Establishment of cell polarity is critical for a variety of cellular processes including proliferation and cell movement, as seen in developing embryos, wound healing, and bud formation during yeast growth. Loss of cell polarity is, therefore, implicated in several disease processes, for example, in epithelial tumors. The Cdc42 GTPase is a central regulator of cell polarity in both yeast and animals and is also a critical regulator f metastasis. Although cell polarization occurs along a single axis that is generally determined by a spatial cue, a large number of recent studies have focused on the mechanisms of polarity establishment in the absence of spatial cues. Thus, how a specific direction of cell polarization is determined and how the process is regulated in a spatial and temporal manner are largely unknown. The proposed work capitalizes on the tractable yeast system to elucidate the mechanisms underlying spatial cue-directed cell polarization. Cells of the budding yeast undergo polarized growth in two spatially programmed patterns. We recently discovered that Cdc42 is activated in two temporal steps in the G1 phase. The first step depends on Bud3, a cortical protein marking the cell division site in haploids, while subsequent activation depends on Cdc24, which has heretofore been the sole GDP-GTP exchange factor (GEF) for Cdc42. This finding led us to hypothesize that sequential activation of Cdc42 in the G1 phase is fundamental to appropriate recognition and readout of cell polarity cues in haploid budding yeast.
Aim 1 seeks to investigate how spatial landmarks and the Rsr1 GTPase module regulate activation of Cdc42 in early G1. Our genetic and biochemical data suggest an unexpected role of Cdc42 in establishment of a proper bud site. Thus, Aim 2 seeks the mechanism of action of Cdc42 and its regulators in establishment of cell polarity at a proper bud site. Finally, Aim 3 explores the molecular basis of the cell-type-specific budding pattern and investigates the assembly of the cortical markers that determines the orientation of the polarity axis. This research plan will employ biochemical methods and quantitative microscopy, as well as computational modeling. This study is expected to establish how sequential activation of Cdc42 governs recognition of spatial landmarks and execution of polarity establishment. Given the ubiquitous importance of Cdc42 in cell polarity events from yeast to humans, findings from this work will be relevant to spatial cue-directed cell polarization in higher eukaryotes, including mammals.

Public Health Relevance

This research concerns the basic mechanisms by which cell polarity is established along a single axis in eukaryotic cells. Cell polarity and asymmetry underlie various cellular processes, including embryonic development and fibroblast movement during wound healing. Thus, understanding the basic mechanisms of polarity establishment may lead to a better understanding of human health as loss of cell polarity is implicated in various diseases including cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM114582-03
Application #
9256508
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Nie, Zhongzhen
Project Start
2015-05-07
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
3
Fiscal Year
2017
Total Cost
$266,805
Indirect Cost
$93,555
Name
Ohio State University
Department
Genetics
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Kang, Pil Jung; Miller, Kristi E; Guegueniat, Julia et al. (2018) The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization. Mol Biol Cell 29:2359-2369
Miller, Kristi E; Lo, Wing-Cheong; Lee, Mid Eum et al. (2017) Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein. Mol Biol Cell 28:3773-3788
Okada, Satoshi; Lee, Mid Eum; Bi, Erfei et al. (2017) Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor. Methods Enzymol 589:171-190
Miller, Kristi E; Kim, Yeonsoo; Huh, Won-Ki et al. (2015) Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies. J Mol Biol 427:2039-2055
Lee, Mid Eum; Lo, Wing-Cheong; Miller, Kristi E et al. (2015) Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast. J Cell Sci 128:2106-17
Kang, Pil Jung; Lee, Mid Eum; Park, Hay-Oak (2014) Bud3 activates Cdc42 to establish a proper growth site in budding yeast. J Cell Biol 206:19-28