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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Löwe, J. & Amos, L. A. Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes. Int. J. Biochem. Cell Biol. 41, 323–329 (2009)
Wickstead, B. & Gull, K. The evolution of the cytoskeleton. J. Cell Biol. 194, 513–525 (2011)
Woese, C. R., Kandler, O. & Wheelis, M. L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl Acad. Sci. USA 87, 4576–4579 (1990)
Baumann, P. & Jackson, S. P. An archaebacterial homologue of the essential eubacterial cell division protein FtsZ. Proc. Natl Acad. Sci. USA 93, 6726–6730 (1996)
Löwe, J. & Amos, L. A. Crystal structure of the bacterial cell-division protein FtsZ. Nature 391, 203–206 (1998)
Margolin, W., Wang, R. & Kumar, M. Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin. J. Bacteriol. 178, 1320–1327 (1996)
Poplawski, A., Gullbrand, B. & Bernander, R. The ftsZ gene of Haloferax mediterranei: sequence, conserved gene order, and visualization of the FtsZ ring. Gene 242, 357–367 (2000)
Wang, X. & Lutkenhaus, J. FtsZ ring: the eubacterial division apparatus conserved in archaebacteria. Mol. Microbiol. 21, 313–320 (1996)
Vaughan, S., Wickstead, B., Gull, K. & Addinall, S. G. Molecular evolution of FtsZ protein sequences encoded within the genomes of Archaea, Bacteria, and Eukaryota. J. Mol. Evol. 58, 19–29 (2004)
Oren, A. Molecular ecology of extremely halophilic Archaea and Bacteria. FEMS Microbiol. Ecol. 39, 1–7 (2002)
Burns, D. G. et al. Haloquadratum walsbyi gen. nov., sp. nov., the square haloarchaeon of Walsby, isolated from saltern crystallizers in Australia and Spain. Int. J. Syst. Evol. Microbiol. 57, 387–392 (2007)
Walsby, A. E. Square bacterium. Nature 283, 69–71 (1980)
Takashina, T., Hamamoto, T., Otozai, K., Grant, W. D. & Horikoshi, K. Haloarcula japonica sp. nov., a new triangular halophilic archaebacterium. Syst. Appl. Microbiol. 13, 177–181 (1990)
Mullakhanbhai, M. F. & Larsen, H. Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch. Microbiol. 104, 207–214 (1975)
Marchler-Bauer, A. et al. CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res. 41, D348–D352 (2013)
Yutin, N. & Koonin, E. V. Archaeal origin of tubulin. Biol. Direct 7, 10 (2012)
Löwe, J., Li, H., Downing, K. H. & Nogales, E. Refined structure of αβ-tubulin at 3.5 Å resolution. J. Mol. Biol. 313, 1045–1057 (2001)
Richards, K. L. et al. Structure-function relationships in yeast tubulins. Mol. Biol. Cell 11, 1887–1903 (2000)
Scheffers, D. J., de Wit, J. G., den Blaauwen, T. & Driessen, A. J. GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers. Biochemistry 41, 521–529 (2002)
Be'er, A., Florin, E. L., Fisher, C. R., Swinney, H. L. & Payne, S. M. Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis. MBio 2, e00069–11 (2011)
Allers, T., Barak, S., Liddell, S., Wardell, K. & Mevarech, M. Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii. Appl. Environ. Microbiol. 76, 1759–1769 (2010)
Large, A. et al. Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene. Mol. Microbiol. 66, 1092–1106 (2007)
Kimble, M., Kuzmiak, C., McGovern, K. N. & de Hostos, E. L. Microtubule organization and the effects of GFP-tubulin expression in Dictyostelium discoideum. Cell Motil. Cytoskeleton 47, 48–62 (2000)
Sun, Q. & Margolin, W. FtsZ dynamics during the division cycle of live Escherichia coli cells. J. Bacteriol. 180, 2050–2056 (1998)
Cooper, S. & Denny, M. W. A conjecture on the relationship of bacterial shape to motility in rod-shaped bacteria. FEMS Microbiol. Lett. 148, 227–231 (1997)
Dusenbery, D. B. Fitness landscapes for effects of shape on chemotaxis and other behaviors of bacteria. J. Bacteriol. 180, 5978–5983 (1998)
Young, K. D. The selective value of bacterial shape. Microbiol. Mol. Biol. Rev. 70, 660–703 (2006)
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004)
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2–approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010)
Miroux, B. & Walker, J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996)
Stock, D., Perisic, O. & Löwe, J. Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology. Prog. Biophys. Mol. Biol. 88, 311–327 (2005)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)
CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Turk, D. MAIN software for density averaging, model building, structure refinement and validation. Acta Crystallogr. D 69, 1342–1357 (2013)
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Allers, T., Ngo, H. P., Mevarech, M. & Lloyd, R. G. Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl. Environ. Microbiol. 70, 943–953 (2004)
Reuter, C. J. & Maupin-Furlow, J. A. Analysis of proteasome-dependent proteolysis in Haloferax volcanii cells, using short-lived green fluorescent proteins. Appl. Environ. Microbiol. 70, 7530–7538 (2004)
