Correlation fluctuation analysis of focal adhesion proteins in cells

MICAELA BIANCHI; LORENA SIGAUT; LÍA PIETRASANTA

Congreso; II Latin American Federation of Biophysical Societies Congreso / xxxVII Congreso of the Brazilian Biophysical Society; 2012

Fluorescence fluctuation analysis allows the measurement of molecular motion and interaction due to its single molecule sensitivity and it is compatible with well-established microscopy techniques used in live cell imaging. Cells can sense and transduce a wide range of mechanical forces into different sets of biochemical signals that ultimately regulate cellular processes, including adhesion, proliferation, differentiation, and apoptosis. The focal adhesions (FAs) are comprised of structural proteins that link integrin receptors to the actin cytoskeleton as well as several signaling molecules (Nat. Rev. Mol. Cell. Biol., 7:265-275, 2006). Many of these molecules are proposed to interact with one another to form an integrated mechanical and biochemical network that regulates the component processes of migration and adhesion.  In this work, we study the molecular diffusion of FAs proteins by determining their diffusion coefficients in living cells using laser confocal imaging techniques (raster-scan mode). For this purpose, we exploited the temporal scales of single-point fluorescence correlation spectroscopy (FCS) and line-FCS, and the advantages of raster-scan image correlation spectroscopy (RICS) in providing spatial information. Zyxin, paxillin and FAK proteins tagged with visible fluorescent proteins (VFPs) were expressed in epithelial mammalian living cells (HC11) and images were acquired in an Olympus spectral confocal microscope FV1000. Correlation analysis was performed using SimFCS software (Laboratory of Fluorescence Dynamics, University of California, Irvine, CA).