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Figure 12


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).





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