Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption
Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption
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DOI：
10.1242/jcs.00284
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发表时间：
2003-03-15
影响因子：
4
通讯作者：
Geiger, B
中科院分区：
文献类型：
--
作者：
Kirchner, J;Kam, Z;Geiger, B
粘着斑成分的酪氨酸磷酸化参与粘着斑形成和周转的调节，但其潜在的分子机制仍不清楚。在本研究中，我们使用定量荧光显微镜来研究新成分掺入生长的粘着斑与这些位点的酪氨酸磷酸化之间的动态关系。为此，开发了一种监测活细胞中磷酸酪氨酸水平的新方法。是基于“磷酸酪氨酸报告基因”而开发的，该报告基因由与源自 pp60(c-Src) 的两个连续磷酸酪氨酸结合 Src 同源 2 (SH2) 结构域融合的黄色荧光蛋白组成。这种 YFP-dSH2 定位于细胞基质粘附，其强度与抗磷酸酪氨酸抗体标记的强度线性相关。通过 CFP-纽蛋白和 YFP-dSH2 的双色延时电影检查活细胞中纽蛋白和磷酸酪氨酸水平的差异增加。在这项研究中，粘着斑生长是由微管破坏引发的，之前的研究表明微管破坏可以通过诱导细胞收缩来刺激粘着斑的发展。我们在此表明，添加微管破坏药物诺考达唑 2 分钟后，粘着斑相关蛋白纽蛋白、桩蛋白和粘着斑激酶 (FAK) 的局部密度显着升高，粘着斑面积增加，而仅几分钟后，生长粘连内的酪氨酸磷酸化就会升高。磷酸酪氨酸和 FAK 密度在治疗 10 分钟后达到最高水平，而纽蛋白和桩蛋白水平以及粘着斑大小继续增长，在约 30 分钟时达到稳定水平。我们的研究结果表明，蛋白质的募集和粘着斑的生长是微管破坏引起的直接且直接的结果或增加的收缩性，而酪氨酸磷酸化是稍后激活的。
Tyrosine phosphorylation of focal adhesion components is involved in the regulation of focal adhesion formation and turnover, yet the underlying molecular mechanisms are still poorly defined. In the present study, we have used quantitative fluorescence microscopy to investigate the dynamic relationships between the incorporation of new components into growing focal adhesions and tyrosine phosphorylation of these sites. For this purpose, a new approach for monitoring phosphotyrosine levels in live cells. was developed, based on a 'phosphotyrosine reporter' consisting of yellow fluorescent protein fused to two consecutive phosphotyrosine-binding Src-homology 2 (SH2)-domains derived from pp60(c-Src). This YFP-dSH2 localized to cell-matrix adhesions and its intensity was linearly correlated with that of an anti-phosphotyrosine antibody labeling. The differential increase in vinculin and phosphotyrosine levels was examined in live cells by two-color time-lapse movies of CFP-vinculin and YFP-dSH2. In this study, focal adhesion growth was triggered by microtubule disruption, which was previously shown to stimulate focal adhesion development by inducing cellular contraction. We show here that, 2 minutes after addition of the microtubule-disrupting drug nocodazole, the local densities of the focal adhesion-associated proteins vinculin, paxillin and focal adhesion kinase (FAK) are significantly elevated and the focal adhesion area is increased, whereas elevation in tyrosine phosphorylation inside the growing adhesions occurs only a few minutes later. Phosphotyrosine and FAK density reach their maximum levels after 10 minutes of treatment, whereas vinculin and paxillin levels as well as focal adhesion size continue to grow, reaching a plateau at about 30 minutes. Our findings suggest that protein recruitment and growth of focal adhesions are an immediate and direct result or increased contractility induced by microtubule disruption, whereas tyrosine phosphorylation is activated later.