Study of binding dynamics of focal adhesion proteins using Fluorescence Microscopy and AFM techniques

CATALINA VON BILDERLING; MARTÍN E. MASIP; ESTEBAN HOIJMAN; ANDREA V. BRAGAS; LÍA I. PIETRASANTA

Salta, Argentina

Congreso; 3rd. Latin American Protein Society Meeting and Annual Meeting of the Argentinean Biophysical Society (SAB 2010).; 2010

Latin American Protein Society - Argentinean Biophysical Society

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 by cells, remodeling presumably governed by the modulation of temporary interactions between its constituent proteins. Our understanding of biophysical and molecular mechanisms underlying these events remains poor. The goal of our work is to study the dynamic response of FAs proteins when a precise and punctual force is applied. In this context, we are applying Fluorescence Microscopy, Atomic Force Microscopy and Force Spectroscopy (FS) on different biomolecular systems in order to optimize force measurements at the intermolecular level and investigate protein dynamics and protein-protein interactions on FAs of live cells. By the fluorescence recovery after photobleaching (FRAP) technique, we studied the kinetic recruitment of different FAs proteins in the presence and in the absence of a mechanical force. We have expressed in epithelial mammalian living cells zyxin, vinculin, FAK and paxilin proteins tagged with green fluorescent protein (GFP) and analyzed the molecular binding interactions, determining the dissociation rate constant parameter koff for the adhesion proteins in situ. We also characterized with FS the interaction forces between chemical functional groups and the well known ligand-receptor pair Biotin-Streptavidin. We acquired arrays of force versus cantilever displacement curves to obtain high-resolution 2-D maps of the specific interactions between cell surface and functionalized AFM probe tips.