Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 5 supplements
FtsZ forms bands of filaments completely encircling C. crescentus and E. coli division sites, as visualised by electron cryotomography.
(A) C. crescentus NA1000/CB15N division site with filaments near the inner membrane IM (top panel, black dots highlighted by arrow, see also Video 1). Bottom panel shows the same cell rotated 90° …
Figure 1—figure supplement 1
The missing wedge problem in cellular electron cryotomography.
Since it is impossible to tilt the sample support (EM grids) from −90° to +90° and because the thickness of the ice film increases at high tilt angles, electron tomograms miss significant amounts of …
Figure 1—figure supplement 2
Electron cryotomograms of wild-type E. coli cells show filaments at the constriction sites.
(A, C) 10-nm thick tomographic slices of two cells showing black dots near the constriction sites corresponding to cross-sections of filaments. Filaments are difficult to discern in this viewing …
Figure 1—figure supplement 3
FtsZ forms bands of filaments at constriction sites in E. coli cells.
(A) 10 nm electron cryotomographic slice of a cell expressing more FtsZ(D212A) protein than in Figure 1E (corresponds to Figure 1G), oriented parallel to the longitudinal axis, showing one layer of …
Figure 1—figure supplement 4
Engineered FtsZ proteins form filaments with altered localisation patterns in E. coli cells.
(A) Extending the C-terminal flexible linker of FtsZ(D212A) makes the protein form filaments further away from the membrane with a distance to IM increased from 16 nm to 21 nm; (B) and (C) are …
Figure 1—figure supplement 5
Overview of FtsZ constructs used for in vivo tomography.
Please also consult Supplementary file 1A,B.
Figure 2
Co-expression of FtsZ and FtsA in E. coli cells leads to extra septa.
(A) A low-magnification 2D electron cryomicrograph (transmission) showing multiple constriction sites (marked with black arrowheads) along the cell. (B–E) 10-nm thick electron cryotomographic slices …
Figure 3 with 2 supplements
In vitro reconstitution of bacterial cell membrane constriction by the FtsZ ring from purified components.
(A) Thermotoga maritima FtsA (TmFtsA) and Thermotoga maritima FtsZ (TmFtsZ) form spirals on a flat lipid monolayer, as indicated by a white dotted line. The filaments tend to appear as double …
Figure 3—figure supplement 1
TmFtsZ and TmFtsA on the outside of liposomes and in the presence of GMPCPP deform liposomes.
(A) Low-magnification (upper panel). More detailed snapshots (lower panel) show that the filaments are on the outside; however, they do not form rings but curved structures that are positioned in …
Figure 3—figure supplement 2
Control experiments showing that both TmFtsA and TmFtsZ form straight filaments when polymerised separately. And liposomes deform mostly after dilution.
(A) When mixed, FtsA and FtsZ form curved filaments (right panel). (B) TmFtsZ does not bind to liposomes on its own. Random electron cryomicroscopy images taken immediately after detergent dilution …
Figure 4 with 2 supplements
Electron cryotomography of liposomes constricted in vitro by rings of TmFtsA and TmFtsZ.
(A) Stereo view of a representative liposome highlighting three different structures made by the enclosed TmFtsA and TmFtsZ proteins. Note that our images derived from tomographic volume data have …
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Figure 4–source data 1
PyMOL (version 1.7) session file showing volume and surface renderings of the liposome in stereo in Figure 4A, top.
Note that this version of the data has been volume edited, removing some of the filaments in the surroundings of the liposome. Nothing has been changed on the surface. These representations have not been segmented (automatically or manually); they show the volume data points as present in the (edited) tomogram. Both surface (threshold) as well as volume data are available as objects ‘surf’ and ‘vol’, respectively.
- https://doi.org/10.7554/eLife.04601.017
Figure 4—figure supplement 1
Constrictions occur only at the site of filament ring formation.
A stereo view of the liposome marked with the black arrowheads in Figure 4A (bottom middle panel). A single helix made of filament doublets is marked with red arrow. Video 6 shows its architecture …
Figure 4—figure supplement 2
A mechanism explaining variable intrinsic FtsA:FtsZ filament curvature.
At some stages of constriction, the ratio of FtsZ to FtsA in the ring may be higher than one. Normally, there is around five times more FtsZ in cells than FtsA, therefore only a few FtsA molecules …
Figure 5
Visualising the FtsZ ring at the molecular level.
(A) A semi-atomic model of the FtsZ ring constricting a liposome. 294 monomers of S. aureus FtsZ have been roughly positioned using a spline-fitting approach (PDB 3VO8 (Matsui et al., 2012)). This …
Videos
Video 1
Tomogram of a wild-type C. crescentus cell showing tomographic slices parallel to the longitudinal axis of the cell.
A single layer of dark dots corresponding to cross-sections of FtsZ filaments is clearly visible at a distance from the membrane on both sides of the septum. The missing wedge is located at top and …
Video 2
Tomogram of a wild-type E. coli cell showing the constriction site along the longitudinal axis of the cell.
FtsZ filaments are visible in certain slices and are likely to be forming continuous helices indicated by its pattern when viewed along the slices. This corresponds to Figure 1C.
Video 3
Tomogram: FtsZ(D212A) expressed in E. coli cell forms doublet FtsZ filaments at the constriction site.
The video shows tomographic slices parallel to the longitudinal axis of the cell. One single layer of dark dots corresponding to cross-sections of FtsZ filaments is clearly visible, and these dark …
Video 4
This video shows a typical field of view from tomographic reconstruction of the in vitro reconstitution specimen.
The filaments present on the water/air interface consist of TmFtsA and TmFtsZ filaments and therefore adopt a curved geometry. This corresponds to Figure 4A.
Video 5
This video shows a volume of the liposome whose stereo view is depicted in Figure 4A.
https://doi.org/10.7554/eLife.04601.021
Video 6
This video shows a volume representation of the liposome that is depicted in Figure 4A (bottom middle panel, black arrowheads) and whose stereo view is shown in Figure 4—figure supplement 1.
https://doi.org/10.7554/eLife.04601.022
Video 7
This video shows the two remaining liposomes that are depicted in Figure 4A.
https://doi.org/10.7554/eLife.04601.023
Video 8
This video shows the two remaining liposomes that are depicted in Figure 4A.
https://doi.org/10.7554/eLife.04601.024
Video 9
This video shows a well-pronounced constriction with spirals being very prominent on lateral sides of the leading membrane edge, which eventually might lead to abscission. Not shown in any other figure.
See also Figure 5D, middle for an explanation.
Video 10
This video runs through a surface representation of the liposome whose stereo view is depicted in Figure 4A, top, with features of interest highlighted along the way.
https://doi.org/10.7554/eLife.04601.026Additional files
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Supplementary file 1
(A) Plasmids used in this study. (B) Exact protein sequences of modified E. coli FtsZ proteins used for in vivo tomography experiments.
- https://doi.org/10.7554/eLife.04601.028
