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: 8349757
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 49.19万
• 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/8349757
• 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; 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, which is an ATP-dependent membrane binding protein, and the two proteins co-localize on the membrane. MinE interacts with MinD and 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 of these two 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, due in part to the absence of a 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, including the establishment and exploitation of a cell-free reaction system that recapitulates the 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 the two proteins in the presence of ATP on the membrane surface. Mechanistic details of the dynamic pattern organization are currently studied. The reaction system studied here is an example of a biomolecular patterning reaction, and the experimental techniques developed here will be exploited for parallel studies of mechanistically related reaction systems.

在细菌如大肠杆菌中，细胞分裂隔膜的中细胞定位。大肠杆菌由一组蛋白质控制，包括MinC，MinD，MinE和FtsZ。FtsZ是细胞开始分裂时第一个附着在中间细胞内膜上的隔片结构成分。FtsZ聚合是有限的中间细胞的三个Min蛋白的作用。MinC是FtsZ聚合的抑制剂，但其本身不表现出特异性膜定位。相反，它与MinD结合，MinD是一种ATP依赖性膜结合蛋白，这两种蛋白质共同定位在膜上。MinE与MinD相互作用并控制MinD ATP酶活性，还影响其膜相互作用，从而影响其膜缔合/解离动力学。体内成像研究已经证明了这两种蛋白质的振荡模式形成，导致MinD的浓度最小值，因此MinC，当随时间平均时，在中间细胞区域。这一观察结果解释了为什么FtsZ聚合仅限于中间细胞。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，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。本项目旨在通过结合各种技术，包括建立和开发一个概括体内系统动力学的无细胞反应系统，研究该反应系统动力学方面的生物化学和生物物理机制。 通过使用灵敏的荧光显微镜/CCD摄像系统，已经开发了在体外研究这些动态反应系统的技术和仪器。 通过使用荧光标记的MinD和MinE蛋白质，这些蛋白质在载玻片表面上的支持的脂质双层上的组装和拆卸在各种反应条件下进行监测。我们成功地重建了一些相互转换模式的自组织的动态模式形成的两种蛋白质在膜表面上的ATP的存在下。目前正在研究动态图案组织的机制细节。 这里研究的反应系统是一个例子的生物分子图案化反应，这里开发的实验技术将被利用的机械相关的反应系统的平行研究。