PhD Proposal Presentation - Dennis Zhou

Advisor:

Andrés J. García, Ph.D. (Georgia Institute of Technology)  

Committee:

Cheng Zhu, Ph.D. (Georgia Institute of Technology)

Jennifer E. Curtis, Ph.D. (Georgia Institute of Technology)

Andrew P. Kowalczyk, Ph.D. (Emory University)

Aránzazu del Campo, Ph.D. (Leibniz Institute for New Materials)

Force-Signaling Coupling at Single Focal Adhesions

Cell adhesion plays a critical role in development, physiology, and disease. Despite significant progress in determining the biochemistry driving cell adhesion assembly and signaling, very little is known about how cell adhesive forces are transduced into biochemical signals. The objective of this project is to analyze how focal adhesions (FAs) sense and transmit force. Our central hypothesis is that mechanosensing at a single FA involves a feedback loop in which force regulates FAK phosphorylation, which controls recruitment of vinculin and paxillin to the FA to tune the local force balance between extracellular matrix-integrin forces and cytoskeletal tension. Through this integrated module, force and FA signaling are coupled.

 We will first analyze the effects of local adhesive force and cytoskeletal tension on FAK phosphorylation and its coupling to force generation and vinculin and paxillin recruitment to FAs. We will establish spatiotemporal profiles for force transmission and FA assembly at single FAs by analyzing live cells expressing fluorescent FA proteins on microfabricated pillar-array-detectors (mPADs), which consist of an array of silicone micropillars, and are commonly used to measure cell traction forces, based on the deflections of the micropillars. The micropillars will also present caged RGD peptide, which can be activated with UV light to initiate FAs at prescribed sites and generate coupled spatiotemporal FA assembly and force generation profiles at single adhesions. We will evaluate these force-signaling responses on mPADs with different elastic moduli and in the presence of contractility modulators. By varying substrate stiffness and contractility state, we will perturb the local force balance between ECM-integrin forces and cytoskeletal tension to examine how adhesive force balance regulates FAK phosphorylation and vinculin-paxillin recruitment at single FAs. We will use also FAK inhibitors to determine how FAK phosphorylation regulates force transmission and vinculin and paxillin recruitment. We hypothesize that adhesive forces at FAs regulate FAK phosphorylation, which then drives paxillin localization and vinculin recruitment. We will next evaluate force-FAK phosphorylation coupling for cells expressing mutant vinculin head and tail domains to dissect the contributions of vinculin head and tail domains to regulating force-dependent FAK phosphorylation at FAs. As outcomes of this project, we will establish how the local balance of adhesive force and cytoskeletal tension regulates coupled force-FAK signaling at single FAs. We will also dissect the roles of FAK and vinculin in this mechanosensing. This research will generate insights into how cell adhesive forces are integrated into biochemical signals. This understanding will provide a framework for mechanotransduction events at cell-ECM junctions, such as adhesion assembly at migratory fronts, force-regulated morphogenesis, and stem cell lineage commitment in response to matrix stiffness.