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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CELLFUSION (Molecular dissection of the mechanisms of cell-cell fusion in the fission yeast)

Teaser

Summary

We aim to depict the process of cell-cell fusion in the simple yeast model Schizosaccharomyces pombe. These haploid cells, which fuse to generate a diploid zygote, use highly conserved mechanisms of cell-cell communication (through pheromones and GPCR signaling), cell polarization (centred around the small GTPase Cdc42) and fusion driven by an actin-based fusion structure, dubbed the actin fusion focus. Five aims probe the molecular nature of, and the links between, signaling, polarization and the fusion machinery from initiation to termination of the process.

1: To define the roles and feedback regulation of Cdc42 during cell fusion
2: To understand the molecular mechanisms of actin fusion focus formation
3: To identify the fusogen(s) promoting membrane fusion
4: To probe the GPCR signal for fusion initiation
5: To define the mechanism of fusion termination

By combining genetic, optogenetic, biochemical, live-imaging, synthetic and modeling approaches, this project will bring a molecular and conceptual understanding of cell fusion. This work will have far-ranging relevance for cell polarization, cytoskeletal organization, cell signalling and communication, and cell fate regulation.

Work performed

We have acquired equipment, recruited personal and initiated the project as planned. Progress has been made in all five aims, with papers published in aims 1, 3, 4 and 5. Briefly, the advances for each aim are as follows:

Aim 1: We have made important progress in understanding the dynamics of Cdc42. Cdc42 exhibits cycles of spontaneous, unstable polarization during sexual differentiation, when cells are exposed to pheromones produced by their sexual partners. This spontaneous polarization dynamics highlights the existence of positive and negative feedback signals, for local amplification of a zone of Cdc42 activity and its consequent disassembly. Through collaboration with Dimitrios Vavylonis, we further demonstrated that these dynamic polarization zones contain both pheromone release and perception machineries, and become stabilized upon perception of opposite type pheromone. Thus, these dynamic polarity zones underlie a â€˜speed-datingâ€™ mechanism for cell pair formation, by probing the environment and becoming locked into place through stimulation. We now understand that they depend on a second 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). Finally, we have now fully implemented an optogenetic system in fission yeast, which we plan to use to dissect Cdc42 regulation.

Aim 2: To understand the formation of the fusion focus, we first concentrated our efforts on the function of capping proteins, identified in a screen (described under aim 3) for fusion defects. Capping proteins are necessary for the normal maturation of the focus. Remarkably, we were able to pinpoint the specific defect to a diversion of the formin Fus1 and other fusion focus components to Arp2/3-nucleated actin patches. This demonstrates that capping proteins protect Arp2/3-nucleated structures against formin activity, thus ensuring the identity of the actin assembly and preventing ectopic formin activity. We are finalising a publication on these findings.

Aim 3: In the hope of identifying potential membrane fusogens, we performed a genome-wide screen for fusion defective mutants (Dudin et al, PLoS Genetics 2017). Though the screen was rich in discoveries (as for instance detailed under aim 2), 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 have re-oriented this aim in three directions: 1) to directly assess the potential fusogenic role of Prm1, a conserved fungal transmembrane protein involved in cell fusion; 2) to extend electron microscopy analysis of the fusion site to gain morphological information on the fusion process; and 3) to 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. The ulstrastructural analysis was spearheaded by my sabbatical stay at the LMB in Cambridge and we now have >100 tomograms of the fusion site. These show that the two cells are not identical and that fusion steps appear asymmetrical, with one cell often penetrating into the other before fusion, while the other display membrane ruffling.

Aim 4: We have 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 sign

Final results

Aim 1: We expect to leverage the optogenetic system we have implemented to dissect the feedback regulation of Cdc42.

Aim 2: In the second half of the grant, we aim to dissect the mechanisms by which Fus1 formin aggregates to form a tight fusion focus.

Aim 3: Our aim here will be to use the information gained through ultrastructural analysis to probe possible mechanisms of membrane fusion. In particular, we aim to test the source and role of membrane ruffling in promoting membrane fusion. We will also probe deeper into the role of Prm1 during membrane fusion.

Aim 4: Our principal aim will be to identify and study the targets of MAPK signaling in the fusion focus.

Aim 5: We are following up on our discovery that a bi-partite transcription factor represses re-fertilization. Our aim will be to study the relative importance of direct blocks and cell cycle control during this step.