Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy

¹ University of California, Irvine
² McGill University
³ University of Virginia

^* To whom correspondence should be addressed. E-mail: mdigman{at}uci.edu.

Submitted on January 22, 2007
Revised on March 26, 2007
Accepted on 21 August 2007

	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 time scales 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 FCS (Fluctuation Correlation Spectroscopy) and Photon Counting Histogram (PCH) analysis. Near adhesions paxillin dynamics are reduced drastically presumably due to binging to protein partners within the adhesions. The PCH 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, while in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning FCS and Raster-scan Image Correlation Spectroscopy (RICS) analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions "appear" to slide transversally due to a tread milling 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.

Key Words: Adhesions, FCS, Fluorescence, Paxillin