Structure and Assembly Dynamics of FtsZ

描述（由申请方提供）：微管蛋白同源物FtsZ是细菌胞质分裂中的主要细胞骨架蛋白。尽管其他十几种蛋白质对E.大肠杆菌，我们最近已经证明，单独的FtsZ是足以重建脂质体中的Z环。此外，这些人工Z环在没有任何其他蛋白质的情况下产生收缩力。我们建议进一步这些研究在一些方向调查的组装和力的产生机制。一个新的方向将是使用TIRF显微镜在体外成像单个FtsZ细丝的生长。这应该可以确定细丝是否经历了动态不稳定性或非稳定性铣削，这是适用于微管和肌动蛋白的两种组装动力学机制。在我们目前的脂质体重建中，FtsZ通过两亲性螺旋（FtsZ-mts）直接拴系到膜上。我们将尝试重建天然的两部分系统，其中FtsZ被FtsA拴在膜上。我们还将研究MinCDE系统，该系统在细菌细胞中从一端振荡到另一端，以将FtsZ环定位到中心。我们将在脂质体中重构MinCDE系统，首先通过其自身（其中它应该显示振荡），然后用FtsZ-mts和FtsZ-FtsA（其中它应该限制Z环的定位）。一个新的问题有关的力量产生是，什么是结构的C-末端尾部的FtsZ？这被认为是FtsZ和膜之间的~50 aa柔性系链，从而将力从FtsZ细丝传递到膜。我们提出了几个研究这个系绳的结构和力学，包括突变和取代，NMR，并将其移动到FtsZ的球状结构域上的不同附着点。在以前的研究中，我们获得了十几个E.大肠杆菌，允许异常FtsZ分裂功能。这些抑制突变可能在未发现的影响胞质分裂的途径。我们建议通过Solexa测序对每个菌株的基因组进行重新测序来识别它们。最后，我们提出了图像的Z环PALM，光学显微镜的“超分辨率”技术，可以给30 nm的分辨率。我们相信这可以成像单个FtsZ原丝，并确定它们如何分布以形成Z环。 公共卫生相关性：我们的总体目标是确定细菌分裂的机制。这首先是一个基础科学的问题，以扩大我们的生物学知识。它也具有潜在的临床意义。FtsZ在细菌中高度保守，是新抗生素的一个有吸引力的靶标。目前正在研究和开发几种靶向FtsZ的先导化合物。

DESCRIPTION (provided by applicant): The tubulin homolog FtsZ is the major cytoskeletal protein in bacterial cytokinesis. Although a dozen other proteins are essential for division in E. coli, we have recently demonstrated that FtsZ alone is sufficient to reconstitute Z rings in liposomes. Furthermore, these artificial Z rings generate a constriction force without any other proteins. We propose to further these studies in a number of directions to investigate the mechanism of assembly and force generation. One new direction will be to image the growth of single FtsZ filaments in vitro using TIRF microscopy. This should determine if the filaments are undergoing dynamic instability or treadmilling, two mechanisms of assembly dynamics that apply to microtubules and actin. In our present liposome reconstitution, FtsZ is tethered directly to the membrane by an amphipathic helix (FtsZ-mts). We will attempt to reconstitute the natural two-part system where FtsZ is tethered to the membrane by FtsA. We will also investigate the MinCDE system, which oscillates from one end to the other in bacterial cells to localize the FtsZ ring to the center. We will reconstitute the MinCDE system in liposomes, at first by itself (where it should show oscillation) and then with FtsZ-mts and with FtsZ-FtsA (where it should restrict the localization of Z rings). A novel question related to force generation is, what is the structure of the C-terminal tail of FtsZ? This is thought to be a ~50 aa flexible tether between FtsZ and the membrane, and thus transmitting the force from the FtsZ filaments to the membrane. We propose several studies of the structure and mechanics of this tether, including mutation and substitution, NMR, and moving it to different attachment points on the globular domain of FtsZ. In a previous study we obtained a dozen suppressor strains of E. coli that permitted aberrant FtsZ to function for division. These suppressor mutations are likely in undiscovered pathways affecting cytokinesis. We propose to identify them by resequencing the genome of each strain by Solexa sequencing. Finally, we propose to image the Z ring by PALM, a light microscope "superresolution" technique that can give 30 nm resolution. We believe this can image single FtsZ protofilaments and determine how they are distributed to make the Z ring. PUBLIC HEALTH RELEVANCE: Our overall goal is to determine the mechanism by which bacteria divide. This is foremost an issue of basic science, to expand our knowledge of biology. It also has potential clinical relevance. FtsZ is highly conserved in bacteria, and is an attractive target for new antibiotics. Several lead compounds targeting FtsZ are already being studied and developed.