Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

Shaoying Lu, Jihye Seong, Yi Wang, Shiou Chi Chang, John Paul Eichorst, Mingxing Ouyang, Julie Y.S. Li, Shu Chien, Yingxiao Wang

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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 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.

Original languageEnglish (US)
Article number5756
JournalScientific reports
Volume4
DOIs
StatePublished - Jul 24 2014

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Lu, S., Seong, J., Wang, Y., Chang, S. C., Eichorst, J. P., Ouyang, M., Li, J. Y. S., Chien, S., & Wang, Y. (2014). Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells. Scientific reports, 4, [5756]. https://doi.org/10.1038/srep05756