Kim Hugh, Sengupta Anita, Glogauer Michael, McCulloch Christopher A
CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Canada.
Exp Cell Res. 2008 Feb 15;314(4):834-46. doi: 10.1016/j.yexcr.2007.11.022. Epub 2007 Dec 4.
Cell spreading and exploration of topographically complex substrates require tightly-regulated interactions between extracellular matrix receptors and the cytoskeleton, but the molecular determinants of these interactions are not defined. We examined whether the actin-binding proteins cortactin, vinculin and filamin A are involved in the formation of the earliest extensions of cells spreading over collagen or poly-L-lysine-coated smooth and beaded substrates. Spreading of human gingival fibroblasts was substantially reduced on beaded or poly-L-lysine-coated substrates. Filamin A, vinculin and cortactin were found in cell extensions on smooth collagen. HEK-293 cells also spread rapidly on smooth collagen and formed numerous cell extensions enriched with filamin A. Knockdown of filamin A in HEK-293 cells by short hairpin RNA reduced spreading and the number of cell extensions. Blocking beta1 integrin function significantly reduced cell spreading and localization of filamin A to cell extensions. Conversely, filamin A-knockdown reduced beta1 integrin-collagen binding as measured by 12G10 antibody, suggesting co-dependence between filamin A and beta1 integrin functions. TUNEL staining showed higher percentages of apoptosis after filamin A-knockdown in spreading cells. Chelation of [Ca2+]i with BAPTA/AM reduced spreading of wild-type and filamin A-knockdown cells, however wild-type cells showed recruitment of filamin A to the subcortex, indicating independent roles of filamin A and [Ca2+]i in cell spreading. We conclude that filamin A integrates with beta1 integrins to mediate cell spreading and prevent apoptosis.
细胞在地形复杂的底物上展开和探索需要细胞外基质受体与细胞骨架之间进行严格调控的相互作用，但这些相互作用的分子决定因素尚未明确。我们研究了肌动蛋白结合蛋白丝状肌动蛋白、纽蛋白和细丝蛋白A是否参与细胞在胶原蛋白或聚-L-赖氨酸包被的光滑和有珠底物上展开时最早延伸的形成。人牙龈成纤维细胞在有珠或聚-L-赖氨酸包被的底物上的展开显著减少。在光滑胶原蛋白上的细胞延伸中发现了细丝蛋白A、纽蛋白和丝状肌动蛋白。HEK-293细胞也能在光滑胶原蛋白上迅速展开，并形成大量富含细丝蛋白A的细胞延伸。通过短发夹RNA敲低HEK-293细胞中的细丝蛋白A可减少细胞展开和细胞延伸的数量。阻断β1整合素功能可显著减少细胞展开以及细丝蛋白A在细胞延伸中的定位。相反，如通过12G10抗体测量所示，敲低细丝蛋白A可降低β1整合素与胶原蛋白的结合，表明细丝蛋白A和β1整合素功能之间存在相互依赖。TUNEL染色显示，在展开的细胞中敲低细丝蛋白A后凋亡百分比更高。用BAPTA/AM螯合细胞内钙离子（[Ca2+]i）可减少野生型和细丝蛋白A敲低细胞的展开，然而野生型细胞显示细丝蛋白A向皮层下募集，表明细丝蛋白A和[Ca2+]i在细胞展开中具有独立作用。我们得出结论，细丝蛋白A与β1整合素结合以介导细胞展开并防止细胞凋亡。
