FtsZ structure and dynamics in Z-rings are not sensitive to increased ring size

As a reference for unmodified division rings, we imaged Z-rings in E. coli cells expressing FtsZ-mNeonGreen as the only source of FtsZ22. Under our experimental conditions, this strain produced normal-looking, sharp Z-rings (Supplementary Figure 1) and grew and divided similarly to wild-type (WT) E. coli (MC4100) (Supplementary Figure 2a-e). We then trapped the cells in a vertical position in micron-sized holes that were produced in agarose pads using silica micron pillar arrays14 (Fig. 1b, Supplementary Figure 3), and imaged the cells using super-resolution time-gated STimulated Emission Depletion (gSTED) nanoscopy. In these standing cells, a heterogeneous Z-ring with distinct FtsZ-mNeonGreen clusters was clearly seen traversing the circumference of the cell (Fig. 1c), similar to what has been observed before12,14.

Previous work has shown that FtsZ clusters generally maintain the same length throughout envelope constriction12,14. We wanted to see if this was also true for unnaturally large cells, i.e., would FtsZ clusters maintain the same dimensions in Z-rings of cells with increased diameter at midcell? In order to increase cell diameter, we treated E. coli cells with A22 and cephalexin (hereafter collectively referred to as “drugs”), in a way similar to what has previously proven successful for cell shape manipulations23. A22 disrupts MreB dynamics and therefore perturbs the characteristic rod-shape of E. coli cells19,24, while cephalexin blocks cell division by inhibiting the transpeptidase activity of FtsI25. The net effect of this dual drug treatment is the growth of cells into shapeable blebs that are unable to divide (Supplementary Figure 4a).

We hypothesized that as long as cell width remains less than cell length, FtsZ molecules should be directed to midcell by the Min system23 and other FtsZ placements systems26, such that a ring-like structure may be observed in the xy-plane of vertically-oriented, standing cells (Fig. 1b). To test this, we exposed E. coli cells expressing FtsZ-mNeonGreen to drugs, and then trapped the cells vertically in holes with a diameter of up to 3.5 μm and a depth of 4.5–6 μm. It has been shown that reshaped E. coli cells longer than 6 μm may revert from pole-to-pole oscillations to high mode oscillations23. Depending on the size of the holes, cells were incubated between 120 and 240 min prior to imaging; over-incubation resulted in cells that outgrew the holes (Supplementary Figure 4b. Allowing cells to grow for longer times (>10 h) produced giant blobs with internalized FtsZ-mNeonGreen chains, see Supplementary Note 1, Supplementary Figures 5 and 6).

The resulting Z-rings in drug-treated cells spanned the midcell circumference for all cell diameters that were imaged (~ 1–3 μm) (Fig. 1d, e). Fluorescence intensity increased as ring size increased (Supplementary Figure 7), possibly indicating an upregulation of cellular FtsZ expression, assuming similar ratios of ring to non-ring associated FtsZ molecules in all cell sizes (~30% of FtsZ molecules are in the Z-ring27). Importantly, confocal Z-stacks showed that each cell contained only one Z-ring (Supplementary Figure 8 and Supplementary Movie 1). Close inspection of STED images revealed that the Z-rings in larger cells were composed of fluorescent clusters (Fig. 1f) with average lengths and radial widths of 132 ± 48 nm and 88 ± 9 nm (mean ± S.D., n = 172), respectively, which were similar to Z-ring clusters in untreated cells (p > 0.05) (Fig. 1g).

After we had established that large Z-rings can form in cells with increased diameter, we proceeded to calculate FtsZ dynamics in these larger rings. However, strains expressing FtsZ-FP (fluorescent protein (FP), e.g., mNeonGreen) as the only source of FtsZ have been shown to have a phenotype similar to that of FtsZ mutants deficient in GTPase activity, with severely impaired treadmilling speed13. Therefore, we chose to image cells that expressed FtsZ-GFP from an ectopic locus on the chromosome, in addition to native FtsZ28. Earlier studies showed that FtsZ-GFP, when expressed at levels below 50% of total cellular FtsZ levels, caused no observable phenotypic changes9,12,28,29. In our experimental setup, FtsZ-GFP was expressed at ~ 30% of total FtsZ levels (Supplementary Figure 2).

