title: Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells.
abstract: 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 αvβ₃, but not in those by integrin α₅β₁. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.
authors: 1] Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093-0435 [2] Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors: Shaoying
authors: S
authors: Lu
authors: 1] Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801 [2].
authors: Jihye
authors: J
authors: Seong
authors: Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors: Yi
authors: Y
authors: Wang
authors: Department of Chemical Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors: Shiou-chi
authors: SC
authors: Chang
authors: Center of Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, Department of Molecular and Integrative Physiology and, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors: John Paul
authors: JP
authors: Eichorst
authors: 1] Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093-0435 [2] Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors: Mingxing
authors: M
authors: Ouyang
authors: Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093-0435.
authors: Julie Y-S
authors: JY
authors: Li
authors: Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093-0435.
authors: Shu
authors: S
authors: Chien
authors: 1] Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093-0435 [2] Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 [3] Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801 [4] Center of Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, Department of Molecular and Integrative Physiology and, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
authors: Yingxiao
authors: Y
authors: Wang
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002448:Cell Adhesion; D002478:Cells, Cultured; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D022001:Focal Adhesions / Q000201:enzymology* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000096:biosynthesis; D051379:Mice; D008856:Microscopy, Fluorescence; D010982:Platelet-Derived Growth Factor / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D005353:Fibronectins; D008164:Luminescent Proteins; D010982:Platelet-Derived Growth Factor; D019061:src-Family Kinases; D000097573:Red Fluorescent Protein