A Preliminary Study For Probing Cellular Mechanics:from Collective Cell Migration To Single Molecular Force Dynamics

During the cell adhesion and migration,mechanical factors such as matrix rigidity and topography have the important impacts on the cell adhesion behaviors.However,the precondition for studying the interplay between physical force and cell adhesion is the measurement of adhesion force exerted by cells.In order to explore the force transduction mechanism of cell adhesion and migration,this article achieved the measurement of adhesion force from collective cells to single-molecule dynamics,and preliminary studied the effect of focal adhesion-related proteins on the cell adhesion mechanics.The main results are summarized as followed:(1)We prepared the fluorescent nanobeads-coated polyacrylamide gels of increasing rigidity.MEF cell were seeded and MDCK cell were confined to monolayer with PDMS on the gel.The traction force exerted by MEF cells plated on polyacrylamide gels of increasing rigidity were measured by traction force microscopy.The traction force dynamics during the process of collective cell migration and wound healing were measured.We showed that the traction force of MEF cell increased with rigidity,and also the cell area,cell polarization,the size of focal adhesion.During the collective cell migration,the highest tractions were localized at the monolayer edge,while the large dynamic fluctuations disappeared as the monolayers approached each other and the wound were closed.(2)Non-uniform hydrogel were prepared by localized illumination method of photopolymerization,and the durotaxis of individual cell and collective cell migration were studied.We showed that MEF cells spread larger on the stiffness hydrogel and rounder on the soft hydrogel.Cell located at the junction of the non-uniform hydrogel migrated from soft region to stiffness region,and collective cells that were seeded on stiffness region migrated faster than soft region,which displayed the features of durotaxis during the rigidity-sensing process.(3)We knocked out the vinculin and talin1 of MEF cells by CRISPR-Cas9 gene editing technique,inhibited the activity of myosin by Blebbistatin and activated the Rho pathway by Rho Activator ? to investigate the effect of focal adhesion proteins on cells exerting traction on the gel of increased rigidity.vinculin-knocked out and talin1-knocked out cells both exerted lower traction force than wild-type cell,but the reduction of traction force caused by the knock-out of talin1 were much more than that of the knock-out of vinculin.MEF cells that inhibited myosin showed a gradual decrease in traction with increasing drug concentration,while activation of the Rho pathway increased the traction of MEF cells on stiffness substrates,but there was no significant change on soft substrates.(4)With the molecular tension sensor for single-molecule imaging,the effect of knocking out vinculin and inhibiting myosin on the adhesion mechanics of the interaction between integrin and substrate were investigated.The single-molecule imaging results by the molecular tension sensor showed that the integrin-sensor binding lifetime of vinculinknocked out cells were much shorter and the bind interaction were more dynamic than wild-type MEF cells.In contrast,the inhibition of myosin did not display the reduction of the binding time,just the number of bind interactions has dropped significantly.