Investigating complexity of protein–protein interactions in focal adhesions

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• 作者：Tanmay P. Lele ; Charles K. Thodeti ; Jay Pendse ; Donald E. Ingber
• 关键词：Focal adhesions ; FRAP ; Kinetics ; Dynamics ; Endothelial cells
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2008
• 出版时间：9 May 2008
• 年：2008
• 卷：369
• 期：3
• 页码：929-934
• 全文大小：973 K

文摘

The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k_OFF ranged over an order of magnitude from 0.009 ± 0.001/s for talin to 0.102 ± 0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.