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%.