Force-signaling coupling at single focal adhesions

dc.contributor.advisor García, Andrés J.
dc.contributor.author Zhou, Dennis Wei
dc.contributor.committeeMember Zhu, Cheng
dc.contributor.committeeMember Curtis, Jennifer E.
dc.contributor.committeeMember Kowalczyk, Andrew P.
dc.contributor.committeeMember del Campo, Aranzazu
dc.contributor.department Biomedical Engineering (Joint GT/Emory Department)
dc.date.accessioned 2020-05-20T16:55:48Z
dc.date.available 2020-05-20T16:55:48Z
dc.date.created 2019-05
dc.date.issued 2019-03-25
dc.date.submitted May 2019
dc.date.updated 2020-05-20T16:55:48Z
dc.description.abstract 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 identify 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 previously showed that vinculin regulates force transmission at FAs. Vinculin residence time in FAs correlated with applied force, supporting a mechanosensitive model in which forces stabilize vinculin’s active conformation to promote force transfer. 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. Lastly, substrate stiffness and cytoskeletal contractility regulated whether vinculin and paxillin turnover dynamics are correlated to each other at single FAs. 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 suggest 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. Our results generate new insights into how cell adhesive forces are integrated into biochemical signals. Furthermore, this understanding provides a framework for mechanotransduction events at cell-ECM junctions, such as cell migration, force-regulated morphogenesis, and stem cell commitment in response to matrix stiffness.
dc.description.degree Ph.D.
dc.format.mimetype application/pdf
dc.identifier.uri http://hdl.handle.net/1853/62651
dc.language.iso en_US
dc.publisher Georgia Institute of Technology
dc.subject Focal adhesions
dc.subject Mechanobiology
dc.subject FAK
dc.subject Mechanotransduction
dc.subject Vinculin
dc.subject YAP
dc.title Force-signaling coupling at single focal adhesions
dc.type Text
dc.type.genre Dissertation
dspace.entity.type Publication
local.contributor.corporatename Wallace H. Coulter Department of Biomedical Engineering
local.contributor.corporatename College of Engineering
relation.isOrgUnitOfPublication da59be3c-3d0a-41da-91b9-ebe2ecc83b66
relation.isOrgUnitOfPublication 7c022d60-21d5-497c-b552-95e489a06569
thesis.degree.level Doctoral
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