Study of the mechanism of septum localization during bacterial cell division

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Mind结合，Mind是一种依赖于ATP的膜结合蛋白，这两种蛋白共同定位在膜上。我与思维和思想相互作用，控制思维的ATPase活性，从而控制其膜结合解离动力学。活体成像研究已经证明了这两种蛋白质的振荡模式形成，导致了最小的精神集中，因此Minc在细胞中部区域，当随着时间的平均。这一观察解释了为什么FtsZ聚合被限制在中间细胞。然而，这一生物图案化反应体系的详细分子机制目前还不清楚，部分原因是缺乏合适的无细胞反应体系来详细研究这一图案化反应。本项目旨在通过结合多种技术来研究该反应系统动力学方面的生化和生物物理机制。 已经开发出了使用灵敏的荧光显微镜/CCD摄像系统在单分子检测水平上研究这些反应的技术和仪器。利用GFP标记和荧光染料偶联的Mind和Mini蛋白，在各种反应条件下监测了这些蛋白在模拟细菌内膜的脂双层上的组装和拆卸。我们了解到：头脑，在三磷酸腺苷存在的情况下，膜具有快速的开关频率。在膜上可以观察到思维的有限聚合。对动力学进行了动力学分析，并研究了Me对心膜缔合的影响。 本文所研究的反应体系是生物分子图案化反应的一个例子，所发展的实验技术将被用于与机理相关的反应体系的平行研究。

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.