In vitro reconstitution of Escherichia coli divisome activation.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
12 05 2022
12 05 2022
Historique:
received:
22
11
2021
accepted:
25
04
2022
entrez:
13
5
2022
pubmed:
14
5
2022
medline:
18
5
2022
Statut:
epublish
Résumé
The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ.
Identifiants
pubmed: 35550516
doi: 10.1038/s41467-022-30301-y
pii: 10.1038/s41467-022-30301-y
pmc: PMC9098913
doi:
Substances chimiques
Bacterial Proteins
0
Escherichia coli Proteins
0
Membrane Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2635Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2022. The Author(s).
Références
Trueba, F. J. On the precision and accuracy achieved by Escherichia coli cells at fission about their middle. Arch. Microbiol. 131, 55–59 (1982).
doi: 10.1007/BF00451499
pubmed: 7039546
McQuillen, R. & Xiao, J. Insights into the structure, function, and dynamics of the bacterial cytokinetic FtsZ-ring. Annu. Rev. Biophys. 49, 309–341 (2020).
doi: 10.1146/annurev-biophys-121219-081703
pubmed: 32092282
pmcid: 8610174
Du, S. & Lutkenhaus, J. Assembly and activation of the Escherichia coli divisome. Mol. Microbiol. https://doi.org/10.1111/mmi.13696 (2017).
Baranova, N. et al. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. Nature Microbiol. https://doi.org/10.1038/s41564-019-0657-5 (2020).
Pichoff, S., Du, S. & Lutkenhaus, J. Disruption of divisome assembly rescued by FtsN–FtsA interaction in Escherichia coli. Proc. Natl. Acad. Sci. USA https://doi.org/10.1073/pnas.1806450115 (2018).
Karimova, G., Dautin, N. & Ladant, D. Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J. Bacteriol. 190, 8248 (2008).
doi: 10.1128/JB.01470-08
pmcid: 2593213
Pichoff, S., Du, S. & Lutkenhaus, J. The bypass of ZipA by overexpression of FtsN requires a previously unknown conserved FtsN motif essential for FtsA- FtsN interaction supporting a model in which FtsA monomers recruit late cell division proteins to the Z ring. Mol. Microbiol. 4, 971–987 (2015).
Geissler, B., Elraheb, D. & Margolin, W. A gain-of-function mutation in ftsA bypasses the requirement for the essential cell division gene zipA in Escherichia coli. Proc. Natl Acad. Sci. USA 100, 4197–4202 (2003).
doi: 10.1073/pnas.0635003100
pubmed: 12634424
pmcid: 153070
Addinall, S. G. & Lutkenhaus, J. FtsA is localized to the septum in an FtsZ-dependent manner. J. Bacteriol. 178, 7167–7172 (1996).
doi: 10.1128/jb.178.24.7167-7172.1996
pubmed: 8955398
pmcid: 178629
Bisson-Filho, A. W. et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science 355, 739–743 (2017).
doi: 10.1126/science.aak9973
pubmed: 28209898
pmcid: 5485650
Szwedziak, P., Wang, Q., Freund, S. M. & Löwe, J. FtsA forms actin-like protofilaments. EMBO J. https://doi.org/10.1038/emboj.2012.76 (2012).
Krupka, M. et al. Escherichia coli FtsA forms lipid-bound minirings that antagonize lateral interactions between FtsZ protofilaments. Nat. Commun. 8, 1–12 (2017).
doi: 10.1038/ncomms15957
Du, S., Pichoff, S. & Lutkenhaus, J. FtsEX acts on FtsA to regulate divisome assembly and activity. Proc. Natl. Acad. Sci. USA https://doi.org/10.1073/pnas.1606656113 (2016).
