Research Projects

Mechanical forces are an integral component of the cellular milieu in tissues and guide processes from individual cells to that of collectives. Cell-matrix and cell-cell interactions, and traction generating mechanisms underlie the complex interplay in cell ensembles to define the mechanical properties of tissues. Cells attach to substrates through transmembrane integrin proteins, located in focal adhesions, and crawl using coordinated and cooperative active traction generating mechanisms to display solid-like behaviors in the short time interval and fluid-like viscous behaviors over longer durations. Cells also synthesize proteins and enzymes in response to mechanical cues through feedback mechanisms to determine self-regulation and tissue organization. Migrating cells in epithelial layers create finger-like projections at the leading edge of the ensemble, called leader cells, that are accompanied by follower cells in the monolayer interior. Leader cells break the monolayer symmetry and direct collective migrations through Rac-dependent actin polymerization processes, in combination with other biochemical signals, to produce synchronized collective behaviors. The primary goals of this study are to quantify cellular behaviors when subjected to mechanical stimuli using experimental and computational methods to explore symmetry breaking mechanisms and generate leader cells. We use Madin–Darby canine kidney (MDCK) epithelial cells cultured on elastomeric substrates of differing stiffness, fabricated using a combination of commercial formulations of poly dimethyl siloxane (Sylgard® 184 and Sylgard® 527), in circular and elliptical shapes, and subjected to cyclic stretching using a custom fabricated microscope mountable stretcher. Cells will be transfected with Clover-Geminin to mark the G2/ G1 phases of the cell cycle, and E-cadherin to help identify cell boundaries in the monolayer, during the stretching experiments. Cyclic uniaxial and equibiaxial stretch will be used to stretch the cell collectives. Cell areas and proliferations will be measured to test the role of density fluctuations in leader cell creation during mechanical stretching. Monolayer stress microscopy will be used to characterize the cell tractions on different substrates. Image processing will yield inputs on velocities, vorticities, and divergence fields during collective migrations to explore the role of mechanical factors in leader cell creation, cell proliferations, and migrations. Knock downs for talin and cytoskeletal inhibitors to stabilize and disrupt actin and myosin motors will be used to measure the roles of stress fiber dynamics in these behaviors. Computational particle based models will be used to explore the role of the individual factors leader cell creation during collective migrations under cyclic loads. These results will be useful to understand how mechanical cues are detected, integrated and manipulated by cells to dictate the overall tissue properties.