SIM of drug-treated E. coli cells expressing FtsZ-GFP showed large heterogeneous rings that were similar to those of FtsZ-mNeonGreen (Fig. 1h–k). Time-lapse imaging revealed that FtsZ clusters moved around the midcell circumference, even in Z-rings with a diameter up to three times larger than that of a WT cell (Supplementary Movie 2). There was no difference in the speed of individual clusters in the rings of untreated cells compared to those in sculpted cells that had a diameter 50–200% larger than WT (26 ± 15 nm s−1 (mean ± S.D., n = 102) and 30 ± 18 nm s−1 (mean ± S.D., n = 102), respectively) (Fig. 1l–m), suggesting that cluster treadmilling speed is not influenced by the length of the cell circumference. ZipA-GFP, an FtsZ membrane anchor, also moved at essentially the same speed in both normal-sized and large-sized rings (26 ± 8 nm s−1, mean ± S.D., n = 10) (Supplementary Figure 9 and Supplementary Movie 3), which is comparable to previously reported speeds14.

Since treadmilling behavior of FtsZ in large cells was very similar to that in WT cells, we were curious to see whether FtsZ subunit exchange in the rings would also be similar. To assess this, we performed Fluorescence Recovery After Photobleaching (FRAP) experiments on both untreated and drug-treated cells. We bleached half of the FtsZ-GFP molecules in the rings of standing cells and monitored fluorescence recovery over time (Fig. 2a). Z-rings in untreated cells had a mean t 1/2 recovery time of 8.4 ± 1.9 s (mean ± S.D., n = 23) (Fig. 2b), consistent with previous studies14,30. We found that the average t 1/2 recovery time was the same for Z-rings with a wide range of diameters (Fig. 2b), indicating that the rate of FtsZ subunit exchange is independent of cell diameter. This further suggests that cell size, and hence membrane curvature, might not be a factor in determining Z-ring dynamics.

Fig. 2 Cell size independent recovery of fluorescence in FtsZ-rings. FRAP measurements on FtsZ-GFP rings in E. coli cells trapped standing in a vertical position. a Representative cells of different diameter, untreated or treated with drugs. White boxes indicate bleached areas. Scale bars = 1 μm. b Quantification of FRAP data from untreated cells (red), and drug-treated cells (other colors), showing that fluorescence recovery time is independent of cell diameter (in the range investigated, i.e., ~ 1–3 μm). n tot = 105. D = cell diameter range. Mean t 1/2 recovery time WT FtsZ-rings was 8.4 ± 1.9 s (mean ± S.D., n = 23, red squares), while for large FtsZ-rings, recovery times were 8.2 ± 1.9 s (n = 7, dark blue circles), 8.4 ± 1.7 s (n = 27, light blue triangles), 8.3 ± 2.8 s (n = 30, green triangles) and 8.2 ± 2.6 s (n = 18, purple diamond) for cells with diameters in the ranges of 1–1.25 μm, 1.25–1.75 μm, 1.75–2.25 μm and > 2.25 μm, respectively. Error bars represent S.D. Note that FRAP measurements on fixed cells indicated that trapped cells were stationary, and not rotating in the holes (Supplementary Figure 10) Full size image

The FtsZ-square

Next, we wanted to know if drug-treated cells placed in deep (5 μm) rectangular volumes would adapt to these shapes and effectively form Z-rectangles or Z-squares instead of Z-rings. Previous work has shown that cells can adapt to rectangular shapes in shallow wells, approximately 1 μm deep23. Here, we produced quadrilateral patterns in agarose pads using silica micron pillar arrays similar to those previously described14, with the exception that the pillars were rectangular and 5.5 ± 0.5 μm in height. Sides of the micron chambers were up to 3.5 μm in length (Supplementary Figure 11), resulting in well volumes up to 80 μm3, roughly 50-fold larger than the volume of a WT cell (assuming a WT cell size of 2 μm in length and 1 μm in width) (Supplementary Figure 12).

Drug-exposed cells expressing FtsZ-mNeonGreen were placed in rectangular micron holes and incubated at room temperature for 300–420 min (longer incubation times were needed due to increased well size). The cells adapted to their new shapes and formed rectangular cuboids with only one Z-square per cell (Fig. 3a, Supplementary Movie 4). Notably, FtsZ clusters were observed both in the sharp corners and along the sides of the rectangles (Fig. 3b, Supplementary Figure 13). Quantification of the FtsZ-mNeonGreen clusters showed that they had similar dimensions to those in untreated cells, with an average length of 105.4 ± 39.6 nm and radial width of 79.6 ± 18.2 nm (n = 147) (Fig. 3c). This suggests that FtsZ cluster dimensions in vivo are insensitive to membrane curvature (or lack thereof).

