The structural protein MreB mediates polarity, chromosome segregation, and cell shape in C. crescentus
(Gitai et al. 2004
; Dye et al. 2005
; Gitai et al. 2005
). Bulk-level imaging and biochemical studies of the cytoplasmic MreB protein are consistent with the formation of a dynamic superstructure made up of actin-like filaments. This structure provides the ma-chinery for key cellular processes, and the dynamics of MreB molecules were investigated on a single-molecule level (Kim et al. 2006
The merodiploid strain LS3813 of C. crescentus
was generated by integrating a single Pxyl::eyfp-mreB
construct into the xylX
locus in a C. crescentus
chromosome already containing a wild-type, unlabeled copy of MreB under its endogenous promoter (Gitai et al. 2004
). Cells with only a small number of isolated EYFP-MreB molecules were prepared by incubating the cells in the M2G minimal medium with 0.0006%–0.003% xylose for 4 hours. The cells were imaged with 15.4-ms imaging frames to resolve the tagged MreB molecules. A1 shows one such frame, in which three single molecules were captured. The image is smoothed 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) in A2 for enhanced visibility. In this cell, two subpopulations of MreB were distinguished from one another: slow-moving molecules and fast-moving molecules (arrowheads and arrow, respectively, in A1). The trajectory of the fast-moving molecule in A1, obtained by fitting the fluorescence image in every frame to a 2D Gaussian function, is plotted in A3. This molecule diffused rapidly and explores a large portion of the cell. 450 successive frames were summed to form the fluorescence image in A4. After this 7-second integration time, the fluorescence from the two slow-moving molecules was still evident, but emission from the fast-moving molecule was smeared out over many pixels and did not appear. The white line in A shows the outline of the C. crescentus
Figure 3. Analysis of motion of EYFP-MreB. (A) 15.4-ms integration time fluorescence images of single EYFP-MreB molecules in a C. crescentus cell. White line shows the cell outline. (A1) 15.4-ms epifluorescence image showing three single EYFP-MreB molecules. The (more ...)
Because the dynamics of these molecules were unchanged in the presence of the MreB-depolymerizing drug A22, the fast-moving EYFP-MreB molecules were ascribed to a free, unpolymerized population. The behavior of the unpolymerized single molecules was further characterized based on 111 trajectories. The observed mean-square displacement (MSD) of the fast-moving molecules is plotted as a function of time lag (Δt) in the open circles of B. Based on the first four points, a diffusion coefficient of D= MSD/(4Δt) of 1.11 ±0.18 µm2/s was extracted. This value of D is smaller than expected for a free, cytoplasmic protein, but is consistent with the motion of a membrane-associated protein. Because the observed motion represents a 2D projection, simulations were again performed to correct for the three-dimensionality of the cell, and the geometry-corrected MSD of the molecules is plotted in the filled circles of B. The geometry-corrected MSD is linear with the time lag, Δt, consistent with diffusion along the cell membrane with D= MSD/(4Δt) of 1.75 ±0.17 µm2/s. The velocity autocorrelation function, CV(τ), was also calculated for these molecules. As shown in C, for the fast-moving molecules, CV(τ) dropped to zero at the very first time step, indicating a nondirected random walk.
Further experiments addressed the behavior of the slow-moving MreB. Because slow-moving EYFP-MreB molecules were not observed in the presence of the MreB-depolymerizing drug A22, the slow-moving molecules represent polymerized MreB. The dynamics of 120 single members of this subpopulation were carefully examined. Because the molecules were stationary within the 30-nm resolution of the measurement over the course of the 7-second integration time shown in A4, time-lapse imaging was used. Specifically, 100-ms imaging frames were separated by 9.9 seconds. In this way, the slow movement of polymerized MreB molecules could be followed over a longer time period before photobleaching. D shows 8 such fluorescence images in reverse contrast, in which a single molecule is tracked for 220 seconds. The molecule moves from left to right, then turns and moves right to left at a downward angle, shown by the red line. The observed MSD of the slow-moving MreB molecules is plotted as a function of Δt in E. Here, the MSD was characterized by a quadratic dependence on time lag, as is typical of directed motion. Also consistent with directed motion is the computed velocity autocorrelation function, CV(τ), shown in F, in which CV(τ) remains positive until τ = 80 s.
The slow, directed motion of polymerized MreB was further explored by analyzing the trajectories of each individual molecule in the context of two putative models for the motion of an MreB molecule in an MreB filament: (1) the filaments into which the monomers are incorporated were moving, or (2) the monomers themselves were treadmilling though largely stationary filaments, analogous to the motion of actin. If the motion of polymerized MreB is because of whole-filament movement, then the observation time for the slow-moving single molecules should be limited by the photobleaching time of the EYFP, independent of experiment timescale. In the inset to A, the total emission time of EYFP-MreB before photobleaching under continuous emission was measured, with an average on-time of 4.6 seconds. However, the true irradiation time before photobleaching of the molecules imaged with 9.9-second time lapses, shown in A, was found to be only 0.8 seconds. Most of the fluorescence from the polymerized MreB molecules therefore disappeared before photobleaching occurred, likely as a result of dissociation from the end of a filament and the onset of fast diffusive motion not resolvable with the 100-ms imaging frames. This result indicates that MreB molecules treadmill through filaments with fixed ends.
Figure 4. Slow movement of MreB molecules through filaments. (A) Distribution of observed true irradiation time of slow-moving MreB molecules with time-lapse imaging (average = 0.8 s) and (inset) average emission time under continuous illumination (average = 4.6 (more ...)
Given that the polymerized MreB molecules showed directed motion, a speed value was extracted from each single-molecule trajectory (B). The average speed was 6.0 ± 0.2 nm/s. From its crystal structure, the length of an MreB monomer is 5.4 nm (van den Ent et al. 2001
); the average speed therefore corresponded to 1.2 monomer additions per second in steady-state fixed MreB filaments. The average length traveled by a polymerized MreB molecule before dissociation, which corresponds to the filament length, was also measured by considering only MreB molecules that became polymerized after the start of imaging and that dissociated before the end of imaging. The MreB filament lengths of 128 trajectories are shown in C, in which the average filament length was 392 ±23 nm—quite small relative to the average cell length of 3.5 µm. A representative subset of these trajectories is plotted on normalized cell shapes in D. Of note, most of the trajectories moved perpendicular to the cell long axis. A smaller number of trajectories were oblique. These nonperpendicular trajectories were characterized as moving toward the swarmer pole (+) or toward the stalked pole (−). No preferred orientation was observed.