Study of the mechanism of septum localization during bacterial cell division

在大肠杆菌等细菌中，细胞分裂间隔的中细胞定位是由一组蛋白质控制的，包括MinC、MinD、MinE和FtsZ。当细胞开始分裂时，FtsZ是隔膜中第一个在细胞膜上聚合的结构成分。FtsZ聚合被三种Min蛋白的作用限制在细胞中。MinC是FtsZ聚合的抑制剂，但它本身不表现出特异性的膜定位。相反，它与MinD结合，这是一种依赖atp的膜结合蛋白，两种蛋白在膜上共定位。MinE与MinD和thought相互作用，控制MinD atp酶活性，从而控制其膜关联解离动力学。体内成像研究表明，这两种蛋白质的振荡模式形成，导致MinD的最低浓度，因此当平均时间时，MinC位于细胞中部区域。这一观察解释了为什么FtsZ聚合仅限于中胞。然而，这种生物模式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细胞自由反应系统来详细研究这种模式形成反应。本项目旨在结合多种技术研究该反应体系动力学方面的生化和生物物理机制。

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.