Fig. 3 FtsZ-squares and FtsZ-rectangles in shaped cells. Drug-treated (A22 and cephalexin) E. coli cells expressing FtsZ-mNeonGreen were sculpted into rectangular shapes and imaged using super-resolution STED nanoscopy. a 3D rendering of a confocal Z-stack of an FtsZ-mNeonGreen square, showing only one band of FtsZ. Note that information along the z-axis is elongated. b Representative STED images of FtsZ-mNeonGreen in square (left) and rectangular (right) cells with perimeters ranging from 8.4 to 11.52 μm (compared to WT ~ 3 μm). Additional examples are provided in Supplementary Figure 13. Close-up images show representative corner angles. BF, brightfield image of corresponding cells. c Quantification of FtsZ cluster dimensions, showing little difference between FtsZ-mNeonGreen (105.4 ± 39.6 nm, 79.6 ± 18.2 nm; mean ± S.D., length and width, respectively. n = 147) and FtsZ-mCitrine (118.3 ± 41.3 nm, 86.3 ± 22.5 nm; length and width, respectively. n = 162. Example images of FtsZ-mCitrine squares are shown in Supplementary Figure 14). Scale bars = 1 μm. Boxes represent S.D., with red lines indicating mean. Whiskers on the box plots encompass 95.5% of the distribution Full size image

To generate a fluorescent FtsZ fusion protein that could be used for both super-resolution STED imaging and examination of cluster dynamics when grown in rich media at 37 °C, we constructed a plasmid-expressed FtsZ-mCitrine fusion. FtsZ-mCitrine was expressed from an IPTG-inducible, medium copy-number plasmid, pTrc99a, at a level approximately equal to 30% of total cellular FtsZ. Under these conditions, FtsZ-mCitrine formed normal-looking, sharp Z-rings (Supplementary Figures 1 and 2). Cells expressing FtsZ-mCitrine were then exposed to drugs, trapped in rectangular micron-sized holes, and incubated for 180–280 min at room temperature before gSTED imaging. We found that FtsZ-mCitrine formed clusters that were 118.3 ± 41.3 nm long and 86.3 ± 22.5 nm wide radially (n = 162), similar to FtsZ-mNeonGreen cluster dimensions (Fig. 3c, Supplementary Figure 14), indicating that fluorophore choice did not influence cluster dimensions in the rings. For consistency, we also imaged rectangular cells expressing FtsZ-GFP from the chromosome using SIM (Supplementary Figure 14). All three strains tested adapted to the rectangular shape, producing sharp-cornered Z-rectangles.

FtsZ dynamics in rectangular-shaped cells

In order to examine the dynamics of FtsZ in rectangular cells, we performed time-lapse imaging on cells expressing either FtsZ-mCitrine or FtsZ-GFP. Although a few fluorescence spots were abnormally bright and immobile (~ 1 spot/5 cells, with a maximum of 2 spots in one cell) (Fig. 4b, Supplementary Movie 7, red arrow), the majority of FtsZ clusters were highly dynamic (Fig. 4a, b, Supplementary Movies 5–6). Note that the bright, immobile spots were excluded from treadmilling analyses. Close inspection of time-lapse sequences suggested that FtsZ clusters in rectangular-shaped cells could treadmill continuously around the perimeter of the cells (Fig. 4a), and importantly, even across sharp corners, without an apparent change in speed (Fig. 4b, c, Supplementary Movie 8). The average treadmilling speed of FtsZ-mCitrine clusters in rectangular cells with perimeter lengths up to 13 μm (more than four times the circumference of a WT cell) was 27.6 ± 12.5 nm s−1 (n = 109), which was consistent with the measured treadmilling speed of FtsZ-GFP in rectangular cells (25.3 ± 11.3 nm s−1, n = 122) (Fig. 4d), large cylindrical cells (30 ± 18 nm s−1, Fig. 1m) and untreated cells (~25 nm s−1)13,14.

