Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin avb3, but not in those by integrin a5b1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.
Bibliographical noteFunding Information:
We thank Drs. Roger Y. Tsien and Ryan G. Holzer (University of California, San Diego), Maria Nemethova and Victor Small (Institute of Molecular Biotechnology, Vienna, Austria), Martin A. Schwartz (University of Virginia) for valuable reagents, constructs, and SYF-/-cell lines. We also thank Dr. Gandauz Danuser (Harvard University) for insightful discussion. We thank Jason Fan (University of Toronto, Canada) for critically proofreading this manuscript. This work is supported by grants from NIH HL098472, HL109142, NS063405, NSF CBET0846429 (Yingxiao W. and S. L.), NIH HL121365 (S.C. and Yingxiao W.), the University of Illinois Research Fund (S.L.) and the Wallace H. Coulter Foundation and Beckman Laser Institute, Inc. (Yingxiao W.). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright 2014 Elsevier B.V., All rights reserved.