Regulation of contractility in mammalian cells by soluble, adhesive and mechanical cues of the microenvironment

Traction forces exerted by adherent cells on their surroundings play an important role in diverse physiological processes including cell migration and endothelial barrier regulation. Intracellular forces are generated by myosin II motors that pull on the actin cytoskeleton, and are transmitted to the extracellular matrix (ECM) via focal adhesions (FAs). The coordination of contractile forces is influenced by extracellular cues such as soluble factors, substrate rigidity and ECM density. In my thesis, I have developed approaches to decouple the effects of different extracellular cues on contractility. To measure cellular contractility, we have fabricated dense arrays of elastomeric microposts (mPADs) on which cells are able to adhere and generate forces that deflect underlying microposts. To determine optimal dimensions for the mPADs, I examined the effect of varying post diameter, center-to-center spacing and height on contractility and cell spreading. Decreasing the micropost spacing beyond a critical threshold had negligible impact on the morphology of spread cells while decreasing micropost diameter significantly decreased the magnitude of forces per post generated by cells. Henceforth, we generated a new library of high-density HD-mPADs in which post spacing and diameter were kept constant but height was varied to manipulate substrate rigidity. With these new devices, I observed that micropost rigidity influenced cell spreading and FA assembly. However, micropost rigidity had a negligible effect on contractility. Beyond these fundamental studies of cell-material interactions, I have used the HD-mPADs primarily to examine how different vasoactive agonists affect contractility. I performed live-cell imaging of cells on HD-mPADs at high temporal resolution to capture dynamic changes in contractility elicited by soluble factors and/or drugs. In this study, I observed a previously uncharacterized contractile response in endothelial cells to vascular endothelial growth factor (VEGF) that differed dynamically and in magnitude, from the responses to known contractility agonists. I characterized the signaling mechanisms regulating this contractile response to VEGF and examined how the extracellular presentation of VEGF influenced the response. Future development of new experimental and computational approaches will further our understanding of the findings presented in this thesis