title: Cross-correlated fluctuation analysis reveals phosphorylation-regulated paxillin-FAK complexes in nascent adhesions.
abstract: We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia. Electronic address: colinc@seas.upenn.edu.
authors.forename: Colin K
authors.initials: CK
authors.lastname: Choi
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia.
authors.forename: Jessica
authors.initials: J
authors.lastname: Zareno
authors.affiliation: Laboratory of Fluorescence Dynamics, University of California, Irvine, California; Department of Biomedical Engineering, University of California, Irvine, California.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.affiliation: Laboratory of Fluorescence Dynamics, University of California, Irvine, California; Department of Biomedical Engineering, University of California, Irvine, California.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia.
authors.forename: Alan Rick
authors.initials: AR
authors.lastname: Horwitz
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D051419:Paxillin; D019000:Phosphotyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases