Hosoya N, Mitsui M, Yazama F, Ishihara H, Ozaki H, Karaki H, Hartshorne D J, Mohri H
Department of Biology, University of Tokyo, Japan.
J Cell Sci. 1993 Aug;105 ( Pt 4):883-90. doi: 10.1242/jcs.105.4.883.
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.
采用间接荧光技术分析了强效1型和2A型蛋白磷酸酶抑制剂花萼海绵诱癌素A（CL-A）对培养的A10平滑肌细胞细胞骨架结构的影响。在存在1×10⁻⁷ M CL-A的情况下，细胞变圆并随后从基质上脱离。CL-A的作用被非选择性激酶抑制剂K-252a抑制，但不被乙二醇双四乙酸（EGTA）抑制。在变圆的细胞中，应力纤维缺失，F-肌动蛋白染色呈斑块状。粘着斑的组成成分之一纽蛋白定位于对照细胞的周边。CL-A处理使粘着斑沿着应力纤维向细胞内部移动，随后细胞变圆。此外，在CL-A处理的细胞中观察到微管结构迅速且显著的变化。在贴壁伸展的细胞中，沿微管观察到许多“缺口”或“间隙”。在脱离的细胞中，即长时间暴露于CL-A后，未观察到微管的丝状网络。这些结果表明，CL-A可能改变粘着斑的结构，导致细胞变圆，并诱导微管切断活性。这些作用与细胞外Ca²⁺浓度无关。细胞骨架结构的变化可能是由于扰乱了细胞内磷酸化和去磷酸化的平衡所致。
