Differential actin isoform reorganization in the contracting A7r5 cell.

In the present study, we investigated the reorganization of alpha- and beta-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (< or =10(-7) mol/L), cells showed an approximately 70% loss in alpha-actin stress fibers with robust transport of this isoform to podosomes. By comparison, beta-actin remained in stress fibers in cells stimulated at low concentrations (< or =10(-7) mol/L) of PDBu. However, at high concentrations (> or =10(-6)mol/L) approximately 50% of cells showed transport of beta-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PDBu-induced (5 x 10(-8) mol/L) loss in alpha-actin stress fibers or reversed podosome formation with re-establishment of alpha-actin stress fibers. By comparison, these inhibitors caused partial loss of beta-actin stress fibers. The results support our earlier conclusion of independent remodeling of alpha- and beta-actin cytoskeletal structure and suggest that the regulation of these structures is different.