Changes in the cytoskeletal structure of cultured smooth muscle cells induced by calyculin-A.

Abstract

Changes in the cytoskeletal structure of cultured A10 smooth muscle cells induced by calyculin-A (CL-A), a potent inhibitor of types 1 and 2A protein phosphatases, were analyzed using indirect fluorescence techniques. In the presence of 1 x 10(-7) M CL-A the cells became round and subsequently detached from the substratum. The effect of CL-A was inhibited by a non-selective kinase inhibitor, K-252a, but not by EGTA. In rounded cells stress fibers were absent and staining for F-actin appeared in patches. Vinculin, one of the components of focal contacts, was localized at the periphery of control cells. CL-A treatment moved the focal contacts towards the inside of the cell along the stress fibers, and this was followed by the rounding up of the cell. In addition, rapid and marked changes in microtubule structure were observed in CL-A-treated cells. Many 'nicks' or 'gaps' were observed along the microtubules in the attached, spread cells. A filamentous network of microtubules was not observed in the detached cells, i.e. after longer exposure to CL-A. These results suggest that CL-A may change the structure of focal contacts, resulting in the rounding up of the cell, and inducing a microtubule-severing activity. These effects were independent of the external Ca2+ concentration. The changes in cytoskeletal structure may be caused by disturbing the balance of phosphorylation and dephosphorylation in the cell.