Our progress is summarized below, with publications indicated.
Aim 1: We made progress in understanding the dynamics of Cdc42, which exhibits unstable polarization during sexual differentiation. This dynamics highlights the existence of positive and negative feedbacks. Through collaboration with Dimitrios Vavylonis, we demonstrated that these dynamic zones contain both pheromone release and perception machineries and become stabilized upon perception of opposite type pheromone. Thus, these dynamic polarity zones underlie a ‘speed-dating’ mechanism for cell pair formation. We understand that they depend on the small GTPase, Ras1, which displays similar behaviour (Merlini et al, JCB 2018; Khalili et al, PLoS Comput Biol 2018), but are independent of regulation by GTPase activating proteins (Gallo Castro and Martin, JCB 2018). We further probed Cdc42 feedback regulation through optogenetics to recruit active Cdc42 to the plasma membrane with exquisite time control. This led to the finding that a complex between active Cdc42 and its activator forms the basis of positive feedback (Lamas et al, PLoS Biology 2020; Lamas et al, Cells 2020). While establishing the optogenetic method, we also made a serendipitous observation, which led us to describe bulk membrane flows as a novel principle of cell patterning (Gerganova et al, Sci Adv 2021).
Aim 2: To understand the formation of the fusion focus, we first concentrated our efforts on capping proteins identified in the screen described under aim 3. Capping proteins compete with formins to bind the barbed ends of actin filaments. Our work showed that cells lacking capping proteins exhibit an aberrant fusion focus, due to the diversion of the formin Fus1 and other fusion focus components to Arp2/3-nucleated actin patches. Thus, capping proteins protect Arp2/3-nucleated structures against formin activity, and ensure structure identity (Billault-Chaumartin and Martin, Curr Biol, 2019). We then conducted a careful structure-function analysis of Fus1 formin, which revealed that 1) rates of actin assembly are tailored to Fus1 function in assembling the fusion focus, and 2) the N-terminal half of Fus1 contains an intrinsically disordered region that mediates focus condensation (2x Billault-Chaumartin et al, BioRxiv 2022).
Aim 3: In the hope of identifying potential fusogens, we performed a genome-wide screen for fusion defective mutants (Dudin et al, PLoS Genetics 2017). Though the screen was rich in discoveries, it did not reveal obvious fusogen candidates. We also directly screened in Sf9 cells for potential fusogenic activities of candidate yeast transmembrane proteins, which also did not yield conclusive results. Therefore, we re-oriented this aim to 1) obtain ultrastructural information of the fusion site and 2) probe the possible role of the lipidic composition of membrane, in particular sterols, in fusion. This was made possible by the development of a live fluorescent reporter of sterol-rich membranes, which also led to the discovery of a novel sterol transport pathway (Marek et al, JCB 2020). The ultrastructural analysis was spearheaded by my sabbatical stay at the LMB in Cambridge. Besides describing the ultrastructure of the fusion focus, an unexpected finding is that cells exhibit asymmetry during fusion, with one cell often penetrating into the other before fusion, while the other display membrane ruffling (Muriel et al, JCB 2021).
Aim 4: We advanced in our understanding of the signal for fusion initiation through study of Ras GTPase and its GTPase activating protein Gap1. Our major findings were that the pheromone MAPK signalling machinery is enriched on the actin fusion focus and promotes its maturation. This early work made the important demonstration that the signal for fusion consists not in physical cell contact, but in the focalization of pheromone signalling driven by a positive feedback between the signalling machinery and the actin cytoskeleton. In follow-up work, we demonstrated that Ras GTPase, which is an activator of the MAPK cascade is maintained is low-activity state by a negative feedback involving Gap1, with Ras activity increasing only upon close proximity between cells when the positive feedback described above increases (Merlini et al, JCB 2018; Khalili et al, PLoS Comput Biol 2018).
Aim 5: We defined a major pathway by which cells block re-fertilization. Indeed, post-fusion, zygotes dismantle the fusion focus and block re-fertilization by signalling through a bi-partite transcription factor formed from a P-cell-specific homeobox protein and a M-cell-specific small peptide (Vjestica et al, Nature 2018). This work also makes the first description of asymmetric gene expression in parental genomes in fungi. We then dissected the downstream signaling pathway and showed that zygotes prevent further mating events in two ways: by rapidly exiting the mating-competent G1 phase and by defining a zygote-specific cell fate (Vjestica et al, PLoS Biology 2021).
