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113015 Ning Dissertation.pdf (20.98 MB)

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Roles of actin motor myosin-V, Rho GEF Gef3, and membrane trafficking in fission yeast cytokinesis

Wang, Ning
http://rave.ohiolink.edu/etdc/view?acc_num=osu1448893835

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Molecular Genetics.
The cell-division cycle is a series of cellular events that partition a mother cell into two daughter cells. The same genetic information has to be maintained during each cell cycle. Cytokinesis, as the last step of the cycle, is precisely regulated to ensure the proper inheritance of both nuclear and cytoplasmic contents. Failure of cytokinesis can lead to tetraploidy and contribute to the transformation of normal cells into cancer cells. Therefore, understanding mechanisms of cytokinesis is fundamental to human health. In this body of work, I use the fission yeast Schizosaccharomyces pombe as a model organism to investigate regulation and cooperation of different stages of cytokinesis because this genetically tractable yeast shares many features and conserved proteins with higher eukaryotes for cytokinesis. Cytokinesis requires actomyosin contractile-ring assembly and constriction, plasma-membrane deposition, extracellular-matrix remodeling/septum formation, and cell separation. The structural and regulatory components of the contractile ring in fission yeast have been largely identified. However, molecular mechanisms for many of these proteins have been barely investigated. For example, the actin motor myosin-Vs (Myo51 and Myo52) have been found to localize to the division site more than a decade ago, but how or if they regulate cytokinesis is still unknown. For my first project, I investigated the roles of the myosin-V called Myo51 during early and late cytokinesis. I found Myo51 enhances contractile-ring assembly efficiency in a motor-dependent manner and stabilizes the contractile ring in later stage of cytokinesis in a motor-independent way. I also identified the regulatory roles of two novel proteins Rng8 and Rng9 on Myo51’s localization and activities in vivo and suggested a novel mechanism that turns the non-processive actin motor into a functional transporter by protein oligomerization. All events of cytokinesis are regulated by Rho GTPases. Rho guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP, thus activate Rho GTPases. Most fission yeast Rho GTPases concentrate at the division site during cytokinesis, but their gene-specific functions have not yet been completely revealed. The cellular functions and the GEF activity of some putative Rho GEFs were untested. My second project was to investigate the cytokinesis-related function of Gef3, which is the smallest putative Rho GEF in fission yeast. I found that Gef3 co-precipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, supporting Gef3 as a GEF for Rho4. I also identified a septin-dependent regulatory mechanism for Gef3 and Rho4 localizations in cells and detected a role for Gef3 and Rho4 in promoting septum formation and cell separation. My third main project focused on plasma-membrane deposition. In late stages of cytokinesis, the ring constricts in coordination with the plasma-membrane insertion and the septum formation. The exocyst complex is an essential player for new membrane insertion by tethering vesicles. The exclusive localization of the exocyst complex at the rim of the division plane seems to limit membrane insertion to this area, therefore contradicting the prevailing model in animal cells that new membrane is predominantly inserted at the leading edge of the cleavage furrow. Through direct observations of membrane trafficking and deposition during cytokinesis, I found secretory vesicles with new membrane are deposited to the entire cleavage furrow rather than exclusively or predominantly at the rim or at the leading edge, suggesting the involvement of other vesicle tethers for membrane deposition. Consistently, several lines of evidence suggested that the Transport Particle Protein II (TRAPP-II) complex and Rab11 GTPase help to tether post-Golgi secretory vesicles along the cleavage furrow for membrane insertion. In addition, I found that endocytosis retrieves and recycle plasma membrane from the cleavage furrow during cytokinesis. In conclusion, myosin-Vs and septin-Rho signaling pathways play roles in regulating cytokinesis. Their molecular functions are precisely controlled spatiotemporally in vivo. Membrane insertion during cytokinesis is a concerted result of targeted exocytosis and endocytosis. Cooperation of multiple vesicle tethers to ensure efficient membrane deposition is essential for fission yeast and likely other organisms.
Jian-Qiu Wu (Advisor)
255 p.
Biochemistry; Cellular Biology; Genetics; Molecular Biology
myosin-V; Rho GEF; exocyst; TRAPP-II; fission yeast; cytokinesis

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Citations

  • Wang, N. (2015). Roles of actin motor myosin-V, Rho GEF Gef3, and membrane trafficking in fission yeast cytokinesis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1448893835

    APA Style (7th edition)

  • Wang, Ning. Roles of actin motor myosin-V, Rho GEF Gef3, and membrane trafficking in fission yeast cytokinesis. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1448893835.

    MLA Style (8th edition)

  • Wang, Ning. "Roles of actin motor myosin-V, Rho GEF Gef3, and membrane trafficking in fission yeast cytokinesis." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1448893835

    Chicago Manual of Style (17th edition)

osu1448893835
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This open access ETD is published by The Ohio State University and OhioLINK.