Fig. 4. Localization of actin and myosin II during cell spreading. Actin was stained with rhodamine-labeled phalloidin, and myosin II was immunolocalized using myosin II antibodies stained with Alexa-488-labeled secondary antibody. The cells shown in the images in the first (A,D,G,J,M), second (B,E,H,K,N) and third (C,F,I,L,O) column from the left are treated with DMSO, KT and STA, respectively. The cells shown in the first/second rows (A-F) and third/fourth rows (G-L) and the last row (M-O) of the images are fixed 10, 30 and 120 minutes after seeding the cells on the dishes, respectively. At 10 minutes after beginning cell spreading, myosin II was localized diffusely with or without STA and KT, yet the samples treated with STA and KT showed many F-actin spikes (A-C). Cross-sectional views of the cells in A-C are shown in D-F, whose cell heights were significantly reduced by myosin II inhibition. At 30 minutes, control cells started to spread asymmetrically, but cells treated with STA and KT were spread into almost circular shapes (G-I). This suggests that myosin might be required for polarization of spreading cells. Localization of myosin II at this stage was not different with or without the KT and STA. Cross-sectional views of cells showed significant differences in flattening of cells due to the increased rate of cell spreading in KT- and STA-treated cells (J-L). The myosin II strongly co-localized with actin filaments at 120 minutes without STA and KT (M). Cells treated with KT also started to show co-localization with actin filaments, which is consistent with the level of myosin RLC phosphorylation shown in Fig. 2 (N). Many cells treated with STA started to break into fragments (O).