Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics
Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics
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DOI：
10.1074/jbc.m113.483016
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发表时间：
2013-12-06
影响因子：
4.8
通讯作者：
Calderwood, David A.
中科院分区：
文献类型：
--
作者：
Brahme, Nina N.;Harburger, David S.;Calderwood, David A.
粘着斑（Focal adhesions，FA）是与细胞外基质紧密粘附的位点，由跨膜整合素粘附受体和将整合素连接到肌动蛋白细胞骨架和信号传导途径的细胞内蛋白质的簇组成。存在于FA中的两种整合素结合蛋白Kindlin-1和Kindlin-2对于整合素活化、FA形成和信号传导是重要的。Migfilin最初是在酵母双杂交筛选kindlin-2相互作用蛋白中鉴定的，是在FA中发现的含有LIM结构域的衔接蛋白，并且涉及控制细胞粘附、扩散和迁移。通过结合细丝蛋白，migfilin提供了kindlin和肌动蛋白细胞骨架之间的联系。在这里，使用kindlin敲除、生化下拉测定、荧光显微镜、荧光共振能量转移（FRET）和光漂白后荧光恢复（FRAP）的组合，我们已经确定了migfilin的C末端LIM结构域决定其FA定位，表明这些结构域介导体外和细胞中与kindlin的相互作用，并证明Kindlin对于细胞中正常的移行因子动力学是重要的。我们还表明，当C-末端LIM结构域区域被删除，然后N-末端细丝蛋白结合区域的蛋白质，这是能够针对肌动蛋白丰富的应力纤维，是主要的驱动程序的migfilin本地化。我们的工作详细说明了驱动kindlin结合的migfilin结构域和驱动FA本地化的migfilin FA招聘和流动性以及kindlin依赖之间的相关性。因此，我们认为kindlin与migfilin LIM结构域的相互作用驱动了migfilin FA的募集、定位和流动性。
Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.