title: Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy.
abstract: Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.forename: Claire M
authors.initials: CM
authors.lastname: Brown
authors.forename: Alan R
authors.initials: AR
authors.lastname: Horwitz
authors.forename: William W
authors.initials: WW
authors.lastname: Mantulin
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D046912:Multiprotein Complexes / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D051419:Paxillin / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D046912:Multiprotein Complexes; D051419:Paxillin