Defining the rate-limiting processes of bacterial cytokinesis

Abstract

Bacterial cytokinesis is accomplished by the essential 'divisome' machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the 'FtsZ-ring' ('Z-ring'). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ's GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome-chromosome coupling. These results challenge a Z-ring-centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.

Keywords: FtsZ; cell wall synthesis; cytokinesis; force generation; superresolution.

Fig. 1.

The E. coli Z-ring in 3D. (A) Two-dimensional projections of iPALM images of three fixed E. coli DH5α cells expressing FtsZ-mEos2. (A, Upper) xz plane. (A, Lower) xy plane. Arrows illustrate cytoplasmic (cyan) and membrane-proximal (white) clusters of FtsZ-mEos2 outside the Z-ring. Cell outlines are approximated by white dotted lines. (B) Cropped midcell regions of DH5α (Left) and BW25113 (Right) cells showing Z-rings projected on yz planes. Fitted ring diameters are shown below each Z-ring. Rings outlined in dashed boxes correspond to those shown in A. (C) Distributions of resolution-deconvolved Z-ring cross-sectional width (blue) and thickness (red) measured from iPALM images of FtsZ-mEos2 in DH5α and BW25113 cells (n = 168). Each histogram bin width is 20 nm. The inset schematic shows the relative orientations of Z-ring thickness (along the cell radius) and Z-ring width (along the cell long axis). (D) Resolution-deconvolved Z-ring width (blue) and thickness (red) plotted against corresponding Z-ring diameter (n = 168). Measurements for individual cells are shown as small, transparent, filled circles. Average measurements for cells binned by ring diameter (bin edges: 0, 300, 400, 500, 600, 700, 800, and 1,000 nm) are shown as larger filled circles with black outlines. Error bars represent SEM. The image axes are as follows: x axis, cell long axis; y axis, cell short axis parallel to the imaging plane; and z axis, cell short axis perpendicular to the imaging plane. (Scale bars, 300 nm.)

Fig. 2.

Time-lapse analysis of Z-ring dynamics during the cell cycle. (A) Time-lapse fluorescence (Upper) and bright-field (Lower) montage of BW25113 cells expressing FtsZ-GFP. Arrows indicate cell cycle landmarks for the top cell: arrow i, cell birth; arrow ii, ring stabilization; arrow iii, onset of visible constriction; arrow iv, beginning of Z-ring disassembly; and arrow v, completion of cell division. The time stamp (Upper Right) is displayed as minutes relative to cell birth. (B) Time-dependent Z-ring fluorescence (percentage of fluorescence in the ring relative to that of the whole cell, p_mid) during the cell cycle of the top cell shown in A. (C) Averaged durations of each cell cycle period for BW25113 cells growing in M9 medium at RT (means ± SE; n = 98). (Scale bar, 1 μm.)

Fig. 3.

Analysis of Z-ring remodeling during constriction and time-dependent septum closure rate for BW25113 cells. (A–C) Bright-field (A), conventional green fluorescence (B), and superresolution PALM (C) images of live BW25113 cells expressing FtsZ-mEos2 grouped by measured Z-ring diameter. Cell outlines are approximated by dashed lines. (D) PALM images of live BW25113 cells expressing mEos2-ZapA grouped by ring diameter as in C. (E–G) Resolution-deconvolved ring width (E), intensity (p_mid) (F), and density (G) in BW25113 cells expressing FtsZ-mEos2 (filled; n = 66) or mEos2-ZapA (empty; n = 82) plotted against and binned by corresponding ring diameter. Bin edges are as follows: 0, 200, 300, 400, 500, and 600 nm. Errors bars represent SEM. (H) Equation describing the monotonic decrease in Z-ring diameter over time (Upper) and table describing different α regimes for the rates of septum diameter closure, ΔD/Δt, and septum surface area addition, ΔA/Δt (Lower). (I) Z-ring intensity, p_mid, plotted against ring diameter as in F overlaid with best-fit (solid black; α = 1.3 ± 0.1), α = 1 (dashed gray), and α = 2 (dotted gray) models generated from time-lapse parameters of BW25113 cells (SI Appendix, Table S2) using the model in H. The best-fit model is consistent with acceleration in septum closure rate. (J and K) Time-dependent change in septum area [A(t)] (J, magenta), septum diameter [D(t)] (J, cyan), rate of septum area addition (ΔA/Δt) (K, magenta), and rate of septum diameter closure (ΔD/Δt) (K, cyan) for the models shown in I, assuming that growing septa resemble hemispherical segments and using an average septum closure time of 51 min (SI Appendix, Table S2): best fit (solid), α = 1 (dashed), and α = 2 (dotted). (Scale bars, 1 μm.)

