Ezzell R M, Goldmann W H, Wang N, Parashurama N, Ingber D E
Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, 02129, USA.
Exp Cell Res. 1997 Feb 25;231(1):14-26. doi: 10.1006/excr.1996.3451.
Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.
与野生型F9细胞或两个转染了纽蛋白的克隆（5.51Vin3和Vin4；塞缪尔斯等人，1993年，《细胞生物学杂志》121卷，909 - 921页）相比，缺乏细胞骨架蛋白纽蛋白的小鼠F9胚胎癌细胞5.51在细胞外基质上的铺展能力较差。在本研究中，我们使用这个模型系统来确定纽蛋白的存在如何促进细胞骨架改变以及相关的细胞形状变化。对早期细胞铺展的显微镜分析表明，5.51细胞保留了形成丝状伪足的能力；然而，它们无法形成片状伪足、组装应力纤维或有效地在培养底物上铺展。去污剂（Triton X - 100）研究表明，细胞形态和细胞骨架组织的这些主要差异并非源于总聚合或交联肌动蛋白水平的差异。生化研究表明，5.51细胞除了缺乏纽蛋白外，其去污剂不溶性细胞骨架中的α - 辅肌动蛋白和桩蛋白水平略有降低。使用细胞磁力测定法测量发现，纽蛋白的缺失与整合素 - 细胞骨架连接的机械刚度降低相关。此外，当在5.51Vin3和5.51Vin4细胞中通过转染替换纽蛋白时，细胞骨架相关的α - 辅肌动蛋白和桩蛋白水平、跨膜机械偶联效率以及肌动蛋白应力纤维的形成均恢复到接近野生型水平。这些发现表明，纽蛋白可能通过稳定黏着斑并传递驱动细胞骨架重塑的机械应力来促进细胞铺展，而不是通过改变肌动蛋白聚合或交联的总水平。
