Interaction revealed by Fluorescence Cross-correlation Spectroscopy of focal adhesion protein in living cell

MICAELA BIANCHI; LORENA SIGAUT; LAURA GASTALDI; LÍA PIETRASANTA

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Focal adhesions (FAs) are comprised of structural proteins that link integrin receptors to the actin cytoskeleton as well as several signaling molecules [1]. 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 adhesion. Many processes in chemistry, physics, and biology, such as molecular diffusion, binding, blinking and rotational motions, have a different time-scale [2]. Fluorescence Correlation Spectroscopy (FCS) is an analytical tool for studying, concentrations, propagation, interaction and internal dynamics of molecules at nanomolar concentrations in living cells [3]. Dual color Fluorescence Cross- correlation Spectroscopy (FCCS) is a powerful multicolor extension of FCS that probe the interaction of two differently labeled molecular species with higher precision than single color FCS [3].  In this work, we analyzed processes of different time scale by FCS and FCCS. With this tool we study binding interaction of two fluorescent pair of FA proteins: EGFP-vinculin and mCherry-paxillin, and EGFP-FAK and mCherry-paxillin. Vinculin, 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).