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。Mine与Mind相互作用，控制Mind的ATPase活性，也影响其膜相互作用，从而影响其膜结合/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的形成，导致了Minc的浓度最低，因此当随着时间的推移平均时，Minc在细胞中部区域。这一观察解释了为什么FtsZ聚合被限制在中间细胞。然而，这种生物图案化反应体系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体系来详细研究这种图案化反应。本项目旨在通过结合多种技术，包括建立和开发概括体内系统动力学的无细胞反应系统，来研究该反应系统动态方面的生化和生物物理机制。 利用灵敏的荧光显微镜/CCD摄像系统在体外研究这些动态反应体系的技术和仪器已经被开发出来。通过使用荧光标记的Mind和Mini蛋白质，在各种反应条件下监测这些蛋白质在载玻片表面的支撑脂质双层上的组装和拆卸。我们成功地重构了在膜表面存在三磷酸腺苷的情况下，两种蛋白质形成自组织动态图案的多种相互转换模式。目前对动态图案组织的机械细节进行了研究。 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被用于机械相关反应系统的平行研究。

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 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.