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The Role of Small GTPase RhoG in Focal Adhesion Dynamics and Contractility.

Hoover, Ashtyn
http://orcid.org/0000-0002-0576-7513
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556712457014336

Abstract Details

2019, Doctor of Philosophy, University of Toledo, Biology (Cell-Molecular Biology).
Cell migration is a critical physiological process that requires the careful cooperation of all cytoskeletal elements within the cell. A multitude of biological events such as embryogenesis, wound healing, tissue maintenance, and cancer metastasis rely upon the ability of the cell to effectively and efficiently migrate. Migration is comprised of four distinct steps: polarization (or reorientation of the cell in the intended direction of migration), protrusion, adhesion, and retraction or contractility. All of these steps are dictated by both internal and external cues, many of which are mechanical in nature. Here, we will primarily focus on the regulation of adhesion and contractility through a structure known as focal adhesions (FA). FA are a complex of proteins formed to allow the cell physical contact between the cytoskeleton and the extracellular matrix (ECM). FA are dynamic structures involved in force transduction and the indirect regulation of the cytoskeleton, including actin and myosin II activity. FA form at the leading edge of cells to stabilize protrusions and disassemble at a later time to allow the cell to retract and progress forward. Many proteins have been identified in the regulation of FA formation; however, the underlying mechanisms that regulate adhesion turnover remain poorly understood. The small family of Rho GTPases are known to play a role in cell migration, including FA dynamics. Several Rho GTPases have been extensively studied in the context of cell migration; however, here we present data showing that the lesser studied RhoG, a Rho GTPase related to Rac, modulates FA dynamics and contractility. Using cell imaging techniques and automated quantification, we have demonstrated that when RhoG expression is silenced (KD), there is a distinct phenotype of increased FA within the cell and a greater number located centrally. Through live imaging, we have shown that this phenotype is the result of increased stability, and therefore longer FA lifetime, in RhoG KD cells. Using specific FA lifecycle markers, we have also shown that these adhesions are compositionally more mature. During our study, it was also noted that FA within RhoG KD cells aligned in a striking manner within individual cells. Along with this, RhoG KD resulted in distinct differences in cytoskeletal structure, with increased number and thickness of contractile stress fibers (SFs). We believed that the cells were producing a greater amount of contractile force, also attributing to their smaller size and rounder shape. To our surprise, using both 2-dimensional (2D) traction force microscopy and 3-dimensional (3D) contractility assays, we found that RhoG KD cells exerted less force upon the substrate despite a FA and cytoskeletal phenotype that suggests the opposite. It is well established that FA formation is dependent upon forces exerted by the actin cytoskeleton, however our data provides a novel role for RhoG in the disassembly of FA, a process that is regulated by microtubules (MTs). Using specific inhibitors of MT dynamics, we found that RhoG KD cells showed a marked difference in their ability to recover after MT inhibition. Taken together, we have provided evidence for two novel roles of RhoG in MT-mediated FA disassembly and contractility.
Rafael Garcia-Mata (Committee Chair)
Tomer Avidor-Reiss (Committee Member)
Kathryn Eisenmann (Committee Member)
Guofa Liu (Committee Member)
Kam Yeung (Committee Member)
102 p.
Biology; Cellular Biology
Rho GTPase, RhoG, Focal Adhesion, Contractility, Microtubules, Cell Migration, Cytoskeleton

Recommended Citations

Citations

  • Hoover, A. (2019). The Role of Small GTPase RhoG in Focal Adhesion Dynamics and Contractility. [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556712457014336

    APA Style (7th edition)

  • Hoover, Ashtyn. The Role of Small GTPase RhoG in Focal Adhesion Dynamics and Contractility. 2019. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556712457014336.

    MLA Style (8th edition)

  • Hoover, Ashtyn. "The Role of Small GTPase RhoG in Focal Adhesion Dynamics and Contractility." Doctoral dissertation, University of Toledo, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556712457014336

    Chicago Manual of Style (17th edition)

toledo1556712457014336
219
© 2019, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.