Hartman, A. L. et al. The complete genome sequence of Haloferax volcanii DS2, a model archaeon. PLoS ONE 5, e9605 (2010)
Wendoloski, D., Ferrer, C. & Dyall-Smith, M. L. A new simvastatin (mevinolin)-resistance marker from Haloarcula hispanica and a new Haloferax volcanii strain cured of plasmid pHV2. Microbiology 147, 959–964 (2001)
Tripepi, M., Imam, S. & Pohlschroder, M. Haloferax volcanii flagella are required for motility but are not involved in PibD-dependent surface adhesion. J. Bacteriol. 192, 3093–3102 (2010)
Strauss, M. P. et al. 3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis. PLoS Biol. 10, e1001389 (2012)
Gustafsson, M. G. L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008)
Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008)
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671–675 (2012)
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005)
Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996)
Landgraf, D., Okumus, B., Chien, P., Baker, T. A. & Paulsson, J. Segregation of molecules at cell division reveals native protein localization. Nature Methods 9, 480–482 (2012)
Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)
Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004)
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).
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 CetZ2–GTP-γS crystals contain protofilaments and 2D sheets.
a, Expansion of crystal symmetry (PDB 4B45) shows lateral association of tubulin/FtsZ-like protofilaments. The lateral repeat distance of 42.4 Å is slightly shorter than the longitudinal distance (43.2 Å). There is similar packing to the lattice of tubulin dimers in the microtubule wall, although a specific M-loop facilitates the lateral contact in tubulin. CetZ2 shows less intimate contacts between GTPase domains (green) in the protofilament compared to tubulin. b, 90°-rotated view along the protofilaments. Protofilaments are arranged in the same orientation, creating two different surfaces of the sheet. The H11 helix and C termini are located on the lower surface as shown, and would have the potential to interact with other molecules or surfaces (as seen with the C-terminal tail of FtsZ).
Extended Data Figure 2 Tubulin-superfamily proteins in Euryarchaeota that contain CetZ.
CetZ1 from H. volcanii (HVO_2204, Uniprot D4GVD7) was used in PSI-BLAST searches to identify tubulin-superfamily members in Euryarchaeota. A representative genome from all genera determined to contain a CetZ or non-canonical CetZ was selected (if available), and then all identified superfamily members from these genomes were aligned for generating the phylogram. Bootstrap support is shown for selected branches. The positions of the H. volcanii CetZ proteins are indicated (HvCetZ). All species identified to contain a CetZ contained at least one FtsZ; most contained two FtsZ proteins from distinct sub-families. CetZ1 orthologues were identified in all species of Halobacteria analysed, and typically shared ∼80% sequence identity. CetZ2 orthologues were identified in many of these. A number of highly diverse sequences containing recognizable tubulin-superfamily signature motifs (Fig. 1b) were identified in Euryarchaeota, including the classes Thermococci, Methanomicrobia and Halobacteria (notably, in the rectangular prism-shaped Haloquadratum walsbyi (Hwa)). These were difficult to classify robustly; however, most were found to branch from near the base of the CetZ-family bough and were referred to as non-canonical CetZs.
Extended Data Figure 3 The role of CetZ1 in H. volcanii motility.
a, b, Motility assays of the ΔcetZ1 strain (ID59+pTA962) were performed with closely-spaced (a) or standard-spaced (b) inoculation sites. Exclusion zones were observed between halos after extended incubation (16 days). Irregularities in halo structures were seen in wild-type and mutant halos, which may be related to colony exclusion (repulsion or inhibition) between local clouds of motile cells. c, Motility agar plates (containing 0.2 mM Trp), were imaged once daily over 4 days after inoculation. Strains containing wild-type CetZ1 (H98+pTA962 and H98+pTA962-cetZ1) show an abrupt development of motile halos from day 3. Strains containing cetZ1 mutations (H98+pTA962-cetZ1(E218A) and ID59+pTA962 (ΔcetZ1)), were strongly inhibited in motility. d, A halo of wild-type H. volcanii (H98+pTA962) is shown in low and high contrast, showing the high-motility fainter outer zone and the denser population of cells growing more centrally. Samples were withdrawn from the indicated positions (1–4) of the motility halo for microscopy. e, f, Cell circularity distributions (e) were then generated from automated computer analysis of images of cells (f) sampled at the four positions.
