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，并且这些可以在没有任何其他蛋白质的情况下产生收缩力。因此，FtsZ集细胞骨架和马达于一身。我们的工作表明，收缩力是由弯曲膜的FtsZ原丝（pfs）的弯曲构象产生的。我们现在提议解决三个最重要和最紧迫的问题。(1)细菌的Z环是什么结构？有两种相互竞争的模式。“带状”模型认为pfs横向结合形成带状，“分散”模型认为pfs更广泛地分散在膜上而不接触。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EM探针。解决这些模型对于探索收缩力的物理特性至关重要。(2) pf动力学的机制是什么？我们已经证明，FtsZ pfs是快速交换亚基，一半时间为5-10秒。机制可能涉及动态不稳定，踩踏或破碎/退火。我们建议用超分辨率光学显微镜和低温和负染色电镜对单个pfs进行成像，以确定其机制。(3)弯曲pfs的结构是什么？我们有强有力的证据表明，收缩力是由采用弯曲构象的pfs产生的，该构象对膜施加弯曲力。然而，有一些结构上的矛盾需要解决，特别是在方向上——C的末端是在曲率的内部还是外部？为了确定这个方向，我们建议开发具有EM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构象，高度弯曲的矿化构象，类似于微管蛋白环，以及中间弯曲形式。我们怀疑这些可能有相反的方向，我们标记的FtsZ应该解决这个问题。

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.