|M.Sc Student||Feld Lea|
|Subject||Cell-scale contractile forces emerge from|
non-mechanosensitive active displacement
|Department||Department of Medicine||Supervisor||Dr. Haguy Wolfenson|
|Full Thesis text|
The extracellular matrix provides multiple signals for cells that interact with it. These can determine cell fate decisions, including proliferation, migration, and differentiation. A critical signal is the stiffness of the matrix, which cells sense by applying contractile force to it. To produce contractile forces, myosin motors pull on actin fibers that are attached to the matrix through adhesion protein complexes. Resistance of the matrix to the force provides the cell the information about its stiffness. This mechanical signal is transferred into the cell to initiate a cascade of events that affect long-term cellular decisions.
Previous studies showed that the maximal values of contractile forces that cells transmit to the matrix are proportional to its stiffness, suggesting that the contractile forces are mechanosensitive in nature, i.e., that the buildup of force over time until it reaches saturation depends on the ability of the cell to measure matrix rigidity. To test this hypothesis not only at force saturation, but at any time t, we used a novel model that describes the variables on which contractility depends. Our model predicts that when the matrix is much softer than the actin fibers (true for physiological conditions), the force equals to the rigidity constant of the matrix times its time-dependent displacement. As the rigidity constant is independent of the mechanosensing process, only the displacement may have a mechanosensitive contribution. We performed experiments using silicone pillar arrays of different rigidities that allow measuring matrix displacement over time. Tracking five cell lines during early spreading on the pillars showed that while forces increase with matrix rigidity, the time-dependent displacement is the same for all rigidities. Thus, displacement is independent of rigidity and contractile forces are nonmechanosensitive.
Notably, whereas the displacement was the same on all rigidities, the displacement function itself differed between cell lines. To find the origin of this cell-type specificity, we focused on the actomyosin network. Simultaneous measurements of pillar displacement and the concentration of fibrous actin around it revealed high correlation on all tested rigidities. The proportionality of this correlation was however cell type specific. To test whether differences in actin spatial organization can underlie this proportionality, we used super-resolution microscopy and advanced image analysis. Indeed, actin density and directionality were significantly different between cell lines. Alignment of actin structures with myosin clusters showed high correlation in all cases, implying that myosin is present in access and supporting the notion that actin concentration and organization are the critical factors in force generation.
Lastly, the displacement did not change with rigidity also in cells that were at steady-state, and the displacement function at steady-state was similar to the one during early spreading, only multiplied by a factor of ~6. This can be attributed to the much more mature and organized actin stress fibers found at steady-state compared those found at early spreading.
Collectively, this study provides a novel framework to address cellular contractility and rigidity sensing. Further studies are needed to identify and define processes that affect cellular responses downstream of the nonmechanosensitive forces.