Fig. 4 FtsZ dynamics in rectangular-shaped cells. The dynamics of FtsZ in rectangular shapes were assessed by time-lapse imaging and FRAP measurements on E. coli cells expressing FtsZ-mCitrine (FtsZ-mCit) or FtsZ-GFP. a–c Snapshot images from time-lapse series of FtsZ-mCitrine or FtsZ-GFP in rectangular shaped cells. Corresponding kymographs are shown next to each image. a Kymographs were taken around the entire perimeter (starting at the upper left corner, moving counter-clockwise, indicated by the yellow arrowheads). b Kymographs were taken along the white line (left kymograph), or over the bright spot indicated by the red arrow (right kymograph). FtsZ can clearly be seen treadmilling continuously across the sharp corner (indicated by the red arrowhead). The red arrows and arrowheads in the images correspond to the arrows and arrowheads on the kymographs. The black dashed line in b indicates the upper left corner of the cell. c Kymograph taken between the yellow arrowheads (top to bottom is left to right in the kymograph). d Average treadmilling speed of FtsZ-mCitrine and FtsZ-GFP in rectangles was 27.6 ± 12.5 nm s−1 (n = 97) and 25.3 ± 11.3 nm s−1 (n = 122), respectively. e Typical FRAP measurement of FtsZ-GFP in a rectangular E. coli cell. Half of the rectangle was bleached. f Average recovery times for FtsZ-mCitrine (dark, n tot = 24) and FtsZ-GFP (light, n tot = 22) in FtsZ-rectangles of various perimeter lengths. FRAP recovery times for rectangular cells with different perimeters: FtsZ-mCitrine t 1/2 recovery times: 9 ± 2.9 s (Circ. 8 ± 1 μm, n = 6), 10 ± 3.2 s (Circ. 10 ± 1 μm, n = 7), 10.4 ± 2.3 s (Circ. 12 ± 1 μm, n = 7), 9.9 ± 1.9 s (Circ. 14 ± 1 μm, n = 4). FtsZ-GFP t 1/2 recovery times: 8.1 ± 1.9 s (Circ. 8 ± 1 μm, n = 5), 8.8 ± 2.6 s (Circ. 10 ± 1 μm, n = 5), 10.1 ± 2.5 s (Circ. 12 ± 1 μm, n = 8), 9.7 ± 3.2 s (Circ. 14 ± 1 μm, n = 4). Circ. = Cell circumference. Values represent mean ± S.D. Dots represent individual data points, bars represent mean with error bars representing S.D. Scale bars = 1 μm Full size image

To determine whether the dynamics of FtsZ subunit exchange are affected by changes to circumferential length and shape, we collected FRAP measurements on FtsZ bundles in rectangular-shaped cells (Fig. 4e, Supplementary Movie 9). The recovery times of half-bleached rectangles of varying sizes matched those of rings, with mean t 1/2 recovery times of 9.85 ± 2.58 s (n = 24) and 9.15 ± 2.55 s (n = 22) for FtsZ-mCitrine and FtsZ-GFP, respectively (Fig. 4f). This suggests that subunit exchange from the cytoplasmic FtsZ pool is independent of circumference length and membrane curvature. The data thus far indicate that the maintenance and dynamics of FtsZ clusters are preserved in both large Z-rings and Z-rectangles of varying size.

FtsZ dimensions and dynamics in heart-shaped cells

To examine whether FtsZ could literally be (at) the heart of cell division, we engineered micron pillar arrays that were heart-shaped (Supplementary Figure 15). Heart shapes were chosen because they would sculpt cells in such a way that highly curved, straight, and angled membrane segments would be present within a single cell. Drug-treated E. coli cells expressing cytoplasmic GFP, FtsZ-mNeonGreen or FtsZ-mCitrine were sculpted into hearts as described above (Fig. 5a). Perhaps not surprisingly, quantification of 155 individual FtsZ clusters from the heart-shaped cells revealed dimensions similar to those in round and rectangular cells (129 ± 44 nm long and 84 ± 9 nm wide) (Fig. 5b). We also found that the average treadmilling speed of FtsZ-mCitrine in heart-shaped cells (22 ± 10 nm s−1, n = 44) was essentially the same as that in untreated cells (Fig. 5c, Supplementary Movie 10).

