Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis

Abstract

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram-negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ-like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ-driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.

Keywords: Caulobacter crescentus; FtsZ; asymmetric division; bacterial cell division; electron cryotomography.

Figure 1. Examples of eight different bacterial species that exhibit asymmetric early‐constriction

1. Representative central slices of tomograms of eight different constricted cells are shown, arranged so that the asymmetric division site is on the right (indicated by white arrows). Scale bars, 100 nm.

2. Table showing the numbers of cells observed constricting asymmetrically and symmetrically for each species.

Figure EV1. Additional examples of cell shapes throughout the constriction process

Central slices of representative 3D tomographic reconstructions of Halothiobacillus. neapolitanus c2, Legionella pneumophila, a Belliella baltica‐related strain, Ralstonia eutropha, and Thiomonas intermedia cells are shown. For each species, cells are arranged in order of presumed cell division progress (from left to right). Scale bars, 100 nm.

Figure 2. Atlas of dividing P. mirabilis cells imaged in this study

Central slices of tomograms or projection images (marked by red asterisks) of P. mirabilis are sorted according to W _mid/W. Numbers and labels, as well as color scheme, correspond to Fig EV2A. White arrows denote indentations detected by the program on the indicated side. The three cells numbered 11, 26, and 31 correspond to cell 11 in Fig 5A and cells 26 and 31 in Fig 6A, respectively. The number of FtsZ‐like filaments observed on each side of the division plane (left: right) is noted below the corresponding image. If no number is given, no filaments were observed, meaning either that there were none or that we could not resolve them. Scale bar, 300 nm.

Figure 3. Atlas of all Caulobacter crescentus cells imaged in this study

Central slices of tomograms of C. crescentus are sorted according to W _mid/W. Cells are arranged with the inner curvature on the left and the outer curvature on the right. Numbers and labels, as well as color scheme, correspond to Fig EV2B. White arrows denote indentations detected by the program. Measurements from the last three cells (47–49) are omitted. Pink arrows indicate filaments formed by CTP synthase (Ingerson‐Mahar et al, 2010). The six cells numbered 17, 24, 32, 34, 38, and 42 correspond to cell 32 in Fig 5B, cells 38 and 42 in Fig 6B, and cells 17, 24 and 34 in Fig EV4, respectively. The number of FtsZ‐like filaments observed on each side of the division plane (left: right) is noted below the corresponding image. Scale bars, 100 nm.

Figure EV2. Indentation length (L) and curvature sharpness (1/r) of P. mirabilis and C. crescentus cells increase as W _mid/W decreases throughout division

1. A, B

   Indentation length (L) and curvature sharpness (1/r) of P. mirabilis (A) and C. crescentus (B) cells increase as W _mid/W decreases throughout division. The circles represent the measured values from the left sides; the triangles denote the measured values from the right sides. Note that these assignments are arbitrary for straight P. mirabilis cells. Colors in (A and B) indicate the corresponding numbers in Figs 2 and 3, respectively. The purple dashed boxes at left highlight cells with detectable indentation only on one side. The red dashed lines at right are the best‐fit line, showing a high degree of correlation. “Unconstricted”, “early”, and “mid” denote the pre‐constriction stage, early‐constriction stage, and mid‐constriction stages in cell division. These stages are roughly determined by W _mid/W.

Figure 4. Localization of new PG incorporation

1. A, B

   Localization of new PG incorporation in different stages of C. crescentus (A) and P. mirabilis (B) cytokinesis. In each panel, the upper row shows the fluorescence (HADA) images of representative cells in different cell division stages overlaid with phase contrast images showing the cell profiles. The bottom rows show fluorescence profiles of the division plane of the indicated early‐constriction and mid‐constriction stage cells. The numbers in (A) and (B) are 1/10 values of the 8‐bit gray values measured using the NIH ImageJ software. Scale bars, 1 μm.

Figure EV3. Cell length versus HADA labeling phenotype

1. A, B

   For C. crescentus (A) and P. mirabilis (B) cells labeled with HADA, the cell length statistics for different stages are shown. For the unconstricted, mid‐constriction, and late‐constriction stages, the cell length was measured in 15 randomly selected cells, and the mean and standard deviation (bars) are plotted using the indicated symbols. The cell lengths of all observed early‐constriction stage cells with one‐sided HADA incorporation are plotted with green diamonds.

Figure 5. Short FtsZ‐like filaments accompany early asymmetric cell constriction

1. A–E

   Short FtsZ‐like filaments accompany early asymmetric cell constriction in P. mirabilis (A), C. crescentus (B), H. neapolitanus c2 (C), L. pneumophila (D), and S. enterica spp. enterica minicells (E). The two cells in (A) and (B) correspond to cells nos. 11 and 32 in Figs 2 and 3, respectively. A central x‐y slice is shown at left. White arrows highlight the asymmetric constriction and indicate the plane of the cross‐sectional x‐z slice shown at right. The limits of visibility or apparent ends of continuous FtsZ‐like filaments are marked by yellow arrows. OM, outer membrane; IM, inner membrane. Scale bars, 100 nm.

Figure 6. FtsZ‐like filament localization in mid‐constriction stages

1. A–D

   FtsZ‐like filament localization in mid‐constriction stages of P. mirabilis (A), C. crescentus (B), H. neapolitanus c2 (C), and S. enterica spp. enterica minicells (D) division. Representative central x‐y slices of 3D tomographic reconstructions of mid‐constriction stage cells are shown on the left. Cell orientation is the same as in Fig 3. White arrows highlight the constriction sites and indicate the cross‐sectional x‐z slice plane. In (B), enlarged views of x‐y slices of both the inner (left) and outer (right) curvature of the division site are shown, with yellow arrows indicating FtsZ‐like filament cross sections. In all panels, the middle column shows an x‐z slice of the division site. Yellow arrows indicate FtsZ‐like filaments. Segmentations are shown at right, highlighting the inner (yellow) and outer (purple) membranes, and all FtsZ‐like filaments observed (red). The two cells in (A) correspond to cells nos. 26 and 31 in Fig 2; the two cells in (B) correspond to nos. 38 and 42 in Fig 3. Scale bars, 50 nm in second column in (B); all other scale bars, 100 nm; scale is the same in upper and lower panels in (A) and (B); segmentations not to scale.

Figure EV4. Additional examples of FtsZ filaments on the outer curvature of C. crescentus cells in the early‐constriction stage of division

The three cells correspond to numbers 17, 24, and 34 in Fig 3. The panel arrangement and color scheme are the same as in Fig 6. The yellow arrows in segmentations indicate FtsZ‐like filaments. Scale bars: 50 nm in Column 2; 100 nm in columns 1 and 3; segmentations in column 4 not to scale.

Figure 7. Model of FtsZ‐driven PG synthesis and constriction in P. mirabilis and C. crescentus cytokinesis

1. A, B

   The diagrams show progression through cell division of P. mirabilis (A) and C. crescentus (B). The cell envelope profiles were drawn according to the experimental images. FtsZ filaments are shown in red, new PG incorporation in green, and outer and inner membranes in purple and yellow, respectively. In the early‐constriction stage, short arc‐like FtsZ filaments assemble on one side of the cell, recruiting PG synthesis and driving initial, asymmetric constriction of the cell envelope. Later, FtsZ filaments accumulate, forming a bundle of overlapping filaments that covers the entire division plane in the mid‐constriction stage. Finally, these highly dynamic filaments drive PG synthesis and constriction symmetrically around the division plane until cytokinesis is complete.