Role of vinculin in regulating focal adhesion turnover
Role of vinculin in regulating focal adhesion turnover
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DOI：
10.1016/j.ejcb.2006.01.014
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发表时间：
2006-06-01
影响因子：
6.6
通讯作者：
Critchley, David R.
中科院分区：
文献类型：
--
作者：
Saunders, Ruth M.;Holt, Mark R.;Critchley, David R.
尽管血管蛋白（-/-）小鼠胚胎成纤维细胞聚集局灶粘连（FAs），但它们比血管蛋白（+/+）细胞传播更慢，范围更小，伤口愈合更快。为了研究这些细胞中FAs的结构和动力学，我们使用了实时干涉反射显微镜（IRM），从而避免了表达外源性gfp标记的FA蛋白的需要，因为这些蛋白可能被错误调节。这表明，与野生型细胞相比，在无血管蛋白的细胞中，FAs更小，丰度更低，转化速度更快。vuculin的表达挽救了扩散缺陷，导致更大更稳定的FAs。磷脂酰肌醇4,5-二磷酸（PIP2）被认为通过缓解掩盖Vh和Vt配体结合位点的血毒蛋白头（Vh）和尾（Vt）之间的分子内结合，在血毒蛋白激活中发挥作用。为了研究血毒蛋白/PIP2相互作用在FA动力学中的作用，我们使用了缺乏C末端臂（残基1053-1066）的血毒蛋白突变体，称为δ C突变体。与野生型相比，该突变减少了PIP2与Vt δ C多肽的结合，而不影响与Vh或f -肌动蛋白的结合。有趣的是，表达血管蛋白δ C突变体的细胞组装了非常稳定的FAs。结果表明，vinculin通过稳定FAs抑制细胞迁移，肌醇磷脂与Vt的结合在FA周转中起重要作用。(c) 2006年Elsevier GmbH出版。
Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the Delta C mutation. This mutation reduced PIP2 binding to a Vt Delta C polypeptide by > 90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin Delta C mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover. (c) 2006 Published by Elsevier GmbH.