Membrane fission during sporulation

 描述（由申请人提供）：膜分裂是细胞分裂和突触囊泡再循环等所需的基本生物学过程。在真核生物中，大多数裂变反应是由发动蛋白和ESCRT-III催化的。相比之下，几乎没有人知道细菌如何实现膜分裂分裂和孢子形成。本项目旨在阐明FisB，最近报道的细菌膜分裂蛋白，介导孢子形成过程中的裂变的机制。当营养缺乏时，细菌喜欢B。枯草芽孢杆菌通过首先不对称分裂产生较大的母细胞和较小的前孢子而形成孢子。母细胞以吞噬作用的方式吞噬前孢子，最终以需要FisB的裂变事件结束。像它的真核对应物，每个相互作用与一个专门的脂质和寡聚化的膜上，FisB形成寡聚体和结合心磷脂（CL），一种脂质的亚细胞定位的变化，在孢子形成。因为没有其他球员被牵连，我们的指导假设是，独特的膜拓扑结构，FisB-CL相互作用，FisB寡聚化，CL动力学的组合单独可以占的聚类，招募裂变网站，和膜裂变活动的FisB。为了验证这一假设，我们的目标是（1）确定控制孢子形成过程中CL动力学的因素。CL被认为是定位到不同的亚细胞位点，以响应几何线索，从而为其他组件的本地化提供地标。我们的实验将测试这一想法，通过创建控制变形的细胞壁缺陷的原生质体和巨大的脂质体使用微量吸管抽吸，拉膜系绳使用光镊，和可视化CL微域。(2)我们将使用定量共聚焦和超分辨率成像来确定支配FisB寡聚化和招募到裂变位点的因素。通过施加受控的几何形状，并通过诱变选择性地破坏FisB-FisB和FisB-CL相互作用，我们将发现这些因素如何驱动FisB簇的形成和动态定位。最后，（3）我们将通过开发新的裂变检测和使用共聚焦成像和电子显微镜观察FisB诱导的膜重塑来确定FisB如何重塑膜。

 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.