FRAP Analysis: mechanical forces alter zyxin binding kinetics within focal adhesions of living cells

M. BIANCHI; L. SIGAUT; C. VON BILDERLING; L. GASTALDI; L. I. PIETRASANTA

Carlos Paz, Córdoba.

Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica

Focal adhesions (FAs) are large, multiprotein complexes that provide a mechanical link between the cytoskeletal contractile machinery and the extracellular matrix, and are involved in cell proliferation, migration and motility. FAs exhibit mechanosensitive properties: in the presence of a mechanical stimulus, FAs are dynamically assembled and disassembled. To investigate the influence of mechanical forces on the molecular binding kinetics of focal adhesion proteins, we use an approach based on Fluorescence Recovery After Photobleaching (FRAP). In this work, we study the kinetics of the FA protein zyxin - a suggested mechanosensitive protein - tagged with green fluorescent protein (GFP) and expressed in epithelial mammalian living cells (HC11), in response to a mechanical stretch. Controlled and repeatable mechanical stimuli were performed using a stretching device that allows both live fluorescence imaging and mechanical equibiaxial stretching of the cells cultured on silicone elastic membranes. From FRAP recovery curves, assuming that the binding kinetics is much slower than the diffusion time, we estimate the dissociation constant and fraction mobile of the protein.