Live Cell Fluorescence Analysis and Mathematical Modeling of Dual Mechanisms of Cdc42 Recycling in Yeast: Relationship of Internalization Rate and Morphology

Abstract

Maintenance of robust cell polarization is a pre-requisite for oriented growth or motility biological systems. Cdc42 is a Rho-GTPase that, once polarized, organizes the deposition of growth materials to the polarized site. Previous studies in yeast have found that after initial polarity establishment, the individual molecules of Cdc42 are rapidly exchanged between the membrane and the cytosol. Despite these rapid dynamics, the site of Cdc42 accumulation is somehow held rigid to allow for polarized growth. We show that after initial symmetry breaking, Cdc42 is dynamically maintained at the polar cap through two recycling mechanisms: actin-mediated transport / endocytosis and cytosolic recycling through the Rho-GDP dissociation inhibitor (GDI) Rdi1. However, the mechanism by which the spatial relationship of these dual recycling pathways is controlled is unclear. We combined detailed live cell fluorescence measurements, including FRAP, iFRAP and fluorescence cross-correlation spectroscopy, with a thorough mathematical model to examine how dual recycling pathways of Cdc42 in yeast work together to shape the Cdc42 membrane distribution. We find that in order to recapitulate the steady-state membrane distribution of WT Cdc42, the dual recycling pathways must employ overlapping delivery windows of similar size. Interestingly, the modeling of live cell fluorescence data reveals that Rdi1-mediated tuning of a single dynamic parameter, internalization rate inside the delivery window, is sufficient to explain differences in the Cdc42 cap distribution between yeast cells destined for different morphogenic fates. These changes in Cdc42 cap distribution are correlated to the observed morphogenic differences under these conditions.