Cross-Correlated Fluctuation Analysis Reveals Phosphorylation-Regulated Paxillin-FAK Complexes in Nascent Adhesions
Cross-Correlated Fluctuation Analysis Reveals Phosphorylation-Regulated Paxillin-FAK Complexes in Nascent Adhesions
复制标题
DOI：
10.1016/j.bpj.2010.12.3719
复制
发表时间：
2011-02-02
影响因子：
3.4
通讯作者：
Horwitz, Alan Rick
中科院分区：
文献类型：
--
作者：
Choi, Colin K.;Zareno, Jessica;Horwitz, Alan Rick
我们使用相关方法来检测和量化迁移细胞中桩蛋白和粘着斑激酶 (FAK) 之间的相互作用。互相关光栅扫描图像相关光谱显示，野生型桩蛋白和磷酸化抑制桩蛋白突变体 Y31F-Y118F 不与胞质中的 FAK 相互作用，但桩蛋白的磷酸模拟突变体 Y31E-Y118E 会与 FAK 相互作用。通过将互相关数和亮度分析扩展到全内反射荧光模式，我们能够证明桩蛋白和 FAK 四聚体在新生粘连中以 1:1 的化学计量比形成复合物。桩蛋白上的拟磷酸化突变增加了复合物的大小和新生粘连的组装率，表明桩蛋白和 FAK 的物理分子聚集调节粘连形成。相反，当磷酸化受到抑制时，相互作用就会减弱，粘附往往会延长而不是翻转。这些直接的体内数据表明，桩蛋白的磷酸化对于粘连具有特异性，并导致与 FAK 形成局部复合物，从而调节新生粘连的动态。
We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.