Abstract

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division1,2. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria3. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

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Primary accessions

Data deposits

Crystal structures have been deposited in the Protein Data Bank (PDB) under the accession codes 4B46 (H. volcanii CetZ1), 4B45 (H. volcanii CetZ2) and 3ZID (M. thermophila CetZ).

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Acknowledgements

We thank T. Allers for discussion, strains and plasmids, F. Pfeiffer, M. Dyall-Smith, R. Cavicchioli, I. Charles, C. Angstmann and P. Curmi for discussion, J. Maupin-Furlow and D. Sherratt for plasmids, M. Johnson and the UTS Microbial Imaging Facility for technical support, F. Gorrec and S. Kühlmann for help at the MRC-LMB crystallization facility, and the European Synchrotron Radiation Facility and Diamond Light Source for service and support. This work was supported by the Medical Research Council, UK (U105184326 to J.L.) and the University of Technology Sydney, Australia. C.B.W. was supported by the NHMRC, Australia (SRF 571905).

Author information

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Iain G. Duggin
    • , Christopher H. S. Aylett
    • , Katharine A. Michie
    • , Qing Wang
    • , Linda A. Amos
    •  & Jan Löwe
  2. The ithree institute, University of Technology Sydney, New South Wales 2007, Australia

    • Iain G. Duggin
    • , James C. Walsh
    • , Lynne Turnbull
    • , Emma M. Dawson
    • , Elizabeth J. Harry
    •  & Cynthia B. Whitchurch
  3. School of Physics, University of New South Wales, Sydney, New South Wales 2052, Australia

    • James C. Walsh

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Contributions

I.G.D., K.A.M. and J.L. initiated the project. I.G.D., C.H.S.A., K.A.M., L.A.A. and J.L. designed the experiments. I.G.D. and E.M.D. carried out molecular phylogeny. C.H.S.A., J.L., L.A.A. and I.G.D. solved or analysed crystal structures. I.G.D. carried out genetic modification and phenotypic analysis. I.G.D., J.C.W., L.T., C.B.W. and E.J.H. carried out light microscopy. J.C.W. and I.G.D. carried out image analysis. Q.W. carried out electron cryotomography. I.G.D. wrote the manuscript, and all authors reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Iain G. Duggin.

Extended data

Supplementary information

Videos

  1. 1.

    Motile cells of H. volcanii

    Cells were prepared for time-lapse imaging (online Methods); after ~18 h incubation at 37 °C, fields containing motile cells apparent in pockets of interstitial liquid (between the base of the dish and the pad) were imaged by phase-contrast microscopy, at 2 frames per second. The motile cells show behaviours such as runs, tumbles and reversals. In some regions of the field, cells remain immobilised. (The strain shown is H98+pIDJL40-cetZ1.)

  2. 2.

    CetZ1-GFP 3D-SIM slice series through H. volcanii cells

    Strain H98+pIDJL40-cetZ1 was grown in mild-induction conditions (Hv-Ca + 0.2 mM Trp), and then cells were placed on an agarose pad. 3D-SIM z-stack images were acquired (125 nm z-intervals between frames). The image sequence begins at the upper surface of the cells as viewed and proceeds down through to the lower surface. Patches of localisation can be seen around the edges of the cell, and on the upper and lower surfaces as viewed, whereas no clear patches of localisation are seen in the interior, which shows consistent fluorescence throughout.

  3. 3.

    CetZ-GFP can form dynamic filaments

    CetZ1-GFP was overproduced in H. volcanii in rich medium (Hv-YPC + 1 mM Trp). The production of CetZ1-GFP increases during the time-lapse experiment (10 min intervals between frames), and then obvious filaments appear in two cells. These CetZ1-GFP filaments are possibly a non-physiological consequence of overproduction; however, these reveal the capacity of CetZ1-GFP to assemble into filaments that appear to show dynamic instability—a common behaviour of tubulin-family proteins. Cells showing these types of filaments were relatively infrequent (~0.1 %). They did not appear to grow substantially compared to surrounding cells, suggesting that they may be moribund (as may be seen, the cell in the right-hand panel eventually erupts).

  4. 4.

    CetZ1.E218A-GFP 3D-SIM slice series through H. volcanii cells

    Strain H98+pIDJL40-cetZ1.E218A was grown in strong induction conditions (Hv-Ca + 2 mM Trp); despite this, fluorescence in the cell’s interior is very low, and there is strong localisation. The sheet-like patches that may be seen in the successive z-intervals (125 nm apart) are consistent with 2D arrays of CetZ1-GFP assembled at the envelope. A stalk-like structure lined with CetZ1.E218A-GFP is indicated by the arrow.

