Date of Award
Doctor of Philosophy (PhD)
Molecular Biophysics and Biochemistry
Membrane fusion and fission are important to all forms of life as they are required for processes such as cellular division, intracellular trafficking, and synaptic vesicle recycling. While membrane fission is extensively studied in eukaryotes, little is known about bacterial membrane fission even though it is required for every cell cycle. Membrane fission also occurs during sporulation. When nutrients are scarce, certain bacteria (e.g. Bacillus subtilis) are able to sporulate, thereby creating resistant endospores that can withstand harsh environmental conditions, such as radiation and drought, for hundreds of years. The first step of sporulation is asymmetric cell division which generates a larger mother cell and a smaller forespore. The mother cell then engulfs the forespore in a process similar to phagocytosis. When engulfment is complete, the leading membrane edge forms a small pore or membrane neck. Membrane fission of this neck connecting the engulfment membrane to the rest of the mother cell membrane, releases the forespore into the mother cell’s cytoplasm. Our lab and collaborators had previously identified that fission protein B (FisB) is required for this membrane fission step. The mother cell nurtures the forespore and once it is mature, lysis of the mother cell releases the spore into the environment. FisB is expressed shortly after asymmetric division and the only dedicated membrane fission machinery described for bacteria so far. It forms small, mobile clusters during engulfment and a large immobile cluster at the engulfment pole where membrane fission occurs. FisB is predicted to have a small N-terminal cytoplasmic domain, one transmembrane domain, and a larger extracellular domain (FisB(ECD)) which binds the phospholipid cardiolipin (CL). CL is a negatively charged lipid with spontaneous, negative curvature and is implicated in membrane fusion and fission reactions. However, the physiological significance of these findings was not clear. Overall, the aims of my thesis were to determine (1) how FisB localizes to the fission site and (2) the mechanism by which it mediates fission. First, we tested if FisB is recruited to the fission site by interaction with another protein, a specific lipid domain, or by membrane curvature. We were unable to identify a protein that interacts with FisB and found that FisB localization and membrane fission do not depend on membrane microdomains of CL or phosphatidylethanolamine (PE), another lipid that previously had been implicated in membrane fission and fusion. Additionally, our results suggest that FisB does not sense or induce membrane curvature, thus localization to the highly curved membrane fission site must rely on a separate mechanism. However, by using mutagenesis, we found that FisB self-oligomerization and binding to acidic lipids is required for targeting of FisB to the fission site. Next, we characterized interactions of FisB(ECD) with artificial membranes and found that FisB(ECD) forms an extended stable network on GUV membranes which is so stable that it persists even when the lipids are subsequently removed with detergent. Moreover, we found that FisB(ECD) bridges membranes of small liposomes and GUVs. Finally, we noticed a strong correlation between forespore inflation during sporulation and membrane fission. We found that forespores of cells that have undergone fission are inflated, while spores of pre-fission cells are not, even if engulfment appears complete. DNA translocation from the mother cell into the forespore by the protein SpoIIIE leads to forespore inflation and requires membrane flow from the peripheral mother cell membrane through the membrane neck into the engulfment membrane. We hypothesize that by forming a stable network in the membrane neck when engulfment is complete, FisB opposes lipid flow, leading to increased stress in the neck and ultimately membrane fission. Future work will test this hypothesis rigorously. Altogether, our results suggest that FisB localizes to the fission site by relying largely on FisB-FisB and FisB-lipid interactions. Since the larger portion of FisB faces into the extracellular space, the membrane geometry at the end of engulfment allows for FisB molecules to interact in trans. Therefore, we propose that a FisB cluster gets trapped in the membrane neck by interacting with other FisB molecules and/or the membrane in trans across the neck. Thus, FisB exploits the high curvature geometry of the membrane fission site without relying on an intrinsic membrane curvature sensing mechanism. Our results also suggest that membrane fission and forespore inflation are linked. We suggest that FisB accumulation in the membrane neck resists membrane flow leading to friction between the FisB network and the membrane which when high enough can lead to membrane fission.
Braun, Martha, "FisB Mediated Membrane Fission during Sporulation in Bacillus Subtilis" (2021). Yale Graduate School of Arts and Sciences Dissertations. 20.