A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions
A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions
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DOI：
10.1074/jbc.m600738200
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发表时间：
2006-06-09
影响因子：
4.8
通讯作者：
Craig, Susan W.
中科院分区：
文献类型：
--
作者：
Cohen, Daniel M.;Kutscher, Brett;Craig, Susan W.
细胞骨架和整合素之间的动态相互作用控制细胞粘附，但调节机制仍然在很大程度上不明确。在这里，我们测试了在何种程度上的自抑制性头-尾相互作用（HTI）在黏着斑蛋白调节形成和寿命的talin-vinculin复合物，一个建议的调解人的整合素cytokinonbonds。在异位募集试验中，HTI的突变减少驱动talin-黏着斑蛋白复合物的组装，而异位复合物在talin和野生型黏着斑蛋白之间没有形成。此外，HTI的减少改变了黏着斑蛋白和talin在局灶性粘连中的动态组装。使用光漂白后的荧光恢复，我们表明，黏着斑的粘附驻留时间增强了3倍的HTI突变。黏着斑蛋白的缓慢动力学与其隐蔽的塔林结合位点的暴露相关，并且塔林结合位点突变拯救了活化黏着斑蛋白的动力学。值得注意的是，HTI缺陷黏着斑蛋白抑制粘着斑动力学的塔林，但不桩蛋白或α-辅肌动蛋白。这些数据表明，塔林构象在细胞中允许粘着斑蛋白结合，而粘着斑蛋白的自抑制构象构成复合物形成的障碍。纽蛋白中HTI下调至类似于10（-7）的K-d足以诱导塔林蛋白结合，并且HTI对于纽蛋白和塔林蛋白在局灶性粘连处的动力学至关重要。因此，我们得出结论，黏着斑蛋白的构象，作为调制的HTI的强度，直接调节细胞中的塔林黏着斑蛋白复合物的形成和寿命。
Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin cytoskeletonbonds. Inanectopicrecruitmentassay, mutationalreduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to K-d similar to 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.