Role and Regulation of Rho-GTPase Cdc42 in Fission Yeast Sexual Reproduction

Diplôme Thèse Role and Regulation of Rho-GTPase Cdc42 in Fission Yeast Sexual Reproduction eng 2026-02-09 2026-02-25 author Saha Sajjita 403510 Département de biologie moléculaire et cellulaire (MOCEL) director Martin Sophie 0000-0002-5317-2557 403314 Département de biologie moléculaire et cellulaire (MOCEL) 132 10.13097/archive-ouverte/unige:193694 urn:nbn:ch:unige-1936946 Sc. Vie 384 Cdc42 is a small Rho-GTPase conserved across eukaryotes which plays essential roles in cell polarization. Cdc42 drives symmetry breaking and polarized growth in unicellular yeast systems by establishing activity zones at cell cortex that locally recruit effectors to remodel the cytoskeleton and direct polarized secretion. In mitotic growth, Cdc42 is locally activated to its GTP-bound form by GEFs and amplified by a scaffold Scd2 mediated positive feedback that assembles complexes with Cdc42-GTP, GEF Scd1 and effector P21-activated kinase Pak1 to drive Cdc42 enrichment at cell tips and bipolar growth. The feedback cooperates with microtubule delivered spatial landmarks, while counteracting GAPs and GDI that restrict Cdc42 activity to cell tips. Unlike mitotic growth, polarity in mating becomes adaptive, reorienting toward extracellular pheromone gradients rather than fixed cell intrinsic landmarks. How polarity is reprogrammed during the transition from vegetative growth to the sexual cycle and how Cdc42, the principal polarity establishment protein, is involved and regulated during this process poses the central question which I aim to address in this project.Here I show that in mating, extracellular pheromone gradients supplant intrinsic cues, allowing external signals to stabilize polarity zones independently of GAP or GDI control. Mutants lacking GAPs and GDI mate efficiently with WT cells and maintain restricted Cdc42-GTP zones during mating—unlike during mitosis, where they exhibit expanded zones. I show a critical role of Cdc42 in cell–cell fusion in S. pombe by analyzing cdc42 mutants with clear genetic separation of function that display very minute detectable defects in mitosis but severe impairment in cell fusion. The cause of fusion defect is traced to lower levels of Cdc42 which I further confirmed by modulating Cdc42 expression over a five-fold range through a promoter exchange allelic series. This series demonstrates that polarized growth during mitosis scales linearly with Cdc42 concentration, whereas sexual reproduction exhibits a switch-like response. By analyzing cdc42 mutants, I show that cell fusion arrests after assembly of the actin fusion focus but before cell wall digestion, indicating that Cdc42 acts during the late stages of fusion where it functions in translating vesicle clustering into polarized cargo delivery. Consistent with these results, experiments using scd1 and scd2 mutants, which disrupt Cdc42 positive feedback, show defective mating. This observation emphasizes crucial role of positive feedback in mating serving to enhance and maintain high levels of active Cdc42. While elevated Cdc42 level is necessary to exceed a threshold critical for initiating mating and completing fusion, hyperactivation or defective turnover can compromise mating efficiency. Constitutive activation of Cdc42 positive feedback, achieved through forced Scd1–Pak1 interactions, or inhibition of autophagic recycling in atg1Δ strains, results in immobile polarity zonesthat fail to reorient, emphasizing that both balanced activation and dynamic turnover are essential for successful mating. Taken together, this project sheds light on how Cdc42 is regulated in mating and describes its essential role in cell fusion. Cdc42 Fission Yeast Polarity Cell fusion Rho-GTPases Dewey 570 7daed2ee-1311-4824-9df9-2e51260a966a Département de biologie moléculaire et cellulaire 16484 false 1410 Groupe Sophie Martin c3d55d63-9232-4268-b360-033e4a6a0651 Thesis Public SAHA_Sajjita_Thesis_2026.pdf 6167104 application/pdf a2fcf65c6b79721218bddc7869f2ea8a7ceafdb9108f9fc108a5b7958752ce72 b3fe3ed0-c615-41e1-9b03-f194bfd1fb26 Imprimatur Public imprimatur.pdf 57270 application/pdf 458e79433b75d4b326c08fa86d09ddd47835543f80fb5b69e45406a8b9a9bf0c openAccess Faculté des sciences / Section de biologie / Département de biologie moléculaire et cellulaire

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Role and Regulation of Rho-GTPase Cdc42 in Fission Yeast Sexual Reproduction				2026	19	19

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Saha, Sajjita

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