Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus

Abstract

The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

Fig. 1.

Unpolymerized single MreB–enhanced YFP molecules exhibit random motion. (A) Fluorescence images of single MreB–YFP proteins in a Caulobacter cell. White line shows the cell outline. (A1) Image showing three fluorescent molecules (MreB–YFP) in a cell at 15.4-ms integration time. The top and bottom molecules are stationary; the middle molecule is moving. (A2) Smoothed image of A1 obtained by applying a low-pass filter (3 × 3 kernels of 0.0625, 0.125, 0.0625, 0.125, 0.25, 0.125, 0.0625, 0.125, and 0.0625). (A3) A representative trajectory of the mobile molecule (middle spot in A1). (A4) Summed image of 450 sequential images. The fluorescence from the two stationary molecules is evident, whereas the middle molecule does not appear. (Scale bar, 1 μm.) (B) MSD of fast-moving MreB molecules versus time lag for both untreated and A22-treated cells (open circles and squares), with geometry-corrected data shown as filled circles and squares. Solid lines represent a linear fit of the corrected data. (C) Distributions of diffusion coefficients from individual molecules, from trajectories truncated to 10 time steps. MreB, n = 81; MreB + A22, n = 84. Solid lines represent the error distribution (39), assuming a homogeneous underlying diffusion coefficient. The arrow shows the expected diffusion coefficient of MreB (62 kDa) in cytoplasm.

Fig. 2.

Polymerized single MreB-enhanced YFP molecules exhibit directed motion consistent with treadmilling. (A) Fluorescence images illustrating the directional movement of fMreB. Time-lapse imaging with 10-s dark intervals, 100-ms exposures, black line showing the cell outline, and inverted contrast. The molecule moves from left to right and then turns and moves right to left at a downward angle, shown by the red line. (B) MSD versus time lag for fMreB with a quadratic fit (smooth line), indicative of directional motion. (C) Velocity autocorrelation for both gMreB (Inset) and fMreB. The autocorrelation of gMreB dropped to near zero at the very first time lag, whereas that for fMreB remained positive over at least 80 s. (D) Distribution of observed true irradiation time of fMreB molecules. Inset shows the distribution of total emission times before photobleaching of fMreB–YFP with continuous illumination (100-ms integration). Although the average “on” time of the fluorophore was 4.6 s (Inset), the average irradiation time of fMreB in the time-lapse experiment was 0.8 s.

Fig. 3.

In vivo assessment of MreB polymer assembly rate, length, and polarity. (A) Speed distribution of polymerized fMreB. The average speed was 6.0 ± 0.2 nm/s. (B) Distribution of end-to-end contour lengths measured from the movement of a single fMreB. The average length was 332 nm, which was quite small compared with the average cell length (3.5 μm). (C) Representative trajectories of fMreB filaments in a ST cell and in a predivisional cell. The single-molecule trajectories were plotted on normalized cell shapes as described in Supporting Text. Examples of global direction assignments are shown as either “+” [toward the swarmer (SW) pole] or “−” (toward the ST pole).