Aleksandar Vještica

Differences between partner gametes, which evolved repeatedly in eukaryotes, can contribute to the evolution of the sexes, sexual selection, and non-Mendelian inheritance. Yet, the empirical evidence for how functional asymmetries arise between initially equivalent gametes is limited. Here, we combine theoretical and experimental approaches in the fission yeast Schizosaccharomyces pombe to show how selective pressures acting concurrently on gametes and zygotes drive the evolution of gamete differences. We find that despite being morphologically identical, P- and M-type partner gametes invest asymmetrically in zygotic development by contributing different amounts of conserved meiotic cohesins. P-gametes preferentially produce the Rec8 cohesin that increases zygotic fitness but reduces gamete viability, revealing a trade-off between reproductive success and gamete survival. We demonstrate that this asymmetry is mediated by partner-specific communication and model its evolutionary dynamics using empirically determined parameters. Our results support classical theoretical predictions for the evolution of gamete differences and provide a mechanistic understanding of how molecular asymmetries between partners can originate from opposing selection pressures acting in species that lack morphologically distinct gametes.

Differences between partner gametes, which evolved repeatedly in eukaryotes, can contribute to the evolution of the sexes, sexual selection and non-Mendelian inheritance ^{1– 4} . Yet, the empirical evidence for how functional asymmetries arise between initially equivalent gametes is limited. Here, we combine theoretical and experimental approaches in the fission yeast Schizosaccharomyces pombe to show how selective pressures acting concurrently on gametes and zygotes drive the evolution of gamete differences. We find that despite being morphologically identical, P- and M-type partner gametes invest asymmetrically in zygotic development by contributing different amounts of conserved meiotic cohesins. P-gametes preferentially produce thve Rec8 ⁵ cohesin that increases zygotic fitness but reduces gamete viability, revealing a trade-off between reproductive success and gamete survival. We demonstrate that this asymmetry is mediated by partner-specific communication and model its evolutionary dynamics using empirically determined parameters. Our results support classical theoretical predictions for the evolution of gamete differences and provide a mechanistic understanding of how molecular asymmetries between partners can originate from opposing selection pressures acting in species that lack morphologically distinct gametes.

Compartmentalized regulation of RNAs is emerging as a key driver of developmental transitions, with RNA-binding proteins performing specialized functions in different subcellular compartments. The RNA-binding protein Mei2, which arrests mitotic proliferation and drives zygotic development in fission yeast, was shown to function in the nucleus to trigger meiotic divisions. Here, using compartment-restricted alleles, we report that Mei2 functions in the cytosol to arrest mitotic growth and initiate development. We found that Mei2 is a zygote-specific component of P-bodies that inhibits the translation of tethered mRNAs. Importantly, we show that P-bodies are necessary for Mei2-driven development. Phosphorylation of Mei2 by the inhibitory Pat1 kinase impedes P-body recruitment of both Mei2 and its target RNA. Finally, we establish that Mei2 recruitment to P-bodies and its cytosolic functions, including translational repression of tethered RNAs, depend on the RNA-binding domain of Mei2 that is dispensable for nuclear Mei2 roles. Collectively, our results dissect how distinct pools of an RNA-binding protein control developmental stages and implicate P-bodies as key regulators of gamete-to-zygote transition.

Compartmentalized regulation of RNAs is emerging as a key driver of developmental transitions, with RNA-binding proteins performing specialized functions in different subcellular compartments. The RNA-binding protein Mei2, which arrests mitotic proliferation and drives zygotic development in fission yeast, was shown to function in the nucleus to trigger meiotic divisions. Here, using compartment-restricted alleles, we report that Mei2 functions in the cytosol to arrest mitotic growth and initiate development. We find that Mei2 is a zygote-specific component of P-bodies that inhibits the translation of tethered mRNAs. Importantly, we show that P-bodies are necessary for Mei2-driven development. Phosphorylation of Mei2 by the inhibitory Pat1 kinase impedes P-body recruitment of both Mei2 and its target RNA. Finally, we establish that Mei2 recruitment to P-bodies and its cytosolic functions, including translational repression of tethered RNAs, depend on the RNA-binding domain of Mei2 that is dispensable for nuclear Mei2 roles. Collectively, our results dissect how distinct pools of an RNA-binding protein control developmental stages and implicate P-bodies as key regulators of gamete-to-zygote transition.

Exocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeastSchizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity. One Sentence Summary: Exocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly two haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe , fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins post-fusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least two mechanisms where the zygotic fate imposed by Mei2 and the cell cycle re-entry triggered by Mei3 synergize to prevent zygotic mating.

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.

Schizosaccharomyces pombe is a widely used model organism to study many aspects of eukaryotic cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.