Fig. 5 FtsZ cluster dimensions and dynamics in heart-shaped cells. FtsZ behavior in E. coli cells sculpted into heart shapes. a Upper left, Cartoon representation of a WT E. coli cell and a heart shape (both colored red for visualization), highlighting the large and complex structural changes of a cell-to-heart transition, approximately to scale. Upper right, Drug-treated cell expressing cytoplasmic GFP, shaped as a heart. Lower, STED image of an FtsZ-heart (FtsZ-mNeonGreen) in a drug-treated E. coli cell. b Lengths and widths of 155 individual FtsZ-mNeonGreen fluorescence clusters in cells shaped as hearts. Average length = 129 ± 44 nm and width = 84 ± 9 nm. Boxes represent S.D., with red lines indicating mean. Whiskers on the box plots encompass 95.5% of the distribution. c Upper row, SIM image from a time-lapse series (epi-fluorescence) of a heart-shaped cell expressing FtsZ-mCitrine. Green arrowhead indicates internal FtsZ clustering. Corresponding kymograph is shown adjacent to the image, and was generated starting at the yellow arrowhead in the SIM image, moving counter-clockwise for the indicated length. The yellow arrow points to an FtsZ trajectory. Lower, average treadmilling speed of FtsZ-mCitrine (Z-mCit) clusters in hearts (22.6 ± 10.4 nm s−1, n = 44). d FRAP measurements of FtsZ-mCitrine in heart-shaped cells. Top row, bleaching of half the FtsZ-mCitrine molecules in a full heart. Bottom row, bleaching of a half-full heart. No difference in recovery times was observed. e Histogram of average t 1/2 recovery times calculated from FRAP measurements. Recovery in full hearts: 7.1 ± 1.1 s (n = 24), recovery in half-hearts: 6.9 ± 0.9 s (n = 9). Scale bars = 1 μm. Dots represent individual data points, bars represent mean with error bars representing S.D Full size image

For about one-third of the heart-shaped cells, we noticed bright spots of internalized FtsZ-FP signal that accumulated close to the cell center (Fig. 5c, green arrowhead). Although we couldn’t distinguish whether these were true FtsZ clusters or aggregated protein, cytoplasmic clustering of FtsZ in WT cells have previously been reported12. Furthermore, although most hearts had FtsZ-FP signal spanning the full perimeter of the cell, approximately 20% were only half-full (Fig. 5d, lower left). We do not fully understand the underlying reason for this, however it is unlikely due to image focus or cell tilt issues, as every cell was scanned in the z-direction prior to imaging. Nevertheless, when we subjected the heart-shaped cells to FRAP, fluorescence recovery rates were equal for both full and half-full hearts (Fig. 5d), with mean t 1/2 recovery times of 7.1 ± 1.1 s (n = 24) and 6.9 ± 0.9 s (n = 9), respectively (Fig. 5e).

FtsZ-rings form in complex cell shapes

To explore if cell geometry plays a role in Z-ring formation, we set out to remodel cells into other complex shapes. Even though highly complex-shaped bacteria occur in nature, such as star-shaped bacteria31, we wanted to test whether rod-shaped E. coli cells would allow themselves to be drastically remodeled. Using micron pillars of various shapes, we produced holes in agarose pads such that drug-exposed cells could be sculpted into complex shapes, such as pentagons, half-moons, stars, triangles and crosses (Fig. 6a, middle row. Supplementary Figure 15). The cells conformed remarkably well to these shapes, forming sharp boundary angles < 70° (Fig. 6a, star). After we confirmed that cells could adapt to these complex shapes, we placed cells expressing FtsZ-mCitrine into the micron holes, allowed for reshaping to occur, and then imaged the cells using STED nanoscopy. Cells of all tested shapes produced FtsZ-shapes at midcell (Fig. 6a, bottom row). Subsequent analysis of the lengths and widths of the FtsZ clusters revealed little difference in dimensions between the different shapes, suggesting a minimal role of cell shape in determining FtsZ cluster dimensions in vivo (Fig. 6b). Additionally, time-lapse imaging of cells expressing FtsZ-mCitrine in various shapes showed similar dynamics to those measured in untreated cells. Specifically, FtsZ clusters treadmilled continuously over sharp corners and severe angles with an average speed of 28.7 ± 11.1 nm s−1 (Supplementary Figure 16 and Supplementary Movie 10), which is similar to the treadmilling speed in both square-shaped and WT cells.