Loren Genrich

Document Type

Honors Thesis

Major

Molecular and Cellular Biology

Advisor(s)

Joshua B Kelley

Committee Members

Julie A Gosse, Karissa Tilbury

Graduation Year

May 2023

Publication Date

2025

Abstract

The yeast Saccharomyces cerevisiae responds to pheromone through a G-protein coupled receptor (GPCR) signaling pathway. Upon receptor activation by pheromone, the heterotrimeric G-protein dissociates and initializes separate downstream signals through the Gα and Gβγ subunits. During this response, cells either elongate towards the pheromone source or form mating projections known as “shmoos.” The kinesin Kar3 is located at the shmoo tip and regulates plus-end microtubule polymerization via interaction with Gα, which negatively regulates microtubule assembly. During pheromone-induced elongation, the nucleus aligns along the axis of cell polarity in a manner dependent on Gpa1 regulation of Kar3. Preliminary data shows that nuclear orientation during gradient tracking in hyperactive Gα mutants varies considerably over time, compared to wild-type. Therefore, we hypothesized that microtubule dynamics contribute to normal gradient tracking through a Gα-dependent mechanism. We are investigating cytoskeletal involvement in gradient tracking using yeast expressing fluorescently tagged tubulin and the polar-cap protein Bem1-Ruby, which we will use to track microtubule localization and abundance during gradient tracking. We are also observing yeast that are expressing Kar3-GFP during gradient tracking. We used a hyperactive Gα mutant that is unable to bind to the regulator of G-protein signaling (RGS), which prevents RGS-induced acceleration of the intrinsic GTPase activity of Gα, the primary negative regulator of G-protein signaling. Using live cell microscopy, microfluidics, and image analysis, I will test the hypotheses that Gα control of Kar3 is important for polarized growth and that microtubules contribute to gradient tracking through a Gɑ-dependent mechanism.

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