Study of the mechanism of septum localization during bacterial cell division

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，并且这两种蛋白质共定位在膜上。 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。 体内成像研究已经证明了这两种蛋白质的振荡模式形成，导致MinD的最小浓度，因此当随时间平均时，MinC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多种技术来研究该反应系统动力学方面的生物化学和生物物理机制。 技术和仪器已经开发，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。 使用GFP标记的和荧光染料偶联的MinD和MinE蛋白，在各种反应条件下监测这些蛋白在模拟固定在载玻片表面上的细菌内膜的脂质双层上的组装和分解。我们了解到：在ATP的存在下，MinD与膜的结合速度很快。 在膜上可以观察到MinD的有限聚合。 动力学分析的动力学，以及MinE对MinD膜协会的影响，进行了研究。 这里研究的反应体系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被用于机械相关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.