Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

37-POS Board 37 ULTRASTRUCTURE OF ACTIN FUSION FOCUS IN S. POMBE DURING CELL- CELL FUSION Olivia Muriel-Lopez 1 ; Laetitia Michon 1 ; Wanda Kukulski 2 ; Sophie G Martin 1 ; 1 University of Lausanne, Fundamental Microbiology, Lausanne, Switzerland 2 Laboratory of Molecular Biology, Cambridge, United Kingdom The fission yeast S. pombe undergoes mating and fusion during its sexual cell cycle. The process is initiated by pheromone release and perception, and followed by a MAPK cascade that induces transcription of specific mating/fusion genes. These include Fus1, a member of the formin family of proteins, which induces actin polymerization. These actin filaments direct he transport of glucanase-containing vesicles to the shmoo tip, where they induce local cell wall digestion necessary for membrane fusion. The extremely precise spatio-temporal regulation of this process occurs thank to the focalization of proteins involved in this process, in particular pheromone receptors and transporter, at a very specific place called actin fusion focus (AFF). We aim to understand how the actin fusion focus is structured, in particular how trafficking vesicles and actin filaments are organized, and how plasma membranes from both partner cells merge. To address this we have optimized a post-embedding correlative light and electron microscopy (CLEM) approach, developed for S. cerevisiae , which successfully works in S. pombe and in mating conditions. With the intention of obtaining all the possible details, we use tomography followed by 3D reconstruction, which allows us to identify many features, such as the two layers of membranes, endocytic events, exocytic vesicles, actin cables, etc. We are currently characterizing all these structures in WT cells. Preliminary observations show plasma membrane waviness and deformation at the site of cell-cell contact, as well as an asymmetric “attack” of the h- into the h+ cell. In order to understand these phenotypes, we are making use of fusion- deficient mutants, such as fus1? or prm1?. With this work we aim to reveal in unprecedented details the organization of the fusion structure.

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