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.
We acknowledge members of the Loose laboratory at IST Austria for helpful discussions—in particular L. Lindorfer for his assistance with cloning and purifications. We thank J. Löwe and T. Nierhaus (MRC-LMB Cambridge, UK) for sharing unpublished work and helpful discussions, as well as D. Vavylonis and D. Rutkowski (Lehigh University, Bethlehem, PA, USA) and S. Martin (University of Lausanne, Switzerland) for sharing their code for FRAP analysis. We are also thankful for the support by the Scientific Service Units (SSU) of IST Austria through resources provided by the Imaging and Optics Facility (IOF) and the Lab Support Facility (LSF). This work was supported by the European Research Council through grant ERC 2015-StG-679239 and by the Austrian Science Fund (FWF) StandAlone P34607 to M.L. and HFSP LT 000824/2016-L4 to N.B. For the purpose of open access, we have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
Radler P, Baranova NS, Dos Santos Caldas PR, et al. In vitro reconstitution of Escherichia coli divisome activation. Nature Communications. 2022;13. doi:10.1038/s41467-022-30301-y
Radler, P., Baranova, N. S., Dos Santos Caldas, P. R., Sommer, C. M., Lopez Pelegrin, M. D., Michalik, D., & Loose, M. (2022). In vitro reconstitution of Escherichia coli divisome activation. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-022-30301-y
Radler, Philipp, Natalia S. Baranova, Paulo R Dos Santos Caldas, Christoph M Sommer, Maria D Lopez Pelegrin, David Michalik, and Martin Loose. “In Vitro Reconstitution of Escherichia Coli Divisome Activation.” Nature Communications. Springer Nature, 2022. https://doi.org/10.1038/s41467-022-30301-y.
P. Radler et al., “In vitro reconstitution of Escherichia coli divisome activation,” Nature Communications, vol. 13. Springer Nature, 2022.
Radler P, Baranova NS, Dos Santos Caldas PR, Sommer CM, Lopez Pelegrin MD, Michalik D, Loose M. 2022. In vitro reconstitution of Escherichia coli divisome activation. Nature Communications. 13, 2635.
Radler, Philipp, et al. “In Vitro Reconstitution of Escherichia Coli Divisome Activation.” Nature Communications, vol. 13, 2635, Springer Nature, 2022, doi:10.1038/s41467-022-30301-y.
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