title: In-situ coupling between kinase activities and protein dynamics within single focal adhesions.
abstract: The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells.
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Yiqian
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Kaiwen
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, South Korea.
authors.forename: Jihye
authors.initials: J
authors.lastname: Seong
authors.affiliation: Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Fan
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.|Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Chien
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.|Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.|Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA.|Center of Computational Mathematics, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Shaoying
authors.initials: S
authors.lastname: Lu
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D022001:Focal Adhesions; D006801:Humans; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*
chemical_list: D011506:Proteins; D011494:Protein Kinases