[en] The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.
Disciplines :
Microbiology
Author, co-author :
Corrales-Guerrero, Laura ; Department of Biology, University of Marburg, 35043 Marburg, Germany
Steinchen, Wieland ; Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany ; Department of Chemistry, University of Marburg, 35043 Marburg, Germany
Ramm, Beatrice ; Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Mücksch, Jonas ; Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Rosum, Julia; Department of Biology, University of Marburg, 35043 Marburg, Germany
Heimerl, Thomas; Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany
Bange, Gert ; Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany ; Department of Chemistry, University of Marburg, 35043 Marburg, Germany ; Max Planck Fellow Group Molecular Physiology of Microbes, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany
Schwille, Petra ; Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Thanbichler, Martin ; Department of Biology, University of Marburg, 35043 Marburg, Germany ; Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany ; Max Planck Fellow Group Bacterial Cell Biology, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany
REFES, Yacine Marc ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM) > Medical Education
External co-authors :
yes
Language :
English
Title :
MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly.
Publication date :
13 December 2022
Journal title :
Proceedings of the National Academy of Sciences of the United States of America
Deutsche Forschungsgemeinschaft Deutsche Forschungsgemeinschaft Max-Planck-Gesellschaft EC | Horizon 2020 Framework Programme
Funding text :
We thank Till Ringel, Jaspara Knopp, and Leonie Tomm for their support during the early phases of this work and Maria Billini for constructing strains. This work was supported by the German Research Foundation (DFG) (TRR 174 \u2013 project 269423233 to G.B., P.S., and M.T. and DFG Core Facility for Interactions, Dynamics and Macromolecular Assembly \u2013 project 324652314 to G.B.) and the Max Planck Society (Max Planck Fellowship; to M.T.). L.C.-G. was supported by the Horizon 2020 Research and Innovation Program of the European Commission (Marie Sklodowska-Curie grant, agreement no. 659174 to L.C-G.).ACKNOWLEDGMENTS. We thank Till Ringel, Jaspara Knopp, and Leonie Tomm for their support during the early phases of this work and Maria Billini for constructing strains. This work was supported by the German Research Foundation (DFG) (TRR 174 \u2013 project 269423233 to G.B., P.S., and M.T. and DFG Core Facility for Interactions, Dynamics and Macromolecular Assembly \u2013 project 324652314 to G.B.) and the Max Planck Society (Max Planck Fellowship; to M.T.). L.C.-G. was supported by the Horizon 2020 Research and Innovation Program of the European Commission (Marie Sklodowska-Curie grant, agreement no. 659174 to L.C-G.).
S. Du, J. Lutkenhaus, At the heart of bacterial cytokinesis: The Z ring. Trends Microbiol. 27, 781–791 (2019).
J. Wagstaff, J. Löwe, Prokaryotic cytoskeletons: Protein filaments organizing small cells. Nat. Rev. Microbiol. 16, 187–201 (2018).
S. Du, J. Lutkenhaus, Assembly and activation of the Escherichia coli divisome. Mol. Microbiol. 105, 177–187 (2017).
J. Errington, L. J. Wu, Cell cycle machinery in Bacillus subtilis. Subcell. Biochem. 84, 67–101 (2017).
E. D. Goley et al., Assembly of the Caulobacter cell division machine. Mol. Microbiol. 80, 1680–1698 (2011).
P. de Boer, R. Crossley, L. Rothfield, The essential bacterial cell-division protein FtsZ is a GTPase. Nature 359, 254–256 (1992).
A. Mukherjee, K. Dai, J. Lutkenhaus, Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein. Proc. Natl. Acad. Sci. U.S.A. 90, 1053–1057 (1993).
D. RayChaudhuri, J. T. Park, Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein. Nature 359, 251–254 (1992).
P. J. Buske, P. A. Levin, Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins. J. Biol. Chem. 287, 10945–10957 (2012).
L. Corrales-Guerrero et al., FtsZ of filamentous, heterocyst-forming cyanobacteria has a conserved N-terminal peptide required for normal FtsZ polymerization and cell division. Front. Microbiol. 9, 2260 (2018).
X. Ma et al., Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring. J. Bacteriol. 179, 6788–6797 (1997).
K. Sundararajan et al., The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction. Nat. Commun. 6, 7281 (2015).
P. J. Buske, P. A. Levin, A flexible C-terminal linker is required for proper FtsZ assembly in vitro and cytokinetic ring formation in vivo. Mol. Microbiol. 89, 249–263 (2013).
M. C. Cohan, A. M. P. Eddelbuettel, P. A. Levin, R. V. Pappu, Dissecting the functional contributions of the intrinsically disordered C-terminal tail of Bacillus subtilis FtsZ. J. Mol. Biol. 432, 3205–3221 (2020).
