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.
Highlights
FtsZ was the first cytoskeleton protein to be identified in prokaryotes (Busiek and Margolin 2015)
single-m olecule localization microscopy (SMLM) combined with indirect immunofluorescent staining was used to visualize FtsZ structures in dividing E. coli cells with DNA visualized by intercalating dye YOYO-1
It is known that immunofluorescence sample preparation could introduce artifacts, especially at the SMLM resolution level (Whelan and Bell 2015), certain degree of reassurance comes from reports that no significant FtsZ fixation artifacts were observed by SMLM (Fu et al 2010) and structured illumination microscopy (SIM) (Rowlett and Margolin 2014)
Summary
FtsZ was the first cytoskeleton protein to be identified in prokaryotes (Busiek and Margolin 2015). Single-molecule localization microscopy (SMLM) of FtsZ-mEos yielded Z-ring images with a resolution of about 35 nm (Fu et al 2010) These results support the model that the Z-ring is a loose irregular assembly of randomly oriented FtsZ filaments that form rings or small-pitch helices that are indistinguishable by conventional FM. Later this method was used to analyze FtsZ, ZapA, ZapB, and MatP colocalization in dividing cells, which allowed a model of their arrangement in the Z-ring to be proposed, suggesting that these proteins form a layered structure, spanning from the cell surface to chromosomes (Buss et al 2013, 2015). DNA staining was performed by incubating cells in a solution of YOYO-1 dye (250 nmol/L in PBS) for 10 min
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