Differential actin isoform reorganization in the contracting A7r5 cell

Abstract

In the present study, we investigated the reorganization of α- and β-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 (≤10−7 mol/L), cells showed an ~70% loss in α-actin stress fibers with robust transport of this isoform to podosomes. By comparison, β-actin remained in stress fibers in cells stimulated at low concentrations (≤10−7 mol/L) of PDBu. However, at high concentrations (≥10−6 mol/L) ~50% of cells showed transport of β-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 × 10−8 mol/L) loss in α-actin stress fibers or reversed podosome formation with re-establishment of α-actin stress fibers. By comparison, these inhibitors caused partial loss of β-actin stress fibers. The results support our earlier conclusion of independent remodeling of α- and β-actin cytoskeletal structure and suggest that the regulation of these structures is different.