Dynamic movement of actin-like proteins within bacterial cells

Abstract

Actin proteins are present in pro- and eukaryotes, and have been shown to perform motor-like functions in eukaryotic cells in a variety of processes. Bacterial actin homologues are essential for cell viability and have been implicated in the formation of rod cell shape, as well as in segregation of plasmids and whole chromosomes. We have generated functional green fluorescent protein fusions of all three Bacillus subtilis actin-like proteins (MreB, Mbl and MreBH), and show that all three proteins form helical filaments underneath the cell membrane, the pattern of which is distinct for each protein. Time-lapse microscopy showed that the filaments are highly dynamic structures. A number of separate filaments of MreB and Mbl continuously move through the cell along helical tracks underneath the cell membrane. The speed of extension of the growing end of filaments is within the range of known actin polymerization (0.1 microm/s), generating a potential poleward or centreward pushing velocity at 0.24 microm/min for MreB or Mbl, respectively. During nutritional downshift and a block in topoisomerase IV activity, the filaments rapidly disintegrated, showing that movement occurs only in growing cells. Contrary to Mbl and MreBH filaments, MreB filaments were generally absent in cells lacking DNA, providing a further distinction between the three orthologues.

Figure 1

Fluorescence microscopy of B. subtilis cells. Exponentially growing cells expressing (A) GFP–MreB, (B) GFP–Mbl or (C) GFP–MreBH. (D) Cells expressing GFP–MreB shifted from exponential growth in nutrient-rich medium to medium lacking nutrients for 5 min. (E) GFP–MreB-expressing cells from stationary phase. (F) Cells in which ParE (a subunit of topo IV) has been depleted for about 2–3 doubling times. GFP–MreB filaments are present only in cells containing DNA, and are absent in anucleate cells (indicated by arrowheads), which have normal rod shape. (G) Cells in which ParE has been depleted for 2–3 doubling times. GFP–Mbl is also present in anucleate cells (indicated by arrowhead). White lines indicate septa between cells (B. subtilis cells do not separate after division and form long chains of cells during exponential growth). Grey arrowheads in (A) point out cellular spaces close to the cell poles, and white arrowheads indicate the brightest signals of GFP–MreB filaments that are mostly close to or over the nucleoids. Grey arrowheads in (B) point out Mbl filaments that extend to the cell poles. Scale bars, 2 μm.

Figure 2

Time-lapse fluorescence microscopy. GFP–MreB filaments are highly dynamic and generally move away from mid-cell, whereas GFP–Mbl filaments move towards mid-cell. Frames are taken every 10 s, and all cells are equally scaled. (A,B,D) Cells expressing GFP–MreB, (A,B) exponentially growing cells, (C) single growing cell expressing GFP–Mbl and (D) fixed cell. White lines in (A) indicate two MreB bundles migrating in parallel towards the cell pole, and a white arrowhead indicates MreB filament visually making a full turn around the inner membrane periphery. White line in (C) indicates Mbl bundle migrating around the cell diameter, from cell pole towards mid-cell. Note that GFP–MreB filaments do not change in fixed cells in (D). Scale bar, 2 μm.

Figure 3

Fluorescence microscopy of B. subtilis cells expressing GFP–MreB, in which ParE (a subunit of topo IV) has been depleted for 5–6 doubling times. The arrow indicates anucleate cell. Scale bar, 2 μm.