Structure and Assembly Dynamics of FtsZ

描述（由申请方提供）：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，并且这些可以在没有任何其他蛋白质的情况下产生收缩力。因此，FtsZ作为细胞骨架和电机都在一个。我们的工作表明，收缩力产生的弯曲构象的FtsZ原丝（pfs）弯曲膜。我们现在建议处理三个最重要和最紧迫的问题。(1)细菌中Z环的结构是什么？有两种相互竞争的模式。“带状”模型提出，PFS横向关联，使一个带，和“分散”模型提出，PFS更广泛地分散在膜上，而不是接触。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个新开发的EM探针。解决这些模型对于探索收缩力的物理学是必不可少的。(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、球磨或碎裂/退火。我们建议通过超分辨光学显微镜和冷冻负染电镜对单个光子晶体进行成像，以解决这一机制。(3)弯曲的pfs的结构是什么？我们有强有力的证据表明，收缩力是由pfs采用弯曲构象，对膜施加弯曲力产生的。然而，有一些结构上的矛盾需要解决，特别是关于方向-C末端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FtsZ亚基与一个大的C-末端标签可见EM。重要的是，我们现在知道有两种弯曲的构象，高度弯曲的迷你环，这是微管蛋白环的模拟物，以及中间弯曲形式。我们怀疑它们可能有相反的方向，我们标记的FtsZ应该可以解决这个问题。

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.