Structure and Assembly Dynamics of FtsZ

描述(申请人提供)：微管蛋白同系物FtsZ是细菌胞质分裂中的主要细胞骨架蛋白。虽然其他十几种蛋白质对大肠杆菌的分裂是必不可少的，但我们最近已经证明，仅FtsZ就足以在脂质体中重建Z环。此外，这些人造Z环在没有任何其他蛋白质的情况下产生一种紧缩力。我们建议在多个方向上进一步进行这些研究，以探讨组装和力产生的机制。一个新的方向将是使用TIRF显微镜对单个FtsZ细丝的体外生长进行成像。这应该确定细丝是经历了动态不稳定还是脚踏球，这两种组装动力学机制适用于微管和肌动蛋白。在我们目前的脂质体重组中，FtsZ通过两亲性螺旋(FtsZ-MTS)直接拴在膜上。我们将尝试重建FtsZ通过FTSA连接到膜上的自然两部分系统。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建MinCDE系统，首先是它本身(它应该显示振荡)，然后是FtsZ-MTS和FtsZ-FTSA(它应该限制Z环的局部化)。一个与力产生相关的新问题是，FtsZ的C末端尾巴是什么结构？这被认为是FtsZ和膜之间的~50aa的柔性系绳，从而将力从FtsZ丝传递到膜。我们提出了对这种系链的结构和机制的几个研究，包括突变和取代，核磁共振，并将其移动到FtsZ球状结构域上的不同连接点。在之前的一项研究中，我们获得了十几株允许异常FtsZ发挥分裂功能的抑制菌株。这些抑制子突变可能存在于未被发现的影响胞质分裂的途径中。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鉴定它们。最后，我们建议用Palm成像Z环，这是一种光学显微镜“超分辨率”技术，可以提供30 nm的分辨率。我们相信这可以成像单个FtsZ原丝，并确定它们是如何分布的，以形成Z环。 与公共卫生相关：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问题，以扩大我们的生物学知识。它还具有潜在的临床相关性。FtsZ在细菌中高度保守，是新抗生素的一个有吸引力的目标。针对FtsZ的几种先导化合物已经在研究和开发中。

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.