Becoming Stable and Strong: The Interplay Between Vinculin Exchange Dynamics and Adhesion Strength During Adhesion Site Maturation
Becoming Stable and Strong: The Interplay Between Vinculin Exchange Dynamics and Adhesion Strength During Adhesion Site Maturation
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DOI：
10.1002/cm.20375
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发表时间：
2009-06-01
影响因子：
--
通讯作者：
Hoffmann, Bernd
中科院分区：
文献类型：
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作者：
Moehl, Christoph C.;Kirchgessner, Norbert;Hoffmann, Bernd
局部粘连的协调形成和释放是细胞附着和迁移所必需的。根据目前的模型，这些过程是由蛋白质组成的时间变化引起的。蛋白质掺入局部粘连被认为是由磷酸化控制的。在此，我们利用光漂白后荧光恢复的方法检测了作为焦点粘连标记蛋白的GFP-vinculin的交换动力学。研究了蛋白质的磷酸化状态、焦点粘连的年龄和作用力之间的相关性。对于稳定的角质形成细胞局灶性粘连，我们测定了可交换纽蛋白的含量为52%，恢复半衰期为57 S，运动细胞的初生灶粘连的交换纽蛋白含量为70%，恢复半衰期为36 S，成熟后的平均饱和值和恢复半衰期分别降至49%和42 S的水平。此外，稳定掺入的纽蛋白的比例随着细胞力量的增加而增加，而随着纽蛋白在这些部位的磷酸化而减少。在磷酸化位点tyr1065处构建的非磷酸化纽蛋白突变体的实验证实，在黏附位点成熟过程中，黏附蛋白的磷酸化和交换动力学之间存在直接的相互作用。细胞运动。《细胞骨架66：350-364》，2009年。(C)2009年Wiley-Liss，Inc.
The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation. Cell Motil. Cytoskeleton 66: 350-364, 2009. (C) 2009 Wiley-Liss, Inc.