Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

• 批准号: 9085296
• 负责人: HAROLD P ERICKSON
• 金额: $ 46.98万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9085296
• 关键词: Address; Adopted; Antibiotics; Bacteria; Basic Science; Biological Assay; Biology; C-terminal; Color; Cryoelectron Microscopy; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; Data; Fluorescence; Funding; Goals; Health; Homologous Gene; Image; Imaging Techniques; In Vitro; Individual; Kinetics; Knowledge; Label; Laboratories; Lateral; Lead; Liposomes; Location; Membrane; Metallothionein; Microtomy; Microtubules; Modeling; Molecular Conformation; Motor; Negative Staining; Peroxidases; Physics; Proteins; Resolution; Rotation; Running; Sampling; Structural Models; Structure; Time; Tubulin; Work; analog; base; clinically relevant; constriction; innovation; light microscopy; reconstitution; treadmill

DESCRIPTION (provided by applicant): The tubulin homolog FtsZ is the major cytoskeletal protein in bacterial cytokinesis. Our recent work has shown that FtsZ can reconstitute Z rings in liposomes, and these can generate a constriction force without any other proteins. FtsZ thus acts as cytoskeleton and motor all in one. Our work suggests that the constriction force is generated by a curved conformation of the FtsZ protofilaments (pfs) bending the membrane. We now propose to address three of the most important and immediate questions. (1) What is the structure of the Z ring in bacteria? There are two competing models. The "ribbon" model proposes that pfs are laterally associated to make a ribbon, and the "scattered" model proposes that the pfs are more widely scattered on the membrane and not in contact. We propose three imaging techniques to resolve this controversy: super-resolution light microscopy and two newly-developed EM probes. Resolving these models is essential to explore the physics of the constriction force. (2) What is the mechanism of pf dynamics? We have shown that FtsZ pfs are rapidly exchanging subunits, with a half time of 5-10 s. The mechanism could involve dynamic instability, treadmilling or fragmentation/annealing. We propose to image single pfs by super-resolution light microscopy and by cryo and negative stain EM to resolve the mechanism. (3) What is the structure of curved pfs? We have strong evidence that the constriction force is generated by pfs adopting a curved conformation that exerts a bending force on the membrane. However, there are structural contradictions that need to be resolved, especially regarding the orientation - is the C terminus on the inside or the outside of the curvature? To determine this orientation, we propose to develop FtsZ subunits with a large C-terminal tag visible by EM. Importantly, we now know that there are two curved conformations, the highly curved miniring, which is an analog of tubulin rings, and an intermediate curved form. We suspect these may have opposite orientations, and our tagged FtsZ should resolve this.

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。我们最近的工作表明，FtsZ 可以在脂质体中重建 Z 环，并且这些环可以在没有任何其他蛋白质的情况下产生收缩力。因此，FtsZ 充当细胞骨架和运动合一的角色。我们的工作表明，收缩力是由 FtsZ 原丝 (pfs) 弯曲膜的弯曲构象产生的。我们现在建议解决三个最重要和最紧迫的问题。 (1)细菌中Z环的结构是什么？有两种竞争模型。 “带”模型提出pfs横向关联以形成带，而“分散”模型提出pfs更广泛地分散在膜上并且不接触。我们提出了三种成像技术来解决这一争议：超分辨率光学显微镜和两种新开发的电磁探针。解决这些模型对于探索收缩力的物理原理至关重要。 （2）pf动力学的机制是什么？我们已经证明 FtsZ pfs 正在快速交换亚基，半衰期为 5-10 秒。该机制可能涉及动态不稳定性、跑步或碎片/退火。我们建议通过超分辨率光学显微镜以及冷冻和负染色电镜对单个 pfs 进行成像，以解决该机制。 (3)弯曲pfs的结构是怎样的？我们有强有力的证据表明收缩力是由 pfs 采用弯曲构象对膜施加弯曲力而产生的。然而，还有一些结构性矛盾需要解决，特别是关于方向——C终点是在曲率的内侧还是外侧？为了确定这个方向，我们建议开发具有电镜可见的大 C 端标签的 FtsZ 亚基。重要的是，我们现在知道有两种弯曲的构象，高度弯曲的微型环（微管蛋白环的类似物）和中间弯曲的形式。我们怀疑它们可能具有相反的方向，我们标记的 FtsZ 应该可以解决这个问题。