A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction dynamics

Abstract

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.

Fig 1. Live-cell PALM imaging of band-like ZapA, ZapB and FtsZ structures.

Images of mEos2-ZapA (pJB051, A-C), ZapB-mEos2 (pJB045, D-F) and FtsZ-mEos2 (pJB042, G-I) in wt cells are shown in the order of bright-field image (i), ensemble fluorescence image (ii) and PALM image displayed in pseudocolor (iii). Approximate cell outlines are indicated by yellow dashed lines. Scale Bars, 500 nm.

Fig 2. Two color-PALM imaging of ZapA-FtsZ, ZapB-FtsZ and ZapA-ZapA pairs.

Cropped PALM images of cells expressing Dronpa-ZapA and FtsZ-PAmCherry1 (Ai-vi), ZapB-Dronpa and FtsZ-PAmCherry1 (Bi-vi), or Dronpa-ZapA and PAmCherry1-ZapA (Ci-v). Approximate cell outlines are indicated in yellow dashed lines. (Cvi) 2C-PALM image of a 100 nm TetraSpeck bead. (D) Histograms of the apparent displacement between the given protein pairs in individual cells. The mean values for the ZapA-FtsZ, ZapB-FtsZ and ZapA-ZapA pairs are 55 ± 50 nm (n = 158), 97 ± 70 nm (n = 132) and 33 ± 33 nm (n = 39), respectively ($\stackrel{-}{x}$ ± sd). (E) The diameters for ZapB-Dronpa and FtsZ-PAmCherry1 structures visualized simultaneously in the same cell (blue circles) were best fit to a line (red) where y = 1.0x - 95. The R² of the fitted line to the data was 0.89. Scale Bars, as labeled in nm.

Fig 3. iPALM imaging and z-position measurements of FtsZ, ZapA, and ZapB.

(A) Cross-sectional views of the midcell region of representative wt cells expressing FtsZ-mEos2, mEos2-ZapA, ZapB-mEos2 or mEos2-MTS_Bs (three cells for each strain) are displayed in respective rows. All images are to scale and share the same z-dimensions as labeled on the left. The dashed rectangle box (gray) in the bottom right-most image is representative of the user-defined box we used to calculate mean z-positions. (B) Histograms (20 nm bins) of the average measured z-positions for each protein structure in individual cells illustrate the internal nature of ZapB. Scale Bar, 250 nm.

Fig 4. PALM imaging of MatP and z-position estimation.

(A) Four representative PALM images for cells expressing MatP-mEos2 in the order of bright-field, ensemble fluorescence, and PALM image. Scale Bars, 500 nm. (B) Distribution of short-axis displacements of MatP-mEos2 clusters from the middle of the cell (yellow bars) fitted with a model (red line) in which a normally-distributed radial displacement is randomly projected to a 2D imaging plane (S1 Text). The fitted radial displacement is 285 ± 120 nm (red line, $\stackrel{-}{x}$ ± sd).

Fig 5. FRAP measurements of FtsZ, ZapA and ZapB turnover rates in BW25113 and ΔmatP cells.

Average fluorescence recovery trajectories for FtsZ (light grey), ZapA (grey) and ZapB (dark grey) are displayed for wt (A) and ΔmatP (B) cells as a fraction of pre-bleached intensity. Trajectories were fit with single exponentials illustrated as dotted lines (FtsZ, red; ZapA, blue; ZapB, black). The respective half-times are listed in Table 2.

Fig 6. The multi-layered organization of the E. coli divisome.

A schematic illustrating the relative radial arrangement of FtsA/ZipA-FtsZ-ZapA-ZapB-MatP. FtsA (green) and ZipA (orange) tether the punctuate FtsZ (red) structure to the inner membrane. ZapA (blue) mimics FtsZ but can deviate, possibly by interacting with ZapB or a number of membrane proteins [50,51]. The large, internal ZapB (grey) structure indirectly associates with FtsZ through ZapA, and is anchored on the chromosome through its associations with MatP (yellow).