Monitoring real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus

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

Varadero

Congreso; XIV Interamerican Congress of Microscopy CIASEM 2017; 2017

Comité Interamericano de Sociedades para Microscopía Electrónica -CIASEM

Mechanical forces have been demonstrated to be essential in the function, organization, growth andmaturation of living tissues [1]. Cells constantly sense and respond to their physical microenvironment through aprocess known as mechanotransduction, by which a mechanical stimulus can be converted into a biochemicalresponse. These specific mechanosensing mechanisms, mediated by the actin cytoskeleton, can be modulated bya wide range of forces. Focal adhesions (FAs), the specialized multiprotein structures that mediatemechanotransduction at the cell-matrix level, are dynamic complexes that can undergo assembly, disassemblyand movement. Their composition involves more than 200 proteins, from which the extracellular matrix (ECM)protein fibronectin is known to trigger a force-driven assembly of focal complexes.The study of mechanotransduction responses requires the correlated measurement of mechanical stimuliand real time live-cell responses. Several approaches have been designed to assess this challenge, using externalmechanical stimuli that range from forces globally applied to the cell (such as fluid shear stress or usingstretching devices) to specific and locally applied forces, generally involving the use of fibronectin (orfibronectin-fragments)-modified beads and tweezers to exert/sense the forces. AFM has become a powerful toolfor nanobiotechnological investigations, capable to exert and probe forces on single cells and molecules [2]. Thecombination of AFM and fluorescence microscopy has been applied successfully to various systems where theresponse to a precise mechanical stimulus must be monitored. However, these studies generally analyze globalcell responses such as cell shape or total protein area, and very few of them quantify the temporal evolution ofthese responses with spatial resolution. In this work, we describe the integration of an AFM with anepifluorescence microscope [3] to investigate the dynamics of adhesion proteins within single FA in response toa controlled local mechanical stimulus. Indeed, AFM combines the possibility of applying forces from pN to nN,localized in a contact area that can be down to a few nm for typical AFM-probes. A mechanical load was exertedon a living cell surface by a fibronectin-modified AFM probe as time-dependent spatial redistribution of theadhesion proteins vinculin, FAK, paxillin and zyxin was monitored in time. With this methodology, we are ableto observe the development of a nascent focal site triggered by a local mechanical stimulus (Figure 1). Aquantitative analysis of the fluorescence intensity allowed us to evaluate characteristic protein recruitment timesin the force induced assembly of focal complexes. In addition, we also observed a spatial distribution remodelingof zyxin (accumulation - loss) within mature FA in response to applied force of different strengths. We foundthat the remodeling is spreading away from the applied stimulus as the force is increased. These results evidencea mechanical connection between the force contact point and mature focal adhesions.