Fig. 1. Morphological and biochemical changes associated with cell attachment and spreading of skeletal muscle cells plated on FN. (A) Myoblasts were plated on FN and photographed at various times after plating. The time in minutes after plating is indicated. The panels show characteristic morphological changes of attachment and spreading, including bleb formation (5 minutes, white arrow), membrane ruffling (5 and 10 minutes, closed arrow) and circumferential lamella (15 minutes, arrowhead). (B) Actin stress fiber formation and FAK localization were determined in myoblasts at different time points after the cells were plated on FN. The upper panels show the development of stress-fiber formation by staining the cells with fluorescently labeled phalloidin. The lower panels show the change in FAK localization from a predominantly diffuse cytosolic localization at 15 minutes to a more focal adhesion site localization (arrows) at later time points. (C) FAK phosphorylation was determined as a function of time after plating on FN. At each time point, the cells were harvested in RIPA buffer. Phosphorylation of FAK was determined by immunoblot analysis using an antiphosphotyrosine antibody. A duplicate blot was probed with an anti-FAK antibody (lower panel) to confirm equal loading of FAK protein in each lane.