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.

Public Health Relevance

Our overall goal is to determine the mechanism by which bacteria divide. This is foremost an issue of basic science, to expand our knowledge of biology. It also has potential clinical relevance. FtsZ is highly conserved in bacteria, and is an attractiv target for new antibiotics. Several lead compounds targeting FtsZ are already being studied and developed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM066014-13
Application #
8697233
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Deatherage, James F
Project Start
2002-07-01
Project End
2018-05-31
Budget Start
2014-07-01
Budget End
2015-05-31
Support Year
13
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Duke University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
City
Durham
State
NC
Country
United States
Zip Code
27705
Erickson, Harold P (2017) How bacterial cell division might cheat turgor pressure - a unified mechanism of septal division in Gram-positive and Gram-negative bacteria. Bioessays 39:
Moore, Desmond A; Whatley, Zakiya N; Joshi, Chandra P et al. (2017) Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins. J Bacteriol 199:
Erickson, Harold P (2017) The discovery of the prokaryotic cytoskeleton: 25th anniversary. Mol Biol Cell 28:357-358
Chen, Yaodong; Huang, Haiyan; Osawa, Masaki et al. (2017) ZipA and FtsA* stabilize FtsZ-GDP miniring structures. Sci Rep 7:3650
Chen, Yaodong; Porter, Katie; Osawa, Masaki et al. (2017) The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments. J Biol Chem 292:5207-5215
Erickson, Harold P; Osawa, Masaki (2017) FtsZ Constriction Force - Curved Protofilaments Bending Membranes. Subcell Biochem 84:139-160
Housman, Max; Milam, Sara L; Moore, Desmond A et al. (2016) FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings. Biochemistry 55:4085-91
Suzuki, Aussie; Badger, Benjamin L; Haase, Julian et al. (2016) How the kinetochore couples microtubule force and centromere stretch to move chromosomes. Nat Cell Biol 18:382-92
Bisson-Filho, Alexandre W; Discola, Karen F; Castellen, PatrĂ­cia et al. (2015) FtsZ filament capping by MciZ, a developmental regulator of bacterial division. Proc Natl Acad Sci U S A 112:E2130-8
Milam, Sara L; Erickson, Harold P (2013) Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2. J Biol Chem 288:23675-9

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