Membrane fission during sporulation

 描述(由申请人提供)：膜分裂是细胞分裂和突触小泡再循环所必需的基本生物学过程。在真核生物中，大多数裂变反应是由Dynamin和ESCRT-III催化的。相比之下，关于细菌如何实现膜裂变以进行分裂和产孢子，几乎一无所知。本项目旨在揭示最近报道的细菌膜分裂蛋白FisB在孢子形成过程中介导分裂的机制。当营养缺乏时，像枯草杆菌这样的细菌通过首先不对称分裂来形成孢子，从而产生更大的母细胞和更小的前孔。母细胞以类似吞噬作用的过程吞噬前孔，最终导致需要FisB的分裂事件。就像真核生物中的同类分子一样，FisB形成低聚体并与心磷脂(CL)结合，心磷脂是一种在产孢子过程中其亚细胞定位发生变化的脂质。由于还没有涉及到其他的参与者，我们的指导性假设是独特的膜拓扑结构、FisB-CL相互作用、FisB寡聚和CL动力学的结合可以解释FisB的聚集、向分裂位点的募集和膜分裂活性。为了验证这一假说，我们的目标是(1)确定控制产孢期CL动态的因素。CL被认为是响应几何提示而定位于不同的亚细胞位置，从而为其他成分的定位提供了里程碑。我们的实验将通过使用微吸管吸入来创建细胞壁缺陷原生质体和巨型脂质体的受控变形，使用光学镊子拉膜系绳，以及可视化CL微域来测试这一想法。(2)我们将使用定量共聚焦和超分辨率成像来确定控制FisB齐聚和重新聚集到裂变位置的因素。通过施加受控制的几何形状，并通过突变选择性地破坏FisB-FisB和FisB-CL的相互作用，我们将发现这些因素是如何驱动FisB簇的形成和动态定位的。最后，(3)我们将通过开发新的分裂分析和利用共聚焦成像和电子显微镜观察FisB诱导的膜重建来确定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.