Mechanical Signaling Induced Cellular Remodeling Studied By Integrated Optical And Atomic Force Microscopy

Vascular wall composition and mechanics are important for cardiovascular physiology and pathology. The reciprocal interaction between cells and their microenvironment influence cellular adaptation to external mechanical cues through the remodeling of cytoskeletal structures and cell?matrix adhesions to ensure normal cell function. We proposed to investigate the relationship between the cytoskeletal tension development and cell adhesion to the matrix in the context of cellular contraction and migration. Our studies aimed to understand how cells sense, respond, and adapt to external mechanical forces in order to induce vascular remodeling in cardiovascular disease.

Integration of atomic force microscopy with total internal reflection fluorescence and spinning-disk confocal microscopy enabled acquisition of complementary structural and functional measurements on live vascular smooth muscle cells expressing key mutant proteins with important roles in defining contractile and migratory cellular properties.

Single ligand?receptor interaction measurements showed that RhoA and c-Src activation have different effects on cytoskeletal tension development, inducing two distinct force?stiffness functional regimes for ?5?1-integrin binding to fibronectin. In addition, c-Src was associated with regulation of myosin light chain phosphorylation, suggesting a c-Src-dependent modulation of RhoA pathway through activation of downstream effectors. These data were in good agreement with fluorescence measurements that showed a modest effect of Src activation on stress fibers formation, in contrast with RhoA activation that had a significant effect. On the other hand, ?-actin null cells exhibited increased FAK activation and cell stiffness. Our results suggest that the absence of ?-actin may induce compensatory effects of up-regulation of other contractile proteins and activation of focal adhesion proteins in order to encourage cell migration and proliferation. In addition, our findings suggest that Nck regulates directional cell migration in part through modulation of cytoskeletal tension and cell-matrix adhesion strength, which has an important role in coordination of cytoskeletal mechanics through a mechanism that also involves the RhoA pathway.

Thus, our findings suggest that the contractile state of the cell is determined by cytoskeletal tension, which is controlled by a regulatory network involving RhoA and activation state of actomyosin apparatus. In turn, the cytoskeletal tension state modulates integrin ?5?1?fibronectin adhesion force. The results of this study suggest a central role for cytoskeletal tension in modulating cytoskeletal dynamics and cell adhesion to the matrix.