Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

• 批准号: 8099656
• 负责人: HAROLD P ERICKSON
• 金额: $ 48.67万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099656
• 关键词: Actins; Affect; Antibiotics; Bacteria; Basic Science; Biological Assay; Biology; C-terminal; Cell division; Cells; Color; Complement; Cytokinesis; Cytoskeletal Proteins; DNA Resequencing; Engineering; Escherichia coli; Filament; Fluorescence Microscopy; Generations; Genome; Goals; Growth; Guanosine Triphosphate; Homologous Gene; Hydrolysis; Image; In Vitro; Knowledge; Label; Lateral; Lead; Length; Life; Light Microscope; Liposomes; Location; Measures; Mechanics; Membrane; Microfilaments; Microscopy; Microtubules; Mind; Mining; Modeling; Mutation; Mycobacterium tuberculosis; Pathway interactions; Peptides; Property; Proteins; Reporting; Resolution; Role; Slide; Structural Models; Structure; Suppressor Mutations; System; Tail; Techniques; Testing; Tubulin; Work; clinically relevant; constriction; flexibility; in vivo; mutant; novel; novel strategies; public health relevance; reconstitution

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.

描述（由申请人提供）：微管蛋白同源性FTSZ是细菌细胞因子的主要细胞骨架蛋白。尽管其他十几种蛋白质对于大肠杆菌中的分裂至关重要，但我们最近证明，仅FTSZ就足以重建脂质体中的Z环。此外，这些人工Z环会产生无蛋白质的收缩力。我们建议在许多方向上进一步研究这些研究，以研究组装和力产生的机理。一个新的方向将是使用TIRF显微镜在体外成像单个FTSZ丝的生长。这应该确定细丝是否正在发生动态不稳定性或踏板，这是适用于微管和肌动蛋白的两种装配动力学机制。在我们目前的脂质体重构中，FTSZ通过两亲螺旋（FTSZ-MTS）直接束缚在膜上。我们将尝试重建FTSA将FTSZ束缚在膜上的天然两部分系统。我们还将研究MinCDE系统，该系统在细菌细胞中从一端到另一端振荡，以将FTSZ环将其定位到中心。我们将首先重建脂质体中的MINCDE系统（应该显示振荡），然后使用FTSZ-MTS和FTSZ-FTSA（在其中限制Z环的定位）。与产生力有关的一个新颖的问题是，FTSZ的C末端尾巴的结构是什么？这被认为是FTSZ和膜之间的〜50 aa柔性系绳，因此将力从FTSZ细丝传递到膜。我们提出了有关该系绳的结构和力学的几项研究，包括突变和取代，NMR，并将其移至FTSZ球状结构域上的不同附着点。在先前的研究中，我们获得了大肠杆菌的十二个抑制菌株，使异常的FTSZ起作用。这些抑制突变可能是在影响细胞因子的未发现的途径中。我们建议通过通过Solexa测序重塑每个菌株的基因组来识别它们。最后，我们建议通过Palm成像Z环，Palm是一种光学显微镜“超分辨率”技术，可以得出30 nm的分辨率。我们认为这可以对单个FTSZ原丝图进行映像，并确定它们的分布方式以制造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。这是基础科学的最重要问题，可以扩展我们对生物学的了解。它还具有潜在的临床相关性。 FTSZ在细菌中高度保守，是新抗生素的有吸引力的靶标。靶向FTSZ的几种铅化合物已经在研究和开发中。