The tubulin homolog FtsZ is the major cytoskeletal protein in bacterial cytokinesis. Although a dozen other proteins are essential for division in E. coli, we have recently demonstrated that FtsZ alone is sufficient to reconstitute Z rings in liposomes. Furthermore, these artificial Z rings generate a constriction force without any other proteins. We propose to further these studies in a number of directions to investigate the mechanism of assembly and force generation. One new direction will be to image the growth of single FtsZ filaments in vitro using TIRF microscopy. This should determine if the filaments are undergoing dynamic instability or treadmilling, two mechanisms of assembly dynamics that apply to microtubules and actin. In our present liposome reconstitution, FtsZ is tethered directly to the membrane by an amphipathic helix (FtsZ-mts). We will attempt to reconstitute the natural two-part system where FtsZ is tethered to the membrane by FtsA. We will also investigate the MinCDE system, which oscillates from one end to the other in bacterial cells to localize the FtsZ ring to the center. We will reconstitute the MinCDE system in liposomes, at first by itself (where it should show oscillation) and then with FtsZ-mts and with FtsZ-FtsA (where it should restrict the localization of Z rings). A novel question related to force generation is, what is the structure of the C-terminal tail of FtsZ? This is thought to be a ~50 aa flexible tether between FtsZ and the membrane, and thus transmitting the force from the FtsZ filaments to the membrane. We propose several studies of the structure and mechanics of this tether, including mutation and substitution, NMR, and moving it to different attachment points on the globular domain of FtsZ. In a previous study we obtained a dozen suppressor strains of E. coli that permitted aberrant FtsZ to function for division. These suppressor mutations are likely in undiscovered pathways affecting cytokinesis. We propose to identify them by resequencing the genome of each strain by Solexa sequencing. Finally, we propose to image the Z ring by PALM, a light microscope """"""""superresolution"""""""" technique that can give 30 nm resolution. We believe this can image single FtsZ protofilaments and determine how they are distributed to make the Z ring.

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 attractive 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 #
5R01GM066014-10
Application #
8099656
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Deatherage, James F
Project Start
2002-07-01
Project End
2014-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
10
Fiscal Year
2011
Total Cost
$486,674
Indirect Cost
Name
Duke University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
044387793
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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