C. Lu, M. Reedy, H. P. Erickson, Straight and curved conformations of FtsZ are regulated by GTP hydrolysis. J. Bacteriol. 182, 164–170 (2000).
Y. Chen, H. P. Erickson, Conformational changes of FtsZ reported by tryptophan mutants. Biochemistry 50, 4675–4684 (2011).
J. F. Diaz et al., Activation of cell division protein FtsZ. Control of switch loop T3 conformation by the nucleotide gamma-phosphate. J. Biol. Chem. 276, 17307–17315 (2001).
G. M. Alushin et al., High-resolution microtubule structures reveal the structural transitions in αβtubulin upon GTP hydrolysis. Cell 157, 1117–1129 (2014).
S. Z. Chou, T. D. Pollard, Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides. Proc. Natl. Acad. Sci. U.S.A. 116, 4265–4274 (2019).
F. Merino et al., Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM. Nat. Struct. Mol. Biol. 25, 528–537 (2018).
J. Fujita et al., Identification of the key interactions in structural transition pathway of FtsZ from Staphylococcus aureus. J. Struct. Biol. 198, 65–73 (2017).
T. Matsui, X. Han, J. Yu, M. Yao, I. Tanaka, Structural change in FtsZ induced by intermolecular interactions between bound GTP and the T7 loop. J. Biol. Chem. 289, 3501–3509 (2014).
E. Ramirez-Aportela, J. R. Lopez-Blanco, J. M. Andreu, P. Chacon, Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations. Biophys. J. 107, 2164–2176 (2014).
K. Natarajan, S. Senapati, Probing the conformational flexibility of monomeric FtsZ in GTP-bound, GDP-bound, and nucleotide-free states. Biochemistry 52, 3543–3551 (2013).
M. Loose, T. J. Mitchison, The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns. Nat. Cell Biol. 16, 38–46 (2014).
D. A. Ramirez-Diaz et al., Treadmilling analysis reveals new insights into dynamic FtsZ ring architecture. PLoS Biol. 16, e2004845 (2018).
A. W. Bisson-Filho et al., Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science 355, 739–743 (2017).
X. Yang et al., GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science 355, 744–747 (2017).
R. McQuillen, J. Xiao, Insights into the structure, function, and dynamics of the bacterial cytokinetic FtsZ-ring. Annu. Rev. Biophys. 49, 309–341 (2020).
J. M. Wagstaff et al., A polymerization-associated structural switch in FtsZ that enables treadmilling of model filaments. mBio 8, e00254-17 (2017).
S. Du, S. Pichoff, K. Kruse, J. Lutkenhaus, FtsZ filaments have the opposite kinetic polarity of microtubules. Proc. Natl. Acad. Sci. U.S.A. 115, 10768–10773 (2018).
E. D. Goley, N. A. Dye, J. N. Werner, Z. Gitai, L. Shapiro, Imaging-based identification of a critical regulator of FtsZ protofilament curvature in Caulobacter. Mol. Cell 39, 975–987 (2010).
F. J. Gueiros-Filho, R. Losick, A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ. Genes Dev. 16, 2544–2556 (2002).
D. Schumacher et al., The PomXYZ proteins self-organize on the bacterial nucleoid to stimulate cell division. Dev. Cell 41, 299–314 (2017).
M. E. Gündoğdu et al., Large ring polymers align FtsZ polymers for normal septum formation. EMBO J. 30, 617–626 (2011).
F. Ramos-Leon et al., A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria. Elife 10, e63387 (2021).
Z. Hu, A. Mukherjee, S. Pichoff, J. Lutkenhaus, The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization. Proc. Natl. Acad. Sci. U.S.A. 96, 14819–14824 (1999).
N. K. Tonthat et al., Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check. EMBO J. 30, 154–164 (2011).
M. Thanbichler, L. Shapiro, MipZ, A spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Cell 126, 147–162 (2006).
D. W. Adams, L. J. Wu, J. Errington, Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane. EMBO J. 34, 491–501 (2015).
N. S. Hill, P. J. Buske, Y. Shi, P. A. Levin, A moonlighting enzyme links Escherichia coli cell size with central metabolism. PLoS Genet. 9, e1003663 (2013).
D. Trusca, S. Scott, C. Thompson, D. Bramhill, Bacterial SOS checkpoint protein SulA inhibits polymerization of purified FtsZ cell division protein. J. Bacteriol. 180, 3946–3953 (1998).
R. B. Weart et al., A metabolic sensor governing cell size in bacteria. Cell 130, 335–347 (2007).
A. Dajkovic, A. Mukherjee, J. Lutkenhaus, Investigation of regulation of FtsZ assembly by SulA and development of a model for FtsZ polymerization. J. Bacteriol. 190, 2513–2526 (2008).
