Structure and Assembly Dynamics of FtsZ

描述(申请人提供)：微管蛋白同系物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，这些Z环可以在没有任何其他蛋白质的情况下产生紧缩力。因此，FtsZ集细胞骨架和运动于一身。我们的工作表明，紧缩力是由FtsZ原丝(PFS)弯曲膜的弯曲构象产生的。我们现在提议解决三个最重要和最紧迫的问题。(1)细菌中Z环的结构是什么？有两个相互竞争的模式。“带状”模型认为PFS是横向缔合而成带状的，而“散布”模型则认为PFS更广泛地分散在膜上，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个新开发的EM探头。解决这些模型对于探索缩缩力的物理特性是至关重要的。(2)PF动力学的机制是什么？结果表明，FtsZ-PFS是一种快速的亚基交换，半衰期为5-10个S，其机制可能涉及动力学不稳定性、跑步机或碎裂/退火。我们建议用超分辨光学显微镜对单个PFS进行成像，并用低温和负染色EM来解决这一机制。(3)曲线型PFS的结构是什么？我们有强有力的证据表明，收缩压力是由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端是在曲率的内部还是外部？为了确定这一方向，我们建议开发具有EM可见的大C-末端标签的FtsZ亚基。重要的是，我们现在知道有两种弯曲的构象，一种是高度弯曲的，类似于微管蛋白环的，另一种是中等弯曲的。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。

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.