Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton
Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton
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DOI：
10.1006/excr.1996.3451
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发表时间：
1997-02-25
影响因子：
3.7
通讯作者：
Ingber, DE
中科院分区：
文献类型：
--
作者：
Ezzell, RM;Goldmann, WH;Ingber, DE
与野生型F9细胞或两种黏着斑蛋白转染的克隆相比，缺乏细胞骨架蛋白黏着斑蛋白的小鼠F9胚胎癌5.51细胞在细胞外基质上的扩散较差（5.51 Vin 3和Vin 4; Samuels等人，121，909-921）。在本研究中，我们使用这个模型系统来确定黏着斑蛋白的存在如何促进细胞骨架的改变和相关的细胞形状的变化。早期细胞铺展的显微镜分析显示，5.51细胞保留了形成丝状伪足的能力;然而，它们不能形成板状伪足，组装应力纤维，或有效地铺展在培养基质上。洗涤剂（Triton X-100）的研究表明，这些主要的差异，细胞形态和细胞骨架组织没有导致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，除了缺乏黏着斑蛋白，表现出轻微减少的α-辅肌动蛋白和桩蛋白在其洗涤剂不溶性细胞骨架的水平。黏着斑蛋白的缺乏与整合素-细胞骨架连接的机械刚度的降低相关，如使用细胞磁力测定法所测量的。此外，当黏着斑蛋白在5.51 Vin 3和5.51 Vin 4细胞中被转染取代时，细胞内相关的α-辅肌动蛋白和桩蛋白的水平、跨膜机械偶联的效率和肌动蛋白应力纤维的形成都恢复到接近野生型的水平。这些研究结果表明，黏着斑蛋白可能通过稳定粘着斑和转移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。(C)北京：科学出版社.
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 (5.51 Vin3 and Vin4; Samuels ct 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 5.51 Vin3 and 5.51 Vin4 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. (C) 1997 Academic Press.