Y. Chen, S. L. Milam, H. P. Erickson, SulA inhibits assembly of FtsZ by a simple sequestration mechanism. Biochemistry 51, 3100–3109 (2012).
A. W. Bisson-Filho et al., FtsZ filament capping by MciZ, a developmental regulator of bacterial division. Proc. Natl. Acad. Sci. U.S.A. 112, E2130–2138 (2015).
K. T. Park, A. Dajkovic, M. Wissel, S. Du, J. Lutkenhaus, MinC and FtsZ mutant analysis provides insight into MinC/MinD-mediated Z ring disassembly. J. Biol. Chem. 293, 5834–5846 (2018).
A. Akhmanova, M. O. Steinmetz, Control of microtubule organization and dynamics: Two ends in the limelight. Nat. Rev. Mol. Cell Biol. 16, 711–726 (2015).
C. E. Walczak, S. Gayek, R. Ohi, Microtubule-depolymerizing kinesins. Annu. Rev. Cell Dev. Biol. 29, 417–441 (2013).
M. Toro-Nahuelpan et al., A gradient-forming MipZ protein mediating the control of cell division in the magnetotactic bacterium Magnetospirillum gryphiswaldense. Mol. Microbiol. 112, 1423–1439 (2019).
D. Kiekebusch, K. A. Michie, L. O. Essen, J. Löwe, M. Thanbichler, Localized dimerization and nucleoid binding drive gradient formation by the bacterial cell division inhibitor MipZ. Mol. Cell 46, 245–259 (2012).
D. Kiekebusch, M. Thanbichler, Spatiotemporal organization of microbial cells by protein concentration gradients. Trends Microbiol. 22, 65–73 (2014).
L. Corrales-Guerrero et al., Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus. Nucleic Acids Res. 48, 4769–4779 (2020).
Y. Abdiche, D. Malashock, A. Pinkerton, J. Pons, Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal. Biochem. 377, 209–217 (2008).
R. Rigler, Ü. Mets, J. Widengren, P. Kask, Fluorescence correlation spectroscopy with high count rate and low background: Analysis of translational diffusion. Eur. Biophys. J. Biophys. Lett. 22, 169–175 (1993).
D. J. Scheffers, J. G. de Wit, T. den Blaauwen, A. J. Driessen, GTP hydrolysis of cell division protein FtsZ: Evidence that the active site is formed by the association of monomers. Biochemistry 41, 521–529 (2002).
S. Arumugam, Z. Petrasek, P. Schwille, MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation. Proc. Natl. Acad. Sci. U.S.A. 111, E1192–1200 (2014).
L. Konermann, J. Pan, Y. H. Liu, Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem. Soc. Rev. 40, 1224–1234 (2011).
C. Lu, J. Stricker, H. P. Erickson, Site-specific mutations of FtsZ – effects on GTPase and in vitro assembly. BMC Microbiol. 1, 7 (2001).
F. Guan et al., Lateral interactions between protofilaments of the bacterial tubulin homolog FtsZ are essential for cell division. Elife 7, e35578 (2018).
S. Du, J. Lutkenhaus, SlmA antagonism of FtsZ assembly employs a two-pronged mechanism like MinCD. PLoS Genet. 10, e1004460 (2014).
R. Evans et al., Protein complex prediction with AlphaFold-Multimer. bioRxiv [Preprint] (2022). https://doi.org/10.1101/2021.10.04.463034 (Accessed 20 November 2022).
B. Monterroso et al., Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA. EMBO Rep. 20, e45946 (2019).
L. Araujo-Bazan, S. Huecas, J. Valle, D. Andreu, J. M. Andreu, Synthetic developmental regulator MciZ targets FtsZ across Bacillus species and inhibits bacterial division. Mol. Microbiol. 111, 965–980 (2019).
D. M. Raskin, P. A. de Boer, Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 96, 4971–4976 (1999).
A. A. Handler, J. E. Lim, R. Losick, Peptide inhibitor of cytokinesis during sporulation in Bacillus subtilis. Mol. Microbiol. 68, 588–599 (2008).
W. Wang et al., Insight into microtubule disassembly by kinesin-13s from the structure of Kif2C bound to tubulin. Nat. Commun. 8, 70 (2017).
D. Wang et al., Motility and microtubule depolymerization mechanisms of the kinesin-8 motor, KIF19A. Elife 5, e18101 (2016).
J. Schindelin et al., Fiji: An open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
J. G. Marblestone et al., Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Sci. 15, 182–189 (2006).
B. Ramm, P. Glock, P. Schwille, In vitro reconstitution of self-organizing protein patterns on supported lipid bilayers. J. Vis. Exp. 137, e58139 (2018).
E. Ingerman, J. Nunnari, A continuous, regenerative coupled GTPase assay for dynamin-related proteins. Methods Enzymol. 404, 611–619 (2005).
Y. Zhang, I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008).
