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

Linz

Workshop; XIII. Annual Linz Winter Workshop and hands-on School: Advances in Single-Molecule Research for Biology & Nanoscience; 2011

Johannes Kepler University Linz. Linz, Austria

In this work we have studied the kinetic recruitment of different proteins of Focal Adhesions (FAs) by the fluorescence recovery after photobleaching (FRAP) technique. We have expressed in epithelial mammalian living cells zyxin, paxilin and FAK proteins tagged with green fluorescent protein (GFP) and analyzed the molecular binding interactions, determining the unbinding rate constant parameter kOFF for the adhesion proteins in situ. We also characterized with AFM-force spectroscopy (AFM_FS) the interaction forces between chemical functional groups and the well known ligand-receptor pair biotin-streptavidin. These results establish the bases for studying the response of the proteins dynamics within intact FAs in living cells under a mechanical stimulus in the process of cellular mechanotransduction.