Park, K.-T., Pichoff, S., Du, S. & Lutkenhaus, J. FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli. Proc. Natl Acad. Sci. USA 118, e2107210118 (2021).
doi: 10.1073/pnas.2107210118
pubmed: 34453005
pmcid: 8536321
Busiek, K. K. & Margolin, W. A role for FtsA in SPOR-independent localization of the essential E scherichia coli cell division protein FtsN. Mol. Microbiol. 92, 1212–1226 (2014).
doi: 10.1111/mmi.12623
pubmed: 24750258
pmcid: 4079119
Liu, B., Persons, L., Lee, L. & de Boer, P. A. J. Roles for both FtsA and the FtsBLQ subcomplex in FtsN-stimulated cell constriction in Escherichia coli. Mol. Microbiol. 95, 945–970 (2015).
doi: 10.1111/mmi.12906
pubmed: 25496160
pmcid: 4428282
Corbin, B. D., Geissler, B., Sadasivam, M. & Margolin, W. Z-Ring-independent interaction between a subdomain of FtsA and late septation proteins as Revealed by a Polar Recruitment Assay. J. Bacteriol. 186, 7736–7744 (2004).
doi: 10.1128/JB.186.22.7736-7744.2004
pubmed: 15516588
pmcid: 524888
Geissler, B., Shiomi, D. & Margolin, W. The ftsA* gain-of-function allele of Escherichia coli and its effects on the stability and dynamics of the Z ring. Microbiology 153, 814–825 (2007).
doi: 10.1099/mic.0.2006/001834-0
pubmed: 17322202
Pichoff, S., Shen, B., Sullivan, B. & Lutkenhaus, J. FtsA mutants impaired for self-interaction bypass ZipA suggesting a model in which FtsA’s self-interaction competes with its ability to recruit downstream division proteins. Mol. Microbiol. https://doi.org/10.1111/j.1365-2958.2011.07923.x (2012).
Bernard, C. S., Sadasivam, M., Shiomi, D. & Margolin, W. An altered FtsA can compensate for the loss of essential cell division protein FtsN in Escherichia coli. Mol. Microbiol. https://doi.org/10.1111/j.1365-2958.2007.05738.x (2007).
Loose, M. & Mitchison, T. J. The bacterial cell division proteins ftsA and ftsZ self-organize into dynamic cytoskeletal patterns. Nat. Cell Biol. 16, 38–46 (2014).
doi: 10.1038/ncb2885
pubmed: 24316672
Rueda, S., Vicente, M. & Mingorance, J. Concentration and assembly of the division ring proteins FtsZ, FtsA, and ZipA during the Escherichia coli cell cycle. J. Bacteriol. 185, 3344–3351 (2003).
doi: 10.1128/JB.185.11.3344-3351.2003
pubmed: 12754232
pmcid: 155373
Caldas, P., López-pelegrín, M., Pearce, D. J. G., Budanur, N. B. & Brugués, J. ZapA stabilizes FtsZ filament bundles without slowing down treadmilling dynamics. bioRxiv https://doi.org/10.1101/580944 (2019).
Caldas, P., Radler, P., Sommer, C. & Loose, M. Computational analysis of filament polymerization dynamics in cytoskeletal networks. Methods Cell Biol. 158, 145–161 (2020).
Wang, H. & Gayda, R. High level expression of the FtsA protein inhibits cell septation in E coli K12. J. Bacteriol. 172, 4736–4740 (1990).
doi: 10.1128/jb.172.8.4736-4740.1990
pubmed: 2198274
pmcid: 213318
Dai, K. & Lutkenhaus, J. The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli. J. Bacteriol. 174, 6145–6151 (1992).
doi: 10.1128/jb.174.19.6145-6151.1992
pubmed: 1400163
pmcid: 207681
Wang, H., Henk, M. C. & Gayda, R. C. Overexpression of ftsA induces large bulges at the septal regions in Escherichia coli. Curr. Microbiol. 26, 175–181 (1993).
doi: 10.1007/BF01577374
Löwe, J. & van den Ent, F. Conserved sequence motif at the C-terminus of the bacterial cell-division protein FtsA. Biochimie 83, 117–120 (2001).
doi: 10.1016/S0300-9084(00)01210-4
pubmed: 11254984
Du, S., Henke, W., Pichoff, S. & Lutkenhaus, J. How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division. Mol. Microbiol. 0, 14324 (2019). mmi.
Verveer, P. J., Rocks, O., Harpur, A. G. & Bastiaens, P. I. H. Imaging protein interactions by FRET microscopy: FRET measurements by acceptor photobleaching. Cold Spring Harb. Protoc. 2006, pdb.prot4598 (2006).
