Abstract
The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.
Highlights
In eukaryotes, the small Rho-family GTPase Cdc42 is a highly conserved regulator of cell morphogenesis, proliferation, and differentiation
Activation of Rho GTPases relies on the activity of guanine nucleotide exchange factors (GEFs), while their intrinsic GTPase activity is enhanced by GTPase activating proteins (GAPs) to return them to the inactive state
We showed that cortical recruitment of a GTP-locked, constitutively active Cdc42 variant (CRY2-Cdc42Q61L,∆CaaX ) led to the Scd2-dependent co-recruitment of its GEF Scd1 and accumulation of endogenous Cdc42, demonstrating feedback amplification [6]
Summary
The small Rho-family GTPase Cdc is a highly conserved regulator of cell morphogenesis, proliferation, and differentiation. Cdc, were shown to become active at the cell cortex upon light-dependent cytosolic clustering [17] In these experiments, the small GTPases were fused to CRY2PHR, the photolyase homology region of. Clustered RhoA induced RhoA signalling-dependent cytoskeletal re-organization and membrane retraction in human cells, suggesting that oligomerization promotes RhoA activation [17]. Cdc forms nanoclusters in Saccharomyces cerevisiae cells [25,26] These nanoclusters show an anisotropic distribution: they accumulate and exhibit larger sizes at cortical sites of polarized growth, in a manner dependent on the scaffold protein Bem and anionic membrane lipids [25,27]. We used an artificial optogenetic strategy to induce the recruitment and clustering of Cdc at the plasma membrane of fission yeast cells. The activation is efficiently countered by Rga GAP-mediated Cdc inactivation, and does not lead to cell shape alteration, showing the robustness of the cell polarization system
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