Structure and Assembly Dynamics of FtsZ

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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.