Periodic Reporting for period 4 - CELLFUSION (Molecular dissection of the mechanisms of cell-cell fusion in the fission yeast)
Berichtszeitraum: 2021-04-01 bis 2022-03-31
The ERC CoG CellFusion aimed to depict the process of cell-cell fusion in the yeast model Schizosaccharomyces pombe. These haploid cells, which fuse to generate a diploid zygote, use conserved mechanisms of cell-cell communication (through pheromones and GPCR signaling), cell polarization and fusion driven by an actin-based fusion structure, dubbed the actin fusion focus. Five aims probed 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 has brought a molecular and conceptual understanding of cell fusion, with relevance for cell polarization, cytoskeletal organization, cell signalling and communication, and cell fate regulation. Our major findings include molecular description of Cdc42 feedback regulation, the unexpected discovery of a general principle of cellular patterning, ultrastructural and molecular understanding of the formation of the actin fusion focus and discovery of a bi-partite transcription factor that forms post-fusion to block further fusion attempts in zygotes.
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 has brought a molecular and conceptual understanding of cell fusion, with relevance for cell polarization, cytoskeletal organization, cell signalling and communication, and cell fate regulation. Our major findings include molecular description of Cdc42 feedback regulation, the unexpected discovery of a general principle of cellular patterning, ultrastructural and molecular understanding of the formation of the actin fusion focus and discovery of a bi-partite transcription factor that forms post-fusion to block further fusion attempts in zygotes.
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).
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).
Our work pushed significantly through the state of the art through implementation of two novel technologies in the fission yeast model. First, we established an optogenetic strategy, which not only allowed to probe the mechanisms of Cdc42 feedback regulation, but also unexpectedly revealed the existence and importance of in-plane membrane flows at sites of polarized secretion. We showed that polarized secretion causes a local in-plane flow of plasma membrane material and that this phenomenon affects the distribution of membrane-associated proteins, which is harnessed by the cell for self-patterning. Second, we established correlative light-electron microscopy, which we used to obtain ultrastructural information of the fusion site. Besides the achieved results described above, a number of follow-up studies are still ongoing on the signaling of cell fusion (through phosphoproteomics analysis) and the mechanisms blocking zygotic mating.