC that lack MYPT1. Bars, 20 µm (a-l); 5 µm (m-o).