Yumura S, Kitanishi-Yumura T
Biological Institute, Faculty of Science, Yamaguchi University, Japan.
Cell Struct Funct. 1990 Dec;15(6):355-64. doi: 10.1247/csf.15.355.
An intact complex that consisted of the cell membrane and cytoskeleton was prepared from Dictyostelium amoebae by an improved version of the method previously used by CLARKE et al. (1975). Proc. Natl. Acad. Sci. USA., 72: 1758-1762. After cells had attached tightly to a polylysine-coated coverslip in the presence of a divalent cation, the upper portions of the cells were removed with a jet of microfilament-stabilizing solution squirted from a syringe. The cell membranes left on the coverslip were immediately stained with tetramethylrhodamine-conjugated phalloidin for staining of actin filaments, and with antibody against myosin from Dictyostelium and a fluorescein-conjugated second antibody for staining of myosin. Networks of actin filaments and numerous rod-like structures of myosin (myosin filaments) aligned along them were observed on the exposed cytoplasmic surfaces of the cell membranes. These networks were similar to those observed in the cortex of fixed whole cells. Addition of ATP to these intact complexes of cell membrane and cytoskeleton caused the aggregation of both actin and myosin into several dot-like structures of actin on the cell membrane. Similar dot-like structures were also seen in the cortex of fixed whole cells, and their changes in distribution correlated with the motile activity of the cells. Transmission electron microscopy showed that these dot-like structures were composed of an electron-dense structure at the center, from which numerous actin filaments radiated outwards. These observations suggest that these novel dot-like structures are organizing centers for cortical actin filaments and may possibly be related to the adhesion of cells to the substratum.
采用CLARKE等人（1975年）之前使用方法的改进版本，从盘基网柄菌变形虫中制备了由细胞膜和细胞骨架组成的完整复合体。《美国国家科学院院刊》，72: 1758 - 1762。在二价阳离子存在的情况下，细胞紧密附着于聚赖氨酸包被的盖玻片后，用从注射器喷出的微丝稳定溶液流去除细胞上部。留在盖玻片上的细胞膜立即用四甲基罗丹明偶联的鬼笔环肽染色以标记肌动蛋白丝，并用抗盘基网柄菌肌球蛋白的抗体和荧光素偶联的二抗染色以标记肌球蛋白。在细胞膜暴露的细胞质表面观察到肌动蛋白丝网络以及沿其排列的众多肌球蛋白杆状结构（肌球蛋白丝）。这些网络与在固定的完整细胞皮质中观察到的相似。向这些细胞膜和细胞骨架的完整复合体中添加ATP会导致肌动蛋白和肌球蛋白都聚集到细胞膜上的几个肌动蛋白点状结构中。在固定的完整细胞皮质中也观察到类似的点状结构，它们分布的变化与细胞的运动活性相关。透射电子显微镜显示这些点状结构由中心的电子致密结构组成，从该结构向外辐射出许多肌动蛋白丝。这些观察结果表明，这些新的点状结构是皮质肌动蛋白丝的组织中心，并且可能与细胞与基质的黏附有关。
