Structure and Assembly Dynamics of FtsZ

描述（由申请人提供）：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。尽管有十几种其他蛋白质对于大肠杆菌的分裂至关重要，但我们最近证明仅 FtsZ 就足以在脂质体中重建 Z 环。此外，这些人造 Z 环无需任何其他蛋白质即可产生收缩力。我们建议在多个方向上进一步开展这些研究，以研究组装和力产生的机制。一个新的方向是使用 TIRF 显微镜对单 FtsZ 丝的体外生长进行成像。这应该确定细丝是否正在经历动态不稳定或跑步，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 通过两亲螺旋 (FtsZ-mts) 直接连接到膜上。我们将尝试重建自然的两部分系统，其中 FtsZ 通过 FtsA 连接到膜上。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建 MinCDE 系统，首先单独使用（它应该显示振荡），然后使用 FtsZ-mts 和 FtsZ-FtsA（它应该限制 Z 环的定位）。与力产生相关的一个新问题是，FtsZ 的 C 端尾部结构是什么？这被认为是 FtsZ 和膜之间的约 50 个氨基酸的柔性系绳，从而将力从 FtsZ 细丝传递到膜。我们提出了对该系链的结构和力学的多项研究，包括突变和取代、NMR，以及将其移动到 FtsZ 球状结构域上的不同附着点。在之前的一项研究中，我们获得了十几种大肠杆菌抑制菌株，它们允许异常的 FtsZ 发挥分裂功能。这些抑制突变可能存在于影响胞质分裂的未被发现的途径中。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识别它们。最后，我们建议通过 PALM 对 Z 环进行成像，PALM 是一种光学显微镜“超分辨率”技术，可以提供 30 nm 的分辨率。我们相信这可以对单个 FtsZ 原丝进行成像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问题，旨在扩展我们的生物学知识。它还具有潜在的临床意义。 FtsZ 在细菌中高度保守，是新型抗生素的有吸引力的靶点。几种针对 FtsZ 的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.