Correlation between focal adhesion protein zyxin aggregation state and response to mechanical stress using an equibiaxial stretching device

CATALINA VON BILDERLING; LORENA SIGAUT; LAURA C. ESTRADA; LIA I. PIETRASANTA

Buzios

Congreso; II Latin American Federation of Biophysical Societies (LAFeBS) Congress and Latin American Postgraduate Program of Biophysics Course 2012; 2012

Latin American Federation of Biophysical Societies

Mechanical forces play an important role in the organization, growth, maturation, and function of living tissues. Mechanotransduction, the process by which cells sense and respond to mechanical signals, is mediated by extracellular matrix, transmembrane integrin receptors, cytoskeletal structures and associated signaling molecules organized in flat, elongated and transient structures known as focal adhesions (FAs). In the presence of a mechanical stimulus, FAs are dynamically assembled and disassembled and this constant remodeling is presumably governed by the modulation of temporary interactions between its constituent proteins [Nat. Rev. Mol. Cell. Biol., 7:265-275, 2006]. The understanding of the biophysical and molecular mechanisms underlying these events requires the development of techniques to identify the organization and dynamics of the FAs proteins in response to a mechanical stimulus. These techniques may have a high spatio-temporal resolution to detect transient and strongly localized events that occur in a region of the cell. In this work, we introduce a stretching device (BMC Cell Biology 10:55-68, 2009) that allows both live fluorescence imaging and mechanical equibiaxial stretching of the cells cultured on silicone elastic membranes. Careful characterization of the device described herein shows its capability of delivering up to 12% of two-dimensional homogeneous strain to silicone membranes. We were able to study the distribution and stoichiometry of the FA protein zyxin in response to stretching. We have expressed in epithelial mammalian living cells (HC11) zyxin protein tagged with green fluorescent protein (GFP) and obtained, as a function of an applied force, the stoichiometry of the zyxin aggregates by Number and Brightness (Biophys. J. 94:2320-2332, 2008) analysis.