BioE PhD Defense Presentation- Dennis Zhou
Andrés J. García, PhD (ME, Advisor)
Aránzazu del Campo, PhD (Leibniz Institute for New Materials)
Jennifer Curtis, PhD (Physics)
Andrew Kowalczyk, PhD (Emory)
Cheng Zhu, PhD (BME)
Force-Signaling Coupling at Single Focal Adhesions
Integrin-mediated adhesion to extracellular matrices (ECM) provides forces and signals that direct cell processes central to tissue organization, homeostasis, and disease. Recent studies show an important relationship between cell adhesive force generation and focal adhesion (FA) assembly, yet it remains unclear how forces are transduced into adhesive signals. Our work seeks to assess coupling between cell adhesive force generation and signaling at FAs. To measure forces, we used Microfabricated Post-Array-Deflectors (mPADs), which are an array of PDMS ~1.8 µm diameter microposts. Based on the micropost deflections, we can calculate the forces exerted by cells. We first examined the relationship between traction force and vinculin-paxillin localization to single FAs in the context of substrate stiffness and actomyosin contractility. Substrate stiffness and contractility regulated vinculin localization to FAs, and vinculin auto-inhibition is a crucial regulatory step in this process that overrides the effects of cytoskeletal tension and substrate stiffness. Vinculin and paxillin FA area did not correlate with traction force magnitudes at single FAs, and this was consistent across different ECM stiffness and cytoskeletal tension states. Vinculin residence time at FAs linearly varied with applied force for stiff substrates, but this coupling was disrupted on soft substrates and in the presence of contractility inhibitors. In contrast, paxillin residence time at FAs was independent of force, substrate stiffness, and cytoskeletal contractility.
We also found that pFAK Y397 levels are linearly coupled to force at single FAs on stiff substrates. On soft substrates, however, this positive relationship is eliminated. We found that talin is required for FAK localization and Y397 phosphorylation at FAs and mediates force-FAK linear coupling at FAs via talin-FAK binding. Furthermore, averaged levels of FAK localization and Y397 phosphorylation at FAs are relatively insensitive to vinculin expression. However, a full-length vinculin molecule that binds talin and actin is required for linear coupling to occur between force-FAK localization and force-FAK Y397 phosphorylation at individual FAs. Lastly, we demonstrate that a full-length vinculin molecule that binds talin and actin is required to promote YAP nuclear accumulation. These findings show that force generation and signaling are coupled at FAs and underscore the role of environmental stiffness, talin, and vinculin in regulating force-signaling coupling at FAs. This understanding provides a framework for mechanotransduction events at cell-ECM junctions, such as force-regulated morphogenesis and stem cell commitment in response to matrix stiffness.
- Workflow Status:Published
- Created By:Laura Paige
- Modified By:Laura Paige