Membrane fission during sporulation

孢子形成过程中的膜裂变

• 批准号: 9036410
• 负责人: ERDEM KARATEKIN
• 金额: $ 32.05万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9036410
• 关键词: Accounting; Area; Bacteria; Binding; Biochemistry; Biological Assay; Biological Process; Cardiolipins; Cell Cycle; Cell Wall; Cell division; Cells; Cellular biology; Collaborations; Cues; Cytoplasm; Dynamin; Electron Microscopy; Electrophysiology (science); Eukaryota; Event; Food; Genetic; Geometry; Health; Human; Image; Link; Lipids; Liposomes; Measurement; Measures; Mediating; Membrane; Methods; Microfabrication; Mitochondria; Modeling; Molecular Biology; Mothers; Mutagenesis; Nutrient; Oral cavity; Phagocytosis; Phase; Process; Prokaryotic Cells; Proteins; Protoplasts; Reaction; Recycling; Reporting; Reproduction spores; Risk; Role; Safety; Site; Synaptic Vesicles; Techniques; Testing; Tube; Virus; laser tweezer; novel; quantitative imaging; research study; response; trafficking

 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.

 描述（由申请人提供）：膜裂变是细胞分裂和突触小泡回收等所需的基本生物过程，大多数裂变反应是由动力和 ESCRT-III 催化的。该项目旨在揭示 FisB（一种最近报道的细菌膜裂变）的机制。当营养缺乏时，枯草芽孢杆菌等细菌首先不对称分裂，产生较大的母细胞和较小的前孢子，母细胞以类似吞噬作用的过程吞噬前孢子。 FisB 与各自与特定脂质相互作用并在膜上寡聚化的真核成员一样，形成寡聚体并结合。心磷脂 (CL)，其脂质亚细胞定位在孢子形成过程中发生变化，因为没有涉及其他参与者，我们的指导假设是独特的膜拓扑、FisB-CL 相互作用、FisB 寡聚和 CL 动力学的组合可以单独解释。 FisB 的聚集、向裂变位点的募集和膜裂变活性 为了检验这一假设，我们的目标是 (1) 定义在孢子形成过程中控制 CL 动力学的因素。我们的实验将通过使用微移液器抽吸产生细胞壁缺陷的原生质体和巨型脂质体的受控变形，使用光镊拉动膜系绳来测试这一想法。 (2) 我们将使用共焦和超分辨率成像定量确定控制 FisB 寡聚和招募到裂变位点的因素。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 簇形成和动态定位。最后，（3）我们将确定 FisB 如何通过新颖的裂变分析重塑膜。使用共焦成像和电子显微镜观察 FisB 诱导的膜重塑。