We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.

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