GDI-Mediated Cdc42 Recycling at Polar Cortex in Dynamic Maintenance of Cell Polarity in Saccharomyces cerevisiae

Das, Arupratan (2012). GDI-Mediated Cdc42 Recycling at Polar Cortex in Dynamic Maintenance of Cell Polarity in Saccharomyces cerevisiae. PhD thesis The Open University.



Cell polarization is a fundamental requirement for development and many physiological processes such as cell motility, stem cell differentiation, immune response and neuronal polarity. Small G-protein Rho GTPase, Cdc42 has long been shown to be the key regulator of the polarization process. In this study we aim to understand how cell maintains an optimum Cdc42 concentration at the polar cortex, and how distinct polarized Cdc42 distribution is achieved based on its recycling pathways taking budding yeast as a model system.

Our in depth study revealed that Cdc42 at yeast polar cortex is dynamically maintained via two redundant pathways of distinct response time. A slow pathway dependent on actin-mediated endocytosis and exocytosis and a fast response pathway dependent on Rdil, yeast GDI (guanine dissociation inhibitor) for Rho GTPases. Quantitative imaging and mathematical modeling found both the pathways to be spatially overlapping in order to have physiological Cdc42 distribution. We further demonstrated Cdc42 GTPase cycle as the common regulator of actin and Rdil mediated pathways, a process supports their concentric localization.

We further focused on gaining mechanistic insight of Rdil-mediated Cdc42 recycling at polar cortex. Using a high throughput genetic screen, imaging, spectroscopy and mathematical modeling we found that phospholipid asymmetry at the polar cortex regulated by lipid flippase complex (Lem3-Dnfl and Lem3-Dnf2) plays a key role in Rdil mediated fast dissociation of Cdc42 from the polar cortex. Our finding suggests that flipping of phosphatidylethanolamine (PE), a phospholipid with a positively charged head group, reduces the charge interaction between a Cdc42 C-terminal cationic region with the inner leaflet of the polar cortex, a key step for fast Rdil mediated Cdc42 extraction. In Δlem3 cells the negatively charged phospholipid phosphatidylserine (PS) is enriched in the inner leaflet of plasma membrane, as demonstrated by analysis of Cdc42 mutants with altered charge properties and biosensor for PS. Increase in PS in the inner leaflet increases Cdc42 and plasma membrane charge interaction antagonizing Rdil-mediated Cdc42 extraction. Using an in vitro assay with reconstituted supported lipid bilayers, we further demonstrated that relative composition of PE versus PS directly modulates the rate of Cdc42 extraction from the membrane by GDI.

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