In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy

Abstract

In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule-based super-resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments.

Figure 1. Scanning electron micrographs of dividing E. coli cells

(A) BW25113 cells show single, midcell septa oriented perpendicular to the cells’ long axes. In ΔzapA (B,C) and ΔzapB (D,E,F) cells, slanted (▲), non-midcell (∆) and multiple (†) septa are observed. Cells were imaged on PLL-treated coverslips (A–E) or on 0.2 µm filters in the absence of PLL (F). Scale Bars, 500 nm.

Figure 2. Localization and dynamics of FtsZ-GFP in wt, Δ zapA and Δ zapB cells

(A) Snapshots of BW25113 (Ai), ΔzapA (Aii) and ΔzapB (Aiii) cells expressing FtsZ-GFP. Brightfield images and corresponding fluorescence images are displayed side by side. (B) Montages from time-lapse movies of BW25113 (Bi), ΔzapA (Bii) and ΔzapB (Biii) cells expressing FtsZ-GFP during cell division. At each time point, the fluorescence image (green) was overlaid with the corresponding brightfield image (gray) with the time (min) indicated in the bottom corner. Corresponding movies are shown in Supplemental Movie 1, 3 and 4, respectively. In wt cells, FtsZ forms a clear band at midcell early and remains there throughout the division process. In contrast, the FtsZ structures in ΔzapA and ΔzapB appear more dynamic, transitioning back-and-forth between multiple active septa. (C) Distributions of cell division times for BW25113 (red) ΔzapA (blue) or ΔzapB (black) cells determined from time-lapse movies. The average division time of wt cells is significantly different from that of ΔzapA and ΔzapB (ks-test, p < 0.01). Subpopulations of ΔzapA and ΔzapB cells in which FtsZ relocalized to visibly constricted sites in newborn daughter cells (for example, Bii 145’, 190’, and 245’ top cells; and Biii 135’, 225’ and 230’ middle cells) divided significantly faster than the general populations with division times of 83 ± 11 min (n = 15) and 77 ± 13 min (n = 6), respectively. Mean ± standard deviation (number of division events). Scale Bars, 1 µm.

Figure 3. Live-cell PALM imaging of ring-like FtsZ structures

Images of live BW25113 (A–C), ΔzapA (D–F) and ΔzapB (G–I) cells expressing FtsZ-mEos2 from pJB042 are shown in the order of brightfield (i), ensemble green fluorescence image (ii), and PALM image (iii). The dotted yellow line is a general indicator of the cell outline. All cells shown here display a single band at midcell, indicative of a normal Z-ring. Scale Bars, 500 nm.

Figure 4. Live-cell PALM imaging of non-ring FtsZ Structures

Images of live BW25113 (A–C), ΔzapA (D–I) and ΔzapB (J–O) cells expressing FtsZ-mEos2 from pJB042 are displayed in the order of brightfield (i), ensemble green fluorescence image (ii), and PALM image (iii). Although the ring-like band conformations shown in Figure 3 are the predominant structures formed in wt cells, PALM imaging reveals that some wt cells possess punctate structures, characterized by a single focus (A), two foci (B) or multiple foci reminiscent of a compact helix (C). For ΔzapA and ΔzapB cells, while the ensemble green fluorescence images show broad, diffusive, or extended multi-band structures, the corresponding PALM images all resolve into various arrangements of clusters. Scale Bars, 500 nm.

Figure 5. Cluster analysis of FtsZ-mEos2 TIR-PALM images

(Ai-iii) Images of a BW25113 cell expressing FtsZ-mEos2 are shown in the order of brightfield (i), ensemble green fluorescence image (ii) and TIR-PALM image (iii). A threshold was applied to the TIR-PALM image to generate a binary image (iv) where the white region indicates an identified cluster. Using the cluster coordinates we determined the number of molecules localized within each identified region and the overall size of the region. (v) Expanded view of the boxed area in iv illustrates additional cluster measurements: major axis length (a), minor axis length (b), displacement of centroid (c) from midcell plane and orientation of major axis relative to midcell plane (d). FtsZ-mEos2 clusters observed in BW25113 (red), ΔzapA (blue) and ΔzapB (black) cells were compared for molecule counts (B), size (C), density (D) location (E) shape (F–G), and orientation (I). Histograms for each measurement were plotted using the same bin size (max/10) and normalized by the total number of counts. These measurements are summarized in Table 2. All the measured properties of clusters in ΔzapA and ΔzapB showed significant differences (p < 1e–5) from wt, with ΔzapB clusters closer to wt values. This observation was true independent of threshold value (Supplemental Table 1). Scale Bars, 500 nm.

Figure 6. Single Molecule Tracking of FtsZ-mEos2

(A) Representative single-molecule trajectories of consecutive frames for a mobile (Ai) and immobile (Aii) FtsZ-mEos2 molecule in ΔzapA. Fluorescence images were acquired every 200 ms. (B) Mean squared displacements (closed circles) are plotted at different time lags for BW25113 (red), ΔzapA (blue) and ΔzapB (black) cells. Error bars indicate Standard Error. The data were fit to a linear equation (y=4Dx+A). The diffusion coefficients for BW25113, ΔzapA and ΔzapB strains determined from the fits are 0.0004 ± 0.00003, 0.0005 ± 0.00001, 0.0006 ± 0.00003 µ;m² s⁻¹, respectively. (C) Histograms of single-step (200 ms) displacements of the three strains showing predominate immobile populations centered around 75 nm. Scale Bars, 1 µm.

Figure 7. FtsZ and SlmA localization under slow and fast growth

(A–B) Representative images of live ΔmatP cells expressing FtsZ-GFP under slow (A) or fast (B) growth conditions. A brightfield image is displayed atop an ensemble fluorescence image. (C–D) Individual ΔslmA, ΔzapA, ΔzapB and ΔmatP cells expressing mEos2-SlmA in the absence of inducer under the slow growth condition (C) and GFP-SlmA under the fast growth condition (D). Each brightfield image is displayed next to the corresponding ensemble fluorescence image (full dynamic range), which is adjacent to an overlaid, intensity-adjusted image. Scale Bars, 1 µm.

Figure 8. Model of how ZapA and ZapB promote Z-ring assembly

1. FtsZ monomers associate longitudinally into FtsZ protofilaments. 2. FtsZ protofilaments laterally associate to form FtsZ clusters, or higher-ordered polymers consisting of multiple FtsZ protofilaments. This process may be mediated by the intrinsic properties of FtsZ and/or other protein factors. ZapA and ZapB may participate but are not required for this step. 3. FtsZ clusters are corralled at midcell through the combined function of ZapA and ZapB, resulting in larger continuous structures. 4. In the absence of ZapA and ZapB, FtsZ clusters scatter throughout the midcell region and some unknown mechanism (dashed arrow) is responsible for proper Z-ring assembly.