New insights into FtsZ rearrangements during the cell division of Escherichia coli from single‐molecule localization microscopy of fixed cells

Abstract

FtsZ – a prokaryotic tubulin homolog – is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so‐called Z‐ring at mid‐cell that guides septum formation. Many approaches were used to resolve the structure of the Z‐ring, however, researchers are still far from consensus on this question. We utilized single‐molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z‐ring dimensions. Analysis of these images demonstrated that Z‐ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half‐way through constriction.

Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

“…This suggests that the initial activity of ZapA is to bridge FtsZ without aligning the protofilaments into bundles. This is in agreement with super resolution images of the FtsZ ring in bacteria [ 16 , 45 , 46 ]. In these studies, the ring is depicted as consisting of short protofilaments of up to 100 nm that are randomly oriented in a structure that has a thickness of about 60 nm and a width of about 100 nm.…”

Beyond force generation: Why is a dynamic ring of FtsZ polymers essential for bacterial cytokinesis?

“…The Z‐ring has been shown to be a patchy, discontinuous structure with FtsZ clusters loosely occupying a three‐dimensional toroidal zone with cross‐sectional dimensions of ∼100 × 60 nm (Fig. D) . As described above, divisome proteins can be concentrated near this zone through direct or indirect interactions with FtsZ, promoting productive interactions between them.…”

“…Furthermore, FtsZ‐dependent cell wall synthesis has been observed at midcell prior to any observed septum invagination , and even along the lateral wall outside the midcell . Interestingly, the axial septum width appears to be similar to axial Z‐ring width: the septum appears to be ∼80 nm wide in AFM images of Gram negative E. coli sacculi and 60–80 nm in electron micrographs of Gram positive B. subtilis , while Z‐ring width has been shown to be 80–100 nm in Gram negative E. coli and C. crescentus , and in Gram positive Streptococcus pneumoniae . Thus, the ultimate output of Z‐ring signaling may be to define the composition and morphology of septal cell wall synthesis, mediated by signaling interactions between FtsZ and other divisome constituents.…”

“…C), but higher resolution studies using electron cryotomography and superresolution fluorescence microscopy have revealed that the ring most likely comprises discontinuous, short filaments that are bundled together into clusters within a toroidal zone of 80–100 nm axial width and 40–60 nm radial width (Fig. D) .…”

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology

“…(b) Native POIs (blue) are either stained in vitro after their expression and isolation before being taken up again into cells [61,62] or in situ by adding a fluorescently labeled interacting compound, e.g. by immunofluorescence using anti-or nanobodies [36,37,39,40,50,51,53,56] (ii), by fluorescently labeled ligands, drug molecules or targeting proteins [15] (iii) or by aptamers (iv). Furthermore, the POI can be modified, either by a FP (v, PAmCherry ( pdb 3KCT)) [13,14,28,40,43,58,59] or by an enzyme/peptide tag (vi, SNAP ( pdb 3L00)) [21,30,35,36,38,39,41,56,63] or by insertion of an unnatural amino acid into the primary structure of the protein (vii), which after protein folding is coupled to a dye by a click chemistry reaction [64,65].…”