Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy

LORENA SIGAUT; MICAELA BIANCHI; CATALINA VON BILDERLING; LÍA I PIETRASANTA

Congreso; 20th IUPAB Congress, 45th Annual SBBf Meeting, and 50th Annual SBBq Meeting; 2021

Cells exert traction forces on the extracellular matrix to which they are adheredthrough the formation of focal adhesions. Spatial-temporal regulation of tractionforces is crucial in cell adhesion, migration, cellular division, and remodeling ofthe extracellular matrix. In this work, we present an approach based on acombination of several microscopies and quantitative data analysis that allowedus to explore the correlation between the generation of traction forces and zyxindynamics at focal adhesions. To this end, we combine techniques such astraction force microscopy (TFM), fluorescence recovery after photobleaching(FRAP) and fluorescence correlation spectroscopy (FCS), in addition to thefabrication of adjustable stiffness polyacrylamide hydrogels and thecharacterization of their elasticity by force spectroscopy using an atomic forcemicroscope (AFM). By cultivating cells on polyacrylamide hydrogels of differentstiffness we were able to investigate the effects of substrate stiffness on thegeneration of cellular traction forces by TFM, and characterize the moleculardynamics of the focal adhesion protein zyxin by FCS and FRAP. As the rigidityof the substrate increases, we observed an increment of both, cellular tractiongeneration and zyxin residence time at the focal adhesions, while its diffusionwould not be altered. Moreover, we found a positive correlation between thetraction forces exerted by cells and the residence time of zyxin at the substrateelasticities studied. This correlation persists at the subcellular level, even ifthere is no variation in substrate stiffness, revealing that focal adhesions thatexert greater traction present longer residence time for zyxin. A key advantagein the approach presented here lies in the possibility of an integral andmultiparametric single cell analysis. Our results provide furtherevidence reinforcing the mechanosensitive properties of zyxin, pointing it out asa key protein for cellular traction forces.