Membrane fission during sporulation



 DESCRIPTION (provided by applicant): Membrane fission is a fundamental biological process required for, e.g. cell division and synaptic vesicle recycling. In eukaryotes, most fission reactions are catalyzed by dynamin and ESCRT-III. In contrast, virtually nothing is known about how bacteria achieve membrane fission for division and sporulation. This project aims to unravel the mechanisms by which FisB, a recently reported bacterial membrane fission protein, mediates fission during sporulation. When nutrients are scarce, bacteria like B. subtilis form spores by first dividing asymmetrically to produce a larger mother cell and a smaller forespore. The mother cell engulfs the forespore in a phagocytosis-like process which ends with a fission event that requires FisB. Like its eukaryotic counterparts that each interact with a specialized lipid and oligomerize on membranes, FisB forms oligomers and binds cardiolipin (CL), a lipid whose subcellular localization changes during sporulation. Because no other players have been implicated, our guiding hypothesis is that the combination of the unique membrane topology, FisB-CL interactions, FisB oligomerization, and CL dynamics alone can account for the clustering, recruitment to the fission site, and membrane fission activity of FisB. To test this hypothesis, we aim (1) to define factors controlling CL dynamics during sporulation. CL is thought to locate to distinct subcellular sites in response to geometric cues, thereby providing landmarks for the localization of other components. Our experiments will test this idea by creating controlled deformations of cell-wall deficient protoplasts and giant liposomes using micropipette aspiration, pulling membrane tethers using optical tweezers, and visualizing CL microdomains. (2) We will determine factors that govern FisB oligomerization and recruitment to the fission site using quantitative confocal and superresolution imaging. By imposing controlled geometries, and selectively disrupting FisB-FisB and FisB-CL interactions through mutagenesis, we will discover how these factors drive FisB cluster formation and dynamic localization. Finally, (3) we will determine how FisB remodels membranes by developing novel fission assays and visualizing FisB-induced membrane remodeling using confocal imaging and electron microscopy.