Fig. 4.

Analysis of Z-ring remodeling during constriction and time-dependent septum closure rate for MC4100 and MCZ84 cells. (A and B) Superresolution PALM images of live MC4100 (A) and MCZ84 (B) cells expressing FtsZ-mEos2 or FtsZ84-mEos2, respectively, grouped by measured Z-ring diameter. Cell outlines are approximated by dashed lines. (C–F) Resolution-deconvolved ring width (C), density (D), and intensity (p_mid) (E and F) in MC4100 (black; n = 83) and MCZ84 (red; n = 154) cells expressing FtsZ-mEos2 or FtsZ84-mEos2, respectively, plotted against and binned by corresponding ring diameter. Bin edges are as follows: 0, 200, 300, 400, 500, and 600 nm. Errors bars represent SEM. Z-ring intensity measurements of FtsZ-mEos2 in MC4100 (E) and FtsZ84-mEos2 in MCZ84 (F) are overlaid with best fit (solid), α = 1 (dashed gray), and α = 2 (dotted gray) using the models in Fig. 3H and corresponding time-lapse parameters (SI Appendix, Table S2). (Scale bar, 1 μm.)

Fig. 5.

Analysis of Z-ring remodeling during constriction and time-dependent septum closure rate for ΔminC and ΔmatP cells. (A and B) Superresolution PALM images of live ΔminC (A) and ΔmatP (B) mutants of BW25113 expressing FtsZ-mEos2, grouped by measured Z-ring diameter. Cell outlines are approximated by dashed lines. (C–F) Resolution-deconvolved ring width (C), density (D), and intensity (p_mid) (E and F) in ΔminC (purple; n = 73), ΔmatP (green; n = 68), or wt (gray; n = 66) BW25113 cells expressing FtsZ-mEos2 plotted against and binned by corresponding ring diameter. Bin edges are as follows: 0, 200, 300, 400, and 500 nm. Errors bars represent SEM. Z-ring intensity measurements in E and F are overlaid with best-fit (solid line) models generated from time-lapse parameters of corresponding strain (SI Appendix, Table S2). Results for wt BW25113 (gray) are replicated from Fig. 3 for comparison. (Scale bar, 1 μm.)

Fig. 6.

Comparison of septum closure and cell elongation rates. (A and B) Average septum closure rate (<v_c>) plotted against cell elongation rate before (<v_ep>) (A) or during (<v_ec>) (B) constriction for all wt E. coli strains (gray circles), with associated linear trend lines (gray lines) [trend line in A: y = 1.14x + 3.75 (r_Pearson = 0.98, P = 0.02); trend line in B: y = 0.91x + 1.33 (r_Pearson = 0.99, P = 0.01)]. (C and D) Average ratio of septum closure rate relative to cell elongation rate before (<v_c/v_ep>) (A) or during (<v_c/v_ec>) (B) constriction for all wt (gray) and mutant (colored) E. coli strains. The dotted line in D illustrates <v_c/v_ec> = 1 ratio. (E) Schematic depicting the correlation between cell elongation rate and septum closure rate in wt cells under different growth conditions (wt BW25113 and MC4100 cells) (Left) and the anticorrelation between cell elongation rate and septum closure rate when the balance between cell wall synthesis and septum closure is perturbed under the same growth condition (Upper, ΔmatP vs. wt BW25113 cells; Lower, MCI23 vs. wt MC4100 cells).