Extended Data Figure 4 H. volcanii cell shape is sensitive to CetZ1 and CetZ1(E218A) concentration.
a, Phase-contrast images of wild-type (H98+pTA962), CetZ1-overproduction (H98+pTA962-cetZ1) and CetZ1(E218A)-overproduction (H98+pTA962-cetZ1(E218A)) strains in mid-log growth in Hv-Ca medium containing the indicated concentrations of Trp. b, c, Images of the strains were analysed for cell circularity (b) and the relative standard deviation (RSD) of curvature (c) of cell outlines. The wild-type (WT) control (H98+pTA962, 4 mM Trp) is shown in both b and c. At greater than 2 mM Trp, the circularity and curvature parameters did not increase further. However, during CetZ1(E218A) overproduction (4 mM Trp) cells appeared to show slightly larger highly distorted cells with more noticeable ruffling and protrusions (a, bottom right image). d, Coulter cell volume analysis of the strains confirmed that CetZ1(E218A) overproduction (4 mM Trp) showed slightly larger cells, indicating delayed cell division—a possible effect of significant cell distortion. e, Western blot analysis of H98+pTA962-cetZ1 (+CetZ1) and H98+pTA962-cetZ1(E218A) (+E218A) sampled during mid-log growth in Hv-Ca medium with the indicated concentrations of Trp. Ponceau S pre-staining of the membrane for total protein, as a loading reference, is shown below the CetZ1 western blot. The levels of CetZ1 increase in response to increasing concentrations of Trp in the medium. CetZ1(E218A) showed consistently higher levels compared to wild-type CetZ1, consistent with protofilament hyper-stability resulting in a slower rate of in vivo degradation of CetZ1(E218A) (see also Fig. 2a).
Extended Data Figure 5 Localization of CetZ1–GFP and variants, CetZ1–GFP(A206K) and CetZ1–mCherry, in H. volcanii.
CetZ1 tagged with the GFP variant used primarily in this study, smRS-GFP40 was compared to CetZ1 tagged with the A206K mutant of smRS-GFP (which blocks GFP self-association51,52) and the mCherry protein51,53 (which shows red fluorescence in H. volcanii). Strains expressing the indicated CetZ1 fusion proteins in pTA962-based plasmids were sampled during mid-log phase growth in Hv-Ca + 0.2 mM Trp (log-phase cells) and from the leading edge of motile halos (0.2 mM Trp).
Extended Data Figure 6 Expression of CetZ1–GFP and CetZ1(E218A)–GFP in H. volcanii.
a, Phase-contrast (left) GFP-fluorescence (middle), and phase/GFP overlay (right) images of strains H98+pIDJL40 (GFP vector only), H98+pIDJL40-cetZ1 (+CetZ1–GFP) and H98+pIDJL40-cetZ1(E218A) (+CetZ1(E218A)–GFP) sampled from mid-log growth in strong-overproduction conditions (Hv-Ca + 4 mM Trp). The GFP-vector-only fluorescence image is high-contrast, showing low-level background GFP fluorescence. The CetZ1–GFP and CetZ1(E218A)–GFP fluorescence images are presented with identical exposure and contrast settings, showing relatively higher background fluorescence for CetZ1–GFP and higher localized fluorescence for CetZ1(E218A)–GFP, indicating that assembly of the localized protein is stabilized by the E218A mutation (in the GTPase domain of CetZ). b, Cell circularity distributions for the indicated strains show no stimulation of rod shape during overproduction of either CetZ1–GFP or CetZ1(E218A)–GFP. c, Cell curvature distributions indicate that overproduction of CetZ1(E218A)–GFP results in blocky, stalked cells, as seen with untagged CetZ1(E218A) (Extended Data Fig. 4). d, Western blot analysis of CetZ1 in the indicated strains, showing increasing expression of CetZ1-GFP fusion proteins in response to Trp concentration. e, Motility assays for the indicated strains (H98 background, 0.2 mM Trp), showing significant inhibition of motility by CetZ1(E218A); this may result from higher level production of this protein (see Extended Data Fig. 4e), or stronger inhibition of native CetZ1 function. f, Cell circularity analysis of cells at the leading edge of the halos in e suggests that strong inhibition of rod shape is needed to see an obvious reduction in motility. (Data for wild-type and +CetZ1(E218A) strains are reproduced from Fig. 2h.) g, Cell outlines resulting from the automated cell detection process in phase-contrast and the corresponding GFP-fluorescence images. Magnified images show the normal lines (3 pixels long) on the inside of the cell outline that were integrated to measure fluorescence near the envelope (the closely spaced normal lines give the appearance of a thick outline of the cell). Sections of high curvature (>2 standard deviations above the mean) are shown in green, whereas the remainder is shown in red. h, A plot of the curvature (red) and the GFP localization (green) of the magnified cell, measured anticlockwise from the arrowhead. In automated analysis of cell populations, the curvature of each whole cell was calculated as the relative standard deviation of this line. Analysis of the strain expressing CetZ1(E218A)–GFP (in Hv-Ca + 2 mM Trp) showed a 73% greater mean fluorescence in areas of high curvature versus the whole cell, 53% greater fluorescence for areas of high curvature versus the whole perimeter, and 10% greater fluorescence for the perimeter versus the whole cell.
