Study of pore expansion and re-sealing during fission yeast cell-cell fusion

Cell-cell fusion is a fundamental process for eukaryotic biology, for example for zygote formation or development. For fertilization, gametes undergo complete cell fusion to form the diploid zygote. The irreversibility of the fusion process is crucial, as it commits the zygote to the next developmental stages. We aim to depict the ultrastructural details of the cell fusion pore and to understand the irreversibility of its expansion using the fission yeast Schizosaccharomyces pombe sexual cycle, a model for cell-cell fusion. In this organism, paired gametes polarize their growth towards each other, then digest their cell wall locally upon cell-cell contact, enabling subsequent plasma membrane (PM) merging. Our prior correlative light-electron microscopy (CLEM) work provided a first view on the ultrastructure of the wild-type fusion zone (1). We also showed that the deletion of the p21-associated kinase (PAK) Pak2, an effector of the conserved small GTPase Cdc42, slows down cell fusion and produces transient fusion events, where cells succeed in opening a fusion pore and effectively merge their cytoplasmic content, but eventually re-seal their PMs (2). We aim to understand the ultrastructural details of the re-sealing pore. Through FLIP-based methods, we showed that over 80% of re-sealing pairs had no open pores. Preliminary cryo-CLEM data suggest that the cell wall is rebuilt post-fusion. This project aims to obtain, for the first time, quantitative data of the 3D full volume of the cell-cell fusion process both in the wild-type situation and the re-sealing mutant by cryogenic soft-X-ray tomography (cryo-SXT), taking advantage of the cryo-epifluorescence microscopy to identify cell pairs that transiently fused before imaging at the MISTRAL beamline in ALBA synchrotron.