Figure 1.

Calyculin A, 5 nM, treatment induces stress fiber formation and increased wrinkling of flexible rubber substrates in serum-starved, quiescent Swiss 3T3 fibroblasts. (A) NIH 3T3 fibroblasts (lanes 1 and 2) or gerbil fibroma cells (lanes 3 and 4) were treated with vehicle alone (lanes 1 and 3) or with calyculin A (lanes 2 and 4) and show increased phosphorylation on Ser19 of MLCs by immunoblotting of whole cell lystaes with an antibody specific for P-Ser19 on MLC. (B) After immunoblotting with the phosphospecific antibody, membranes were stripped and either stained with Coomassie blue or reprobed with antiactin antibody to confirm equal protein loading. (C) Quiescent cells have lost stress fibers as shown by fluorescent labeling with FITC-conjugated phalloidin. (D) Ten mintes after addition of calyculin A, stress fibers have reformed. (E-G) Time series images of a single cell shown before stimulation (E), and at 10 (F) and 20 min (G) after stimulation with 5 nM calyculin, which induces wrinkling in the rubber substrate underlying the cell. Bar, 10 μm.