Conformational changes of focal adhesion protein vinculin in response to mechanical stress using an equibiaxial stretching device

LORENA SIGAUT; CATALINA VON BILDERLING; MICAELA BIANCHI; LAURA GASTALDI; LÍA PIETRASANTA

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

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 a complex network of different proteins. Evidence from cells in culture suggests that vinculin functions in transducing force across cell membranes [1,2] in regulating cell adhesion and motility [3,4]. When cells adhere and spread on the extracellular matrix (ECM) a portion of the cytoplasmic vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FA). At these sites, dynamic connections are made between the actin cytoskeleton and ECM through transmembrane integrin receptors. In the presence of a mechanical stimulus, FAs are dynamically assembled and disassembled.  In this work, we introduce a stretching device [5] 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. Using a Forster resonance energy transfer probe that reports on changes in vinculin’s conformation [6] expressed in epithelial mammalian living cells (HC11) cultured in the silicone membrane we were able to study conformational changes of vinculin in response to stretching.