Tilghman Robert W, Slack-Davis Jill K, Sergina Natalia, Martin Karen H, Iwanicki Marcin, Hershey E Daniel, Beggs Hilary E, Reichardt Louis F, Parsons J Thomas
Department of Microbiology, University of Virginia Health System, Charlottesville, 22908, USA.
J Cell Sci. 2005 Jun 15;118(Pt 12):2613-23. doi: 10.1242/jcs.02380. Epub 2005 May 24.
The process of cell migration is initiated by protrusion at the leading edge of the cell, the formation of peripheral adhesions, the exertion of force on these adhesions, and finally the release of the adhesions at the rear of the cell. Focal adhesion kinase (FAK) is intimately involved in the regulation of this process, although the precise mechanism(s) whereby FAK regulates cell migration is unclear. We have used two approaches to reduce FAK expression in fibroblasts. Treatment of cells with FAK-specific siRNAs substantially reduced FAK expression and inhibited the spreading of fibroblasts in serum-free conditions, but did not affect the rate of spreading in the presence of serum. In contrast with the wild-type cells, the FAK siRNA-treated cells exhibited multiple extensions during cell spreading. The extensions appeared to be inappropriately formed lamellipodia as evidenced by the localization of cortactin to lamellipodial structures and the inhibition of such structures by expression of dominant-negative Rac. The wild-type phenotype was restored by reexpressing wild-type FAK in the knockdown cells, but not by expression of FAK containing a point mutation at the autophosphorylation site (FAK Y397F). In wound-healing assays, FAK knockdown cells failed to form broad lamellipodia, instead forming multiple leading edges. Similar results were obtained using primary mouse embryo fibroblasts from FAK-flox mice in which Cre-mediated excision was used to ablate the expression of FAK. These data are consistent with a role for FAK in regulating the formation of a leading edge during cell migration by coordinating integrin signaling to direct the correct spatial activation of membrane protrusion.
细胞迁移过程始于细胞前缘的突出、外周黏附的形成、对这些黏附施加力，最终是细胞后部黏附的释放。黏着斑激酶（FAK）密切参与这一过程的调节，尽管FAK调节细胞迁移的确切机制尚不清楚。我们采用了两种方法来降低成纤维细胞中FAK的表达。用FAK特异性小干扰RNA（siRNA）处理细胞可显著降低FAK表达，并在无血清条件下抑制成纤维细胞的铺展，但不影响有血清存在时的铺展速率。与野生型细胞相比，经FAK siRNA处理的细胞在铺展过程中表现出多个延伸。这些延伸似乎是异常形成的片状伪足，这可通过皮层肌动蛋白定位于片状伪足结构以及显性负性Rac的表达对这些结构的抑制来证明。通过在敲低细胞中重新表达野生型FAK可恢复野生型表型，但在自磷酸化位点携带点突变的FAK（FAK Y397F）的表达则不能恢复。在伤口愈合实验中，敲低FAK的细胞无法形成宽阔的片状伪足，而是形成多个前沿。使用来自FAK-flox小鼠的原代小鼠胚胎成纤维细胞（其中采用Cre介导的切除来消除FAK的表达）也获得了类似结果。这些数据与FAK在细胞迁移过程中通过协调整合素信号传导以指导膜突出的正确空间激活来调节前沿形成的作用一致。
