Study of the mechanism of septum localization during bacterial cell division

细菌细胞分裂过程中隔膜定位机制的研究

• 批准号: 8741432
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 49.29万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741432
• 关键词: ATP phosphohydrolase; Anti-Bacterial Agents; Bacteria; Binding; Binding Proteins; Biochemical; Cell division; Cells; Development; Dissociation; Escherichia coli; Exhibits; Fluorescence; Foundations; Glass; Goals; Image; In Vitro; Label; Lipid Bilayers; Membrane; Mind; Mining; Molecular; Monitor; Pattern; Pattern Formation; Protein Family; Proteins; Reaction; Set protein; Slide; Surface; System; Techniques; Time; charge coupled device camera; dimer; fluorescence microscope; in vivo; inhibitor/antagonist; instrument; mind control; novel; polymerization; reconstitution

Mid-cell localization of the cell division septum in bacteria such as E. coli is controlled by a set of proteins including MinC, MinD, MinE, and FtsZ. FtsZ is the first structural component of the septum to polymerize on the inner membrane at the mid-cell when the cell starts to divide. FtsZ polymerization is limited to mid-cell by the action of the three Min proteins. MinC is an inhibitor of FtsZ polymerization, but on its own, it does not exhibit specific membrane localization. Instead, it binds to MinD dimers that form on the membrane in the presence of ATP: MinD is an ATP-dependent membrane binding protein. The two proteins generally co-localize on the membrane. MinE also interacts with MinD dimer and can displace MinC from MinD. More importantly, MinE controls MinD ATPase activity and also influences its membrane interaction, and hence its membrane association/dissociation dynamics. In vivo imaging studies have demonstrated the oscillating pattern formation by MinD and MinE proteins, resulting in a concentration minimum of MinD, and hence MinC, at the mid-cell region when averaged over time. This observation explained why FtsZ polymerization is restricted to mid-cell. However, a detailed molecular mechanism of this bio-patterning reaction system is still poorly understood. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of this reaction system by combining a variety of techniques, including the exploitation of a cell-free reaction system we established recently that recapitulates aspects of in vivo system dynamics. Techniques and instruments have been developed to study these dynamic reaction systems in vitro by using a sensitive fluorescence microscope/CCD camera system. By using fluorescence-labeled MinD and MinE proteins, assembly and disassembly of these proteins on a supported lipid bilayer on the slide glass surface are monitored under a variety of reaction conditions. We successfully reconstituted a number of inter-converting modes of self-organized dynamic pattern formation by MinD and MinE proteins in the presence of ATP on the membrane surface. Mechanistic details of the dynamic pattern organization are currently studied.

细菌中细胞分裂隔膜的中间细胞定位由一组蛋白质控制，包括Minc、Mind、Mine和FtsZ。FtsZ是当细胞开始分裂时，在细胞中部的内膜上聚合的第一个隔膜结构成分。FtsZ聚合受这三种Min蛋白的作用限制在细胞中部。MinC是FtsZ聚合的抑制剂，但它本身并不表现出特异性的膜定位。相反，它与在有ATP存在的情况下在膜上形成的Mind二聚体结合：Mind是一种依赖于ATP的膜结合蛋白。这两种蛋白质通常共定位在细胞膜上。我的也可以与思维二聚体相互作用，并可以将Minc从思维中取代。更重要的是，MINE控制着Mind ATPase的活性，也影响其膜的相互作用，从而影响其膜的结合/解离动力学。活体成像研究证明了Mind和Mini蛋白质形成的振荡模式，导致Mind浓度最低，因此Minc，当随着时间的推移平均时，在细胞中部区域。这一观察解释了为什么FtsZ聚合被限制在中间细胞。然而，这种生物图案化反应系统的详细分子机制仍然知之甚少。本项目旨在通过结合多种技术，包括开发我们最近建立的概括体内系统动力学方面的无细胞反应系统，来研究该反应系统动力学方面的生化和生物物理机制。 利用灵敏的荧光显微镜/CCD摄像系统在体外研究这些动态反应体系的技术和仪器已经被开发出来。通过使用荧光标记的Mind和Mini蛋白质，在各种反应条件下监测这些蛋白质在载玻片表面的支撑脂质双层上的组装和拆卸。在膜表面有ATP存在的情况下，我们成功地重构了由Mind和Mini蛋白质形成的多种自组织动态模式的相互转换模式。目前对动态图案组织的机械细节进行了研究。