Verkhovsky A B, Surgucheva I G, Svitkina T M, Tint I S, Gelfand V I
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Exp Cell Res. 1987 Nov;173(1):244-55. doi: 10.1016/0014-4827(87)90349-1.
Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and alpha-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.
用Triton X-100提取小鼠和鹌鹑胚胎成纤维细胞，并用凝溶胶蛋白样肌动蛋白封端蛋白（从牛脑中分离出的90 kDa蛋白-肌动蛋白复合物）处理所得的细胞骨架。用罗丹明偶联鬼笔环肽或抗肌动蛋白抗体对细胞进行染色，在用90 kDa蛋白-肌动蛋白复合物处理后未发现任何含肌动蛋白的结构。通过SDS-凝胶电泳证实了肌动蛋白的提取。免疫荧光显微镜显示，纽蛋白和α-辅肌动蛋白与肌动蛋白一起从细胞骨架中释放出来。然而，在用90 kDa蛋白-肌动蛋白复合物处理后，肌球蛋白仍与细胞骨架相关。处理后细胞中肌球蛋白的分布与对照细胞无显著差异：在两种情况下，肌球蛋白主要定位于应力纤维中。双重荧光染色显示处理后细胞含肌球蛋白的应力纤维中不存在肌动蛋白。通过铂复型的透射电子显微镜研究了肌动蛋白缺失的应力纤维的超微结构组织。在电子显微镜照片上，这些纤维表现为含有球状物质簇的细丝束。得出的结论是，肌球蛋白在应力纤维中的定位不依赖于肌动蛋白。
