Using optogenetics to interrogate cell polarity signaling in budding yeast
- Author(s): Jost, Anna Payne-Tobin
- Advisor(s): Weiner, Orion
- et al.
This thesis presents several novel approaches to studying yeast polarity signaling. Cell polarity is required for a wide variety of cell functions, from oocyte polarization in early development to the directed motility of neutrophils. Budding yeast polarize growth in order to bud and therefore reproduce. While genetics and biochemistry have given us a deep understanding of the components involved in cell polarity in yeast, many of these tools lack the temporal or spatial resolution needed to perturb the dynamics of this fast process. My work focuses on developing optogenetic tools for perturbing signaling in yeast, and applying them to key questions in yeast polarity signaling.
In the first project, I used the Phy-PIF light-gated dimerization system to recruit activators of the key polarity protein Cdc42 to the membrane, attempting to generate a synthetic pole and measure the strength of the positive feedback loops involved in polarization (Chapter 2). This gain-of-function use of the system proved difficult and I ultimately ended the project, but the infrastructure and skills I developed contributed to my success in future projects.
In the second project, I collaborated with Xiaojing Yang to adapt the Phy-PIF system to inducibly control protein localization in yeast (Chapter 3). I then applied this system to GAPs for Cdc42 (Chapter 4) and the scaffold protein Bem1 (Chapter 5). I showed that acute depletion of Bem1 using the light-gated system generated a stronger phenotype than genetic deletion of Bem1. Future work will examine the role of Bem1 over the course of polarization, determining when during polarization Bem1 is required.