Loose, M., Fischer-Friedrich, E., Herold, C., Kruse, K. & Schwille, P. Min protein patterns emerge from rapid rebinding and membrane interaction of MinE. Nat. Struct. Mol. Biol. 18, 577–583 (2011).
doi: 10.1038/nsmb.2037
pubmed: 21516096
Hernández-Rocamora, V. M. et al. Real-time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin-binding proteins. Elife 10, e61525 (2021).
Gerganova, V. et al. Cell patterning by secretion-induced plasma membrane flows. Sci. Adv. 7, 1–19 (2021).
doi: 10.1126/sciadv.abg6718
Schoenemann, K. M. et al. Gain-of-function variants of FtsA form diverse oligomeric structures on lipids and enhance FtsZ protofilament bundling. Mol. Microbiol. 109, 676–693 (2018).
doi: 10.1111/mmi.14069
pubmed: 29995995
pmcid: 6181759
Nierhaus, T. et al. The bacterial actin-like cell division protein FtsA forms curved antiparallel double filaments upon binding of FtsN. bioRxiv https://doi.org/10.1101/2021.11.08.467742 (2021).
Du, S. & Lutkenhaus, J. Assembly and activation of the Escherichia coli divisome. Mol. Microbiol. 105, 177–187 (2017).
doi: 10.1111/mmi.13696
pubmed: 28419603
pmcid: 5517055
Shannon, C. E. Communication in the presence of noise. Proc. IRE 37, 10–21 (1949).
doi: 10.1109/JRPROC.1949.232969
McCausland, J. W. et al. Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism. Nat. Commun. 12, 609 (2021).
doi: 10.1038/s41467-020-20873-y
pubmed: 33504807
pmcid: 7840769
Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: A versatile method for protein labeling. Nat. Chem. Biol. https://doi.org/10.1038/nchembio.2007.31 (2007).
Baranova, N. & Loose, M. Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. Methods Cell Biol. 137, 355–370 (2017).
Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods https://doi.org/10.1038/nmeth.2019 (2012).
Dunn, K. W., Kamocka, M. M. & McDonald, J. H. A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300, 723–742 (2011).
doi: 10.1152/ajpcell.00462.2010
Tinevez, J. Y. et al. TrackMate: An open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).
doi: 10.1016/j.ymeth.2016.09.016
pubmed: 27713081
Renner, M., Wang, L., Levi, S., Hennekinne, L. & Triller, A. A Simple and powerful analysis of lateral subdiffusion using single particle tracking. Biophys. J. 113, 2452–2463 (2017).
doi: 10.1016/j.bpj.2017.09.017
pubmed: 29211999
pmcid: 5738498
Lagardère, M., Chamma, I., Bouilhol, E., Nikolski, M. & Thoumine, O. FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins. Sci. Rep. 10, 1–14 (2020).
doi: 10.1038/s41598-020-75814-y
Caldas, P. & Radler, P. In vitro reconstitution of Escherichia coli divisome activation: Transient Confinement Analysis. GitHub https://doi.org/10.5281/zenodo.6397261 (2022).
Gebhardt, J. C. M. et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat. Methods 10, 421–426 (2013).
doi: 10.1038/nmeth.2411
pubmed: 23524394
pmcid: 3664538
Sorkin, A., McClure, M., Huang, F. & Carter, R. Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Curr. Biol. 10, 1395–1398 (2000).
doi: 10.1016/S0960-9822(00)00785-5
pubmed: 11084343
Sommer, C. In vitro reconstitution of Escherichia coli divisome activation: FrapDiff. GitHub https://doi.org/10.5281/zenodo.6400639 (2022).
Sobrinos-Sanguino, M., Vélez, M., Richter, R. P. & Rivas, G. Reversible membrane tethering by ZipA determines FtsZ polymerization in two and three dimensions. Biochemistry 58, 4003–4015 (2019).
doi: 10.1021/acs.biochem.9b00378
pubmed: 31390865
Eisele, N. B., Frey, S., Piehler, J., Görlich, D. & Richter, R. P. Ultrathin nucleoporin phenylalanine-glycine repeat films and their interaction with nuclear transport receptors. EMBO Rep. 11, 366–372 (2010).
doi: 10.1038/embor.2010.34
pubmed: 20379223
pmcid: 2868541