  5. 5.

    Overproduction of CetZ1.E218A produces an extra sheet-like layer on the inside of the H. volcanii envelope

    (Strain H98+pTA962-cetZ1.E218A, grown in Hv-YPC + 4 mM Trp.) The video shows an electron cryotomography slice series. The extra envelope layer covers a larger surface area than what could be explained by single filaments, suggesting a 2D-array of subunits (scale bar: 100 nm). (Note: a region of this cell is also shown in Fig. 4.)

  6. 6.

    Short-term CetZ1-GFP localisation dynamics in H. volcanii

    Strains expressing cetZ1-gfp or cetZ1.E218A-gfp were grown in low (0.2 mM Trp, panels a and c) and high (2 mM Trp, panels b and d) expression conditions, and then cells were imaged at 10-second intervals at 37 °C by OMX deconvolution microscopy. The panels, except panel a, have identical imaging settings (1 ms exposures); panel a was obtained with longer exposures (5 ms), as it showed fainter localised fluorescence. CetZ1-GFP shows localisation dynamics over this time frame, with more stable fluorescence at the edges near mid-cell, and fluorescence throughout the interior. CetZ1.E218A-GFP shows very little fluorescence in the cell interior, whereas the strongly localised material at the envelope is very stable (See also Supplementary Video 7.). Some much fainter areas of fluorescence from CetZ1.E218A-GFP show dynamic behaviour, suggesting that this is not dependent on the GTPase activation domain (E218).

  7. 7.

    Stable localisation of CetZ1.E218A to cell protrusions and high-curvature regions

    Cells producing CetZ.E218A-GFP (Hv-Ca + 2 mM Trp) were imaged in time-lapse by phase contrast (left) and fluorescence microscopy (right) at 15-second intervals with a Nikon Ti system. The localised material is stably associated with structural features of cells attributed to the E218A mutation. Fluorescence fades over time, which was attributed to photobleaching.

  8. 8.

    Transition from plate to rod cell shapes in H. volcanii

    Cells producing CetZ1-GFP were grown in Hv-YPC + 0.2 mM Trp liquid medium and visualised with both differential interference contrast (DIC) and GFP-fluorescence time-lapse microscopy. In this media, cetZ1-dependent rod-shaped cells form during exponential growth; in this field two cells can be seen undergoing transition or rod elongation. (The lower cell is also shown in Fig. 4g.) Note that cells shift positions somewhat over time as they are not immobilised on a gel pad. CetZ1-GFP shows dynamic patches and filamentous localisation along the length of the rod during development (15 min frame intervals). The strongest localisations are seen near mid-cell, perpendicular to potential division sites, along the edges of the rod. We speculate that the potential division site and cell poles might be specific sites of activity of CetZ1 (and any associated molecules) for generating and maintaining rod shape.

  9. 9.

    Transition from plate to rod cell shapes in H. volcanii

    A sample was withdrawn from the central inoculation site of a motility agar plate (Hv-Ca + 0.3 % agar + 1 mM Trp, day 3) and then prepared for time-lapse microscopy (10 min intervals). Under these conditions, the fluorescence from CetZ1-GFP in some rod-shaped cells (such as this one) was observed to alternate suddenly between irregular periods of dynamic localisation and uniform fluorescence throughout the cell. During the localisation periods, patches of CetZ1-GFP and filaments moving along the cell edges around the mid-cell region may be seen, as the cell narrows and elongates.

  10. 10.

    Stability of polar CetZ1-GFP localisation in motile rods of H. volcanii

    Cells producing CetZ1-GFP (in Hv-Ca + 0.2 mM Trp) were incubated at 37 °C for ~18 h on a 0.3 % agarose pad in a microscopy dish (see online Methods), and then a field showing motile cells at the edge of a pocket of interstitial liquid was imaged by both phase-contrast and fluorescence microscopy at 10 min intervals. The field becomes increasingly infiltrated by rods over time, which eventually become immobilised, apparently due to overcrowding. The immobilised rods eventually show stable polar localisation of CetZ1-GFP, suggesting that CetZ1 recognises a feature of the pole after initial rod development has occurred, and might therefore have an ongoing role there. Also evident are plate cells undergoing division, showing dynamic CetZ1-GFP localisation around the cells and at the division furrow before and during constriction. Note that fluorescence from CetZ1-GFP increases over time, as seen in Supplementary Video 3—this might reflect increased expression from the tnaA promoter under these conditions.

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https://doi.org/10.1038/nature13983

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