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）：膜裂变是一种基本的生物过程，例如，细胞分裂和突触小泡回收。在真核生物中，大多数裂变反应是由动力和 ESCRT-III 催化的。相比之下，对于细菌如何实现细胞分裂和产孢子的膜裂变几乎一无所知。该项目旨在揭示 FisB（一种最近报道的细菌膜裂变蛋白）在孢子形成过程中介导裂变的机制。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，从而形成孢子。母细胞在类似吞噬作用的过程中吞噬前孢子，该过程以需要 FisB 的裂变事件结束。与真核生物中的对应物一样，FisB 与特定的脂质相互作用并在膜上寡聚化，FisB 形成寡聚体并结合心磷脂 (CL)，心磷脂是一种在孢子形成过程中其亚细胞定位发生变化的脂质。由于没有涉及其他参与者，我们的指导性假设是，独特的膜拓扑结构、FisB-CL 相互作用、FisB 寡聚化和 CL 动力学的组合可以单独解释 FisB 的聚集、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) 定义控制孢子形成过程中 CL 动态的因素。 CL被认为响应几何线索而定位到不同的亚细胞位点，从而为其他成分的定位提供标志。我们的实验将通过使用微量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、使用光镊拉动膜系链以及可视化 CL 微域来测试这一想法。 (2) 我们将使用定量共焦和超分辨率成像来确定控制 FisB 寡聚化和招募到裂变位点的因素。通过施加受控几何形状，并通过诱变选择性破坏 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）我们将通过开发新的裂变测定法并使用共焦成像和电子显微镜可视化 FisB 诱导的膜重塑来确定 FisB 如何重塑膜。