CIHR Group in Matrix Dynamics, University of Toronto, Room 239, 150 College Street, Toronto, Ontario, Canada M5S 3E2.
Exp Cell Res. 2010 Jul 1;316(11):1829-44. doi: 10.1016/j.yexcr.2010.02.007. Epub 2010 Feb 17.
Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by beta1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate beta1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of beta1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase Cvarepsilon bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of beta1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cepsilon is an essential regulatory step for the trafficking and activation of beta1 integrins and cell spreading on collagen.
细胞黏附和铺展是哺乳动物发育和伤口愈合过程中的基本过程，其由β1 整合素和肌动蛋白及中间丝细胞骨架介导。这些独立的细胞骨架系统相互作用以调节β1 整合素和细胞铺展的机制尚未完全确定。我们之前的研究报道称，肌动蛋白交联蛋白细丝蛋白 A 与中间丝蛋白波形蛋白结合，并且这两种蛋白共同调节细胞铺展。在这里，我们使用细丝蛋白 A 的缺失突变体来定义细丝蛋白 A-波形蛋白相互作用，以及在细胞铺展到胶原蛋白上时波形蛋白的随后磷酸化和重新分布。固定和活细胞制剂的成像显示，磷酸化的波形蛋白在铺展过程中被转运到细胞膜上。细丝蛋白 A 的敲低抑制了细胞铺展以及波形蛋白的磷酸化和重新分布。细丝蛋白 A 和/或波形蛋白的敲低减少了β1 整合素的细胞表面表达和激活，如通过质膜相关蛋白的免疫印迹和剪切力测定所指示的。使用细丝蛋白 A 突变体进行的体外下拉测定表明，波形蛋白和蛋白激酶 Cε都结合到细丝蛋白 A 的重复 1-8。用全长细丝蛋白 A 或细丝蛋白 A 重复 1-8 重建缺乏细丝蛋白 A 的细胞恢复了细胞铺展、波形蛋白磷酸化以及β1 整合素的细胞表面表达。我们得出结论，细丝蛋白 A 与波形蛋白和蛋白激酶 Cε 的结合是β1 整合素的运输和激活以及细胞在胶原蛋白上铺展的必要调节步骤。
