Katoh Kazuo, Kano Yumiko, Ookawara Shigeo
Department of Anatomy, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-city, Tochigi 329-0498, Japan.
Genes Cells. 2007 May;12(5):623-38. doi: 10.1111/j.1365-2443.2007.01073.x.
The activation of Rho-kinase is known to modulate the organization of the actin-based cytoskeletal systems, including the formation of stress fibers and focal adhesions. Rho-kinase likely plays a more crucial and complex role in the organization of actin-based cytoskeletal systems than in that of myosin light chain kinase (MLCK). In order to understand the role of Rho-kinase in the organization of stress fibers and focal adhesions, we treated cultured fibroblasts with a Rho-kinase inhibitor and analyzed the stress fiber and focal adhesion organization under conventional fluorescence microscopy and replica electron microscopy. Some of the cells were transfected with GFP-labeled paxillin, actin or alpha-actinin, and the effects of the inhibitor were monitored in the living cells. The Rho-kinase inhibitor caused disassembly of the stress fibers and focal adhesions in the central portion of the cell within 1 h. However, the stress fibers and focal adhesions located in the cell periphery were not as severely affected by the Rho-kinase inhibitor. The time-lapse video recording revealed that when these cells were washed with a fresh medium in order to remove the Rho-kinase inhibitor, the stress fibers and focal adhesions located in the center of the cell gradually reorganized and, within 1.5-2 h, the cells completely recovered. This observation strongly suggests that the activation of Rho-kinase plays an important role in the organization of the central stress fibers and focal adhesions.
已知Rho激酶的激活可调节基于肌动蛋白的细胞骨架系统的组织，包括应力纤维和粘着斑的形成。与肌球蛋白轻链激酶（MLCK）相比，Rho激酶在基于肌动蛋白的细胞骨架系统的组织中可能发挥更关键和复杂的作用。为了了解Rho激酶在应力纤维和粘着斑组织中的作用，我们用Rho激酶抑制剂处理培养的成纤维细胞，并在传统荧光显微镜和复型电子显微镜下分析应力纤维和粘着斑的组织情况。一些细胞用绿色荧光蛋白标记的桩蛋白、肌动蛋白或α辅肌动蛋白进行转染，并在活细胞中监测抑制剂的作用。Rho激酶抑制剂在1小时内导致细胞中央部分的应力纤维和粘着斑解体。然而，位于细胞周边的应力纤维和粘着斑受Rho激酶抑制剂的影响并不严重。延时视频记录显示，当用新鲜培养基洗涤这些细胞以去除Rho激酶抑制剂时，位于细胞中央的应力纤维和粘着斑逐渐重新组织，并且在1.5 - 2小时内，细胞完全恢复。这一观察结果强烈表明，Rho激酶的激活在中央应力纤维和粘着斑的组织中起重要作用。