Extended Data Figure 7 Electron cryotomography of H. volcanii.
Cells growing in Hv-YPC + 4 mM Trp were frozen in vitreous ice to maintain native structure, and then tomographic data were acquired with a 300 kV cryo-transmission electron microscope. A 10 nm section from the tomographic reconstruction of the whole cell is shown for each. a, H98+pTA962 (wild-type) cell, showing cell envelope layers with some structural detail of the outer S-layer visible, a dense granule, particles consistent with ribosomes visible in the outer ∼25% of the cytoplasmic region, and fibres that may be seen underlying the cell envelope. b, Wild-type cell, with detail of the cell envelope region (inset as per Fig. 4f). c, Cells overproducing CetZ1(E218A) (H98+pTA962-cetZ1(E218A)), show extensive sections of the envelope with underlying fibres (inset as per Fig. 4f). (Supplementary Video 5 shows a segment image series through a cell, showing the sheet-like structure of the extra layer.) d, Example showing subtle ruffling of the cell envelope associated with the additional envelope fibres in a cell producing CetZ1(E218A). Scale bars, 100 nm for all panels, and 50 nm for all insets.
Extended Data Figure 8 Expression of CetZ2(E212A) affects H. volcanii cell shape and motility.
The T7-loop mutation CetZ2(E212A) is equivalent to CetZ1(E218A). a, Expression of CetZ2(E212A) in motility assays, by addition of increasing concentrations of Trp, inhibits motility, but to a lesser degree than CetZ1(E218A). b, Cells were withdrawn from the leading edge of the indicated motile halos. Moderate expression of CetZ2(E212A) (0.2 mM Trp) partially inhibits rod development, and higher expression (1 mM Trp) strongly inhibits rod formation and produces cells with sharp corners and regions of high curvature, similar to cells expressing CetZ1(E218A) (Fig. 4). CetZ2(E218A) is therefore dominant-inhibitory to rod development. c, Coulter cell volume analysis of cells withdrawn from mid-log cultures of wild-type (H98 + pTA962), CetZ2-overproducing (H98 + pTA962-cetZ2) and CetZ2(E212A)-overproducing (H98 + pTA962-cetZ2(E212A)) strains shows that overexpression of CetZ2 or CetZ2(E212A) does not affect cell division. d, Phase-contrast images of cells from the same cultures shown in c. Data obtained from automated analysis of images for each strain is shown in the plots in d. Unlike CetZ1 (Fig. 4), overexpression of CetZ2 in liquid culture did not stimulate rod morphology, indicating that CetZ2 is not a dominant driver of rod formation. However, analysis of cell curvature (bottom right) showed that expression of CetZ2(E212A) produced a high-curvature phenotype, similar to CetZ1(E218A) (Fig. 4), indicating that this mutant specifically interferes with shape regulation in H. volcanii.
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.) (MOV 1697 kb)
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. (MOV 462 kb)
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). (MOV 1035 kb)
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. (MOV 93 kb)
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.) (MOV 6237 kb)
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). (MOV 282 kb)
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. (MOV 1893 kb)
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. (MOV 278 kb)
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. (MOV 635 kb)
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. (MOV 8102 kb)
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Duggin, I., Aylett, C., Walsh, J. et al. CetZ tubulin-like proteins control archaeal cell shape. Nature 519, 362–365 (2015). https://doi.org/10.1038/nature13983
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