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The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns

Nature Cell Biology volume 16, pages 3846 (2014) | Download Citation

Abstract

Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other’s assembly dynamics is not known. Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and negative regulation of FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures.

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Acknowledgements

We thank the members of the laboratories of T.J.M., T. Bernhardt and T. Rapoport for discussions and support; J. Werbin (Harvard Medical School, USA) for the plasmid for sortase expression; H. Erickson (Duke University, USA) for the plasmid for FtsZ–YFP–mts expression; J. Brugues for valuable discussions and advice; and E. Garner, K. Kruse and S. Grill for discussions and comments on the manuscript. We would also like to thank the Nikon Imaging Center at Harvard Medical School for their excellent service. M.L. is supported by fellowships from EMBO (ALTF 394-2011) and HFSP (LT000466/2012). Cytoskeleton dynamics research in the T.J.M. group is supported by NIH-GM39565.

Author information

Affiliations

  1. Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Martin Loose
    •  & Timothy J. Mitchison

Authors

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Contributions

M.L. designed and performed experiments and analysed results. M.L. and T.J.M. discussed results and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Martin Loose.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Information

  2. 2.

    Supplementary Table 2

    Supplementary Information

Videos

  1. 1.

    FtsZ and FtsA self-organize into a rapidly reorganizing filament network.

    Video of FtsZ–FtsA filament pattern formation after addition of GTP (FtsZ = 1.5 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.5 μM. Note that Video was normalized to have a constant overall intensity, thus compensating for the increasing intensity over time due to protein binding to the membrane. As a consequence of this normalization, non-specific bound fluorescent particles appear disproportionately bright at the beginning of a Video. Scale bar is 10 μm.

  2. 2.

    Close-up of video 1, FtsZ and FtsA form chirally rotating rings.

    Close up of Supplementary Video 1. Scale bar is 1 μm.

  3. 3.

    FtsZ and FtsA form chirally rotating rings.

    Video corresponds to Fig. 1c (FtsZ = 1.5 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.5 μM). Scale bar is 10 μm.

  4. 4.

    FtsZ–FtsA filament network formation depends on ATP.

    Video corresponds to Supplementary Fig. 2a. Scale bar is 5 μm.

  5. 5.

    Cytoskeletal dynamics depend on GTP hydrolysis.

    The FtsZ–FtsA filament network does not show rapid rearrangements in the presence of GMPCPP, a non-hydrolyzable analog of GTP (FtsZ = 1.5 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.5 μM). Scale bar is 5 μm.

  6. 6.

    Single molecule dynamics of FtsZ in a FtsZ–FtsA cytoskeletal network.

    While the protein network is reorganizing, individual FtsZ proteins remain at the same position (FtsZ, 1.25 μM with 30 mol-% FtsZ-Alexa488 and 0.4 mol-% FtsZ-Cy5; FtsA, 0.4 μM). Scale bar is 10 μm.

  7. 7.

    Close up of Supplementary Video 6 showing single FtsZ monomers in a rotating ring of FtsZ filaments.

    Scale bar is 3 μm.

  8. 8.

    Single molecule dynamics of FtsZ in a FtsZ–FtsA cytoskeletal network.

    Dynamics of individual Cy5-labeled FtsZ proteins in a dynamic network at higher frame-rate (360 ms/frame) (FtsZ, 1.25 μM with 0.4 mol-% FtsZ-Cy5; FtsA, 0.4 μM). Scale bar is 5 μm.

  9. 9.

    Dynamics of single FtsZ filaments.

    (FtsZ = 0.45 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.2 μM). Scale bar is 5 μm.

  10. 10.

    Dynamics of FtsZ-YFP-mts filament network.

    A polymer network formed with autonomously binding FtsZ does not show large-scale reorganizations (mts  =  membrane targeting sequence, FtsZ-YFP-mts  = 1.25 μM). Scale bar is 5 μm.

  11. 11.

    Dynamics of FtsZ filament network with ZipA as membrane anchor.

    In contrast to FtsA, ZipA does not allow for rapid rearrangement of the cytoskeletal pattern. Instead FtsZ forms a static network of dynamic FtsZ filaments (FtsZ = 1.5 μM with 30 mol-% FtsZ-Alexa488; His-Δ22-ZipA 0.5 μM, supported membrane contained 4 mol-% Ni-chelating lipids). Scale bar is 5 μm.

  12. 12.

    Co-assembly of FtsZ and FtsA on the supported membrane.

    Video corresponds to Fig. 4a (FtsZ = 1.5 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.5 μM with 10 % Cy5-GG-FtsA). Left: merged channels; center: FtsZ-Alexa488, right: Cy5-GG-FtsA. Scale bar is 5 μm.

  13. 13.

    Dual color Video of single filament dynamics of FtsZ and FtsA.

    FtsZ = 0.45 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.2 μM with 10% Cy5-GG-FtsA. Left: merged channels; center: FtsZ-Alexa488, right: Cy5-GG-FtsA. Scale bar is 5 μm.

  14. 14.

    FtsZ filaments facilitate FtsA binding to the membrane.

    Video corresponds to graph in Fig. 4c (FtsZ = 1 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.3 μM with 10 % Cy5-GG-FtsA) (final concentrations). FtsZ was added at 19 min (at change in xy-position). Left: merged channels; center: FtsZ-Alexa488, right: Cy5-GG-FtsA. Scale bar is 5 μm.

  15. 15.

    FtsZ filament dynamics do not depend on reversible binding of FtsA.

    Video of FtsZ filament network formation with FtsA-Δ15-6xHis as membrane anchor (FtsZ = 1.5 μM with 30 mol-% FtsZ-Alexa488; FtsA-Δ15-6xHis 0.5 rmmuM, supported membrane contained 4 mol-% Ni-chelating lipids). Scale bar is 5 μm.

  16. 16.

    In contrast to ZipA, FtsA can disassemble preformed FtsZ filaments.

    Top: Video corresponding to graph in Fig. 5a showing the disassembly of preformed FtsZ filaments after addition of FtsA (FtsZ = 1.2 μM with 30 mol-% FtsZ-Alexa488, FtsA = 0.3 μM with 10 % Cy5-GG-FtsA, final concentrations). Left: merged channels; center: FtsZ-Alexa488, right: Cy5-GG-FtsA. Bottom: Video corresponding to graph in Fig. 5b showing the assembly of an FtsZ filament network after addition of ZipA. (FtsZ = 1.2 μMwith 30 mol-% FtsZ-Alexa488, His-Δ22-ZipA = 0.3 μM, final concentrations, supported membrane contained 4 mol-% Ni-chelating lipids). Scale bar is 5 μm.

  17. 17.

    In contrast to ZipA, FtsA destabilizes the FtsZ filaments network.

    Videos of rapid dilution experiment showing disassembly of FtsZ filaments after 5-fold volume increase of the buffer. For the Videos in the top row, the intensities of each frame were normalized to visualize the architecture of FtsZ filaments after dilution. The bottom row shows the raw data. Left column: Disassembly of FtsZ-ZipA. Sample was diluted between 5 and 10 sec. Right column: Disassembly of FtsZ–FtsA. Sample was diluted between 45 and 50 sec. Scale bar is 2 μm.

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https://doi.org/10.1038/ncb2885

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