Differential GAP requirement for Cdc42-GTP polarization during proliferation and sexual reproduction

Abstract

The formation of a local zone of Cdc42 GTPase activity, which governs cell polarization in many cell types, requires not only local activation but also switch-off mechanisms. In this study, we identify Rga3, a paralog of Rga4, as a novel Cdc42 GTPase-activating protein (GAP) in the fission yeast Schizosaccharomyces pombe. Contrary to Rga4, Rga3 localizes with Cdc42-GTP to sites of polarity. Rga3 is dispensable for cell polarization during mitotic growth, but it limits the lifetime of unstable Cdc42-GTP patches that underlie cell pairing during sexual reproduction, masking a partly compensatory patch-wandering motion. In consequence, cells lacking rga3 hyperpolarize and lose out in mating competition. Rga3 synergizes with the Cdc42 GAPs Rga4 and Rga6 to restrict Cdc42-GTP zone sizes during mitotic growth. Surprisingly, triple-mutant cells, which are almost fully round, retain pheromone-dependent dynamic polarization of Cdc42-GTP, extend a polarized projection, and mate. Thus, the requirement for Cdc42-GTP hydrolysis by GAPs is distinct during polarization by intrinsic or extrinsic cues.

Fission Yeast Polarization: Modeling Cdc42 Oscillations, Symmetry Breaking, and Zones of Activation and Inhibition

“…Here we associated the nonlinear negative feedback necessary for emergence of oscillations with the function of tip-localized GAPs ("GAP I "). However we note that deletion of the tip-localized Rga3 did not significantly change Cdc42 oscillations compared to wild-type cells [40]. While it is also conceivable that negative regulation occurs by diffusion-limited supply of Cdc42-GDP at activated cell tips, this depletion is relatively small in Figure 2.…”

“…(iii) The two Cdc42 GEFs, Scd1 and Gef1, localize at cell tips, together with Cdc42-GTP [22,[30][31][32][33][34][35]. By contrast, three known Cdc42 GAPs establish an intriguing pattern, with Rga4 [36][37][38] and Rga6 [39] decorating primarily the cell sides while Rga3 accumulates primarily at the cell tips [40]. Fluorescence recovery after photobleaching (FRAP) studies indicate different dynamics of Rga6 at cell tips as compared to cell sides: the percent recovery at the cell sides is smaller than the tips over the same time period [39].…”

Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase

“…Thus, in contrast to its ubiquitous localization, Cdc42 activity is strictly polarized. The importance of local Cdc42 activity is illustrated by the observation that not only does loss of Cdc42 result in small, dense, round cells, but also its constitutive activation, or lack of inactivation, causes the formation of large, round cells [6,9,10]. Thus, a key question is what controls the local activation of Cdc42.…”

“…GTP hydrolysis for Cdc42 inactivation is catalyzed by the 3 GAPs Rga3, Rga4, and Rga6. [5,10,18]. Whereas Rga3 colocalizes with active Cdc42 at cell tips, Rga4 and Rga6 are present at cell sides.…”

Clustering-mediated activation of Cdc42 GTPase antagonized by GAPs in fission yeast

“…We focused our attention on the three Cdc42 GAPs Rga3, Rga4 and Rga6, the GDI protein Rdi1 and the Ras1 GAP Gap1. Rga3, Rga4 and Rga6 directly promote Cdc42-GTP hydrolysis (Das et al, 2007;Gallo Castro and Martin, 2018;Revilla-Guarinos et al, 2016). Rdi1 may promote Cdc42 extraction from the membrane, although it is largely dispensable for Cdc42 dynamics Nakano et al, 2003).…”