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.
Brown
D
D
Department of Physiology, The Joan Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA.
Dykes
A
A
Black
J
J
Thatcher
S
S
Fultz
M E
ME
Wright
G L
GL
D000199:Actins / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000502:physiology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D020033:Protein Isoforms / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology
D000199:Actins; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D020033:Protein Isoforms; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D019311:Staurosporine; C088060:bisindolylmaleimide