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. 2011 Apr 22;407(4):650-5.
doi: 10.1016/j.bbrc.2011.03.062. Epub 2011 Mar 16.

Long Helical Filaments Are Not Seen Encircling Cells in Electron Cryotomograms of Rod-Shaped Bacteria

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Long Helical Filaments Are Not Seen Encircling Cells in Electron Cryotomograms of Rod-Shaped Bacteria

Matthew T Swulius et al. Biochem Biophys Res Commun. .
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Abstract

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

Figures

Figure 1
Figure 1. No long filaments are seen encircling rod shaped bacteria
A) 45-nm thick tomographic slice through a C. crescentus swarmer cell along the cell envelope. The inner membrane (IM), outer membrane (OM) and surface layer (S) are visible, but there are no filaments near the inner membrane. B) Projections onto the inner membrane of all the density from the periplasmic face of the inner membrane to 13 nm into the cytoplasm. Red represents higher density. C) Line-segment-based search for filaments 21 nm into the cell counted from the center of the inner membrane. Red pixels are those identified as potentially belonging to filaments. The position of the slice shown in A is marked with a dashed line. D) Analogous 45-nm slice, E) density projection, F) and filament search of a predivisional C. crescentus "positive control" cell possessing 6 native ("a" – "f") and 10 simulated ("1" – "10") filaments. The insert in D shows a side view including native filament “b”(arrowheads). The simulated filaments of different lengths (#'s 1–4, which are 40, 80, 120, and 180 nm long, respectively) show that filaments 80 nm or longer are clear. Simulated filaments #4–7 exhibit different pitches with respect to the long axis of the cell, but are all equally visible. Filaments #1–7 are immediately adjacent to the membrane, but filaments #8–10 cannot be seen as they range from partially to fully embedded, positioned successively one pixel (1.3 nm) each deeper into the membrane. To the left of the dashed white line in E, the projected shell was optimized to show the filaments next to the membrane, and extended from the cytoplasmic face of the inner membrane to 13 nm into the cytoplasm. To the right, the projection is of the same shell shown in B (including the membrane). Scale bars represent 50 nm for (A & D) and 100 nm for (B, C, E and F).
Figure 2
Figure 2. Long helixes are not seen encircling rod shaped bacteria
Shown are tomographic slices (A–F) and density projections onto the inner membrane of all the densities between the periplasmic edge of the inner membrane to 13 nm into the cytoplasm (G–L) of all the cell types where MreB has been reported to form long helical filaments encircling the cell (E. coli, B. subtilis, V. cholera, with C. crescentus shown in Fig.1 of the main text), plus C. crescentus treated with A22 (a small molecule known to depolymerize MreB filaments), plus B. burgdorferi and A. longum, which are especially slender and therefore yield the highest resolution cryotomograms. All scale bars represent 200 nm.
Figure 3
Figure 3. Cytoplasmic MreB filaments are clearly visible in cryotomograms
A) Tomographic slice through a V. cholerae cell overexpressing GFP-MreB. The inset shows the image taken by cryo-fLM. Red is the membrane-dye FM4-64 and green is GFP-MreB. Dashed lines (red and green) represent the thresholded boundaries of each signal, respectively. The dashed lines generated from the cryo-fLM image were rescaled to match the much higher magnification of the EM slice and superimposed to show the GFP signal appears in the same location as a large filament bundle running through the cytoplasm (arrows), proving upon repetition that these bundles are composed of MreB. B)15-nm thick tomographic slice though an MreB bundle (not fused to GFP). Arrows point to filaments within the bundle. Scale bars represent 1 µm in the fLM inset of A, 200 nm in the ECT slice of A and 50 nm in B.

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