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: 7967402
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 31.28万
• 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/7967402
• 关键词: ATP phosphohydrolase; Anti-Bacterial Agents; Bacteria; Binding; Binding Proteins; Biochemical; Cell division; Cells; Coupled; Detection; Development; Dissociation; Escherichia coli; Exhibits; Fluorescent Dyes; Foundations; Glass; Goals; Image; Kinetics; Learning; 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; fluorescence microscope; in vivo; inhibitor/antagonist; instrument; mind control; novel; polymerization; single molecule

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, which is an ATP-dependent membrane binding protein and the two proteins co-localize on the membrane. MinE interacts with MinD and thought to control MinD ATPase activity and hence its membrane association dissociation dynamics. In vivo imaging studies have demonstrated oscillating pattern formation of these two proteins, resulting in the minimum concentration of MinD, hence MinC at the mid-cell region when averaged over time. This observation explained why FtsZ polymerization is restricted to mid-cell. However, detailed molecular mechanism of this bio-patterning reaction system is still poorly understood, due in part to the absence of suitable cell free reaction system to study this pattern formation reaction in detail. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of this reaction system by combining a variety of techniques. Techniques and instruments have been developed to study these reactions at the single molecule detection level by using a sensitive fluorescence microscope/CCD camera system. Using GFP-tagged and fluorescent dye coupled MinD and MinE proteins, assembly and disassembly of these proteins on lipid bilayer that mimics bacterial inner membrane that is immobilized on a slide glass surface was monitored under a variety of reaction conditions. We learned that: MinD, in the presence of ATP associates with membrane with rapid on- and off-rates. Limited polymerization of MinD could be observed on the membrane. Kinetic analysis of the dynamics, as well as the influence of MinE on MinD membrane association, has been investigated. The reaction system studied here is an example of biomolecular patterning reaction, and the experimental techniques developed here will be exploited for the parallel studies of mechanistically related reaction systems.

大肠杆菌等细菌中细胞分裂隔膜的细胞中定位由一组蛋白质控制，包括 MinC、MinD、MinE 和 FtsZ。 当细胞开始分裂时，FtsZ 是隔膜的第一个结构成分，在细胞中部的内膜上聚合。 FtsZ 聚合通过三种 Min 蛋白的作用仅限于细胞中部。 MinC 是 FtsZ 聚合的抑制剂，但其本身并不表现出特定的膜定位。 相反，它与 MinD 结合，MinD 是一种 ATP 依赖性膜结合蛋白，并且这两种蛋白在膜上共定位。 MinE 与 MinD 相互作用，并被认为控制 MinD ATP 酶活性，从而控制其膜缔合解离动力学。 体内成像研究表明，这两种蛋白质形成振荡模式，导致 MinD 浓度最低，因此当随时间平均时，MinC 位于细胞中部区域。 这一观察结果解释了为什么 FtsZ 聚合仅限于细胞中部。 然而，这种生物图案反应系统的详细分子机制仍然知之甚少，部分原因是缺乏合适的无细胞反应系统来详细研究这种图案形成反应。 该项目旨在通过结合多种技术来研究该反应系统动态方面的生化和生物物理机制。 已经开发出通过使用灵敏的荧光显微镜/CCD相机系统在单分子检测水平上研究这些反应的技术和仪器。 使用 GFP 标记和荧光染料偶联的 MinD 和 MinE 蛋白，在多种反应条件下监测这些蛋白在模拟固定在载玻片表面的细菌内膜的脂质双层上的组装和分解。我们了解到：MinD 在 ATP 存在的情况下与膜结合，具有快速的结合和解离速率。 在膜上可以观察到 MinD 的有限聚合。 研究了动力学的动力学分析以及 MinE 对 MinD 膜缔合的影响。 这里研究的反应系统是生物分子图案化反应的一个例子，这里开发的实验技术将用于机械相关反应系统的并行研究。