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.bacteria ͉ cytoskeleton ͉ single-molecule fluorescence I n both eukaryotic and prokaryotic cells, actin mediates essential cellular processes. A quantitative understanding of the kinetic dynamics and ultrastructural architecture of actin's polymerized filaments has helped elucidate the mechanisms by which eukaryotic actin functions. For example, high-resolution imaging and the in vivo and in vitro dissection of the kinetics of its assembly have demonstrated how actin polymerization at the tips of a rigid, crosslinked actin meshwork can drive cell motility at the leading edge of Dictyostelium (1, 2). In budding yeast, the polarized assembly of actin cables provides both a road and direction signs for the directed transport of proteins to the tip of growing buds (3).There are two known bacterial actin homologs, the widely conserved, chromosomally encoded MreB family of proteins and the plasmid-specific ParM family of proteins. ParM functions to partition plasmid DNA by polymerizing in between two sister plasmids, thereby generating a tension rod that physically pushes them apart (4). MreB is essential in most bacteria and has been shown to form a lengthwise spiral that contributes to cell shape, chromosome segregation, and polar protein localization in multiple species, including Caulobacter crescentus, Escherichia coli, and Bacillus subtilis (5-10). The mechanism by which MreB executes its functions remains largely unknown (11).In vitro studies of the dynamics of eukaryotic actin filament assembly have demonstrated th...

Single-molecule imaging in live bacteria cells

“…protein (YFP) in C. crescentus [26]. Using an observation rate of 65 Hz and correcting the results for the shape of the C. crescentus cells, Kim et al [26] found that the MreB-YFP construct had a diffusion coefficient of 1.75 + 0.17 mm 2 s -1 .…”

Analytical tools for single-molecule fluorescence imaging in cellulo

“…Directed diffusion has a typically parabolic MSD versus time interval trace, seen during active diffusive processes such as those of translocating molecular motors. 59,60 Anomalous or sub-diffusive behavior 61 is usually modeled as MSD proportional to time interval to the power of an exponent  where  is a coefficient between 0 and 1, indicative of percolation through the disordered media of the cell as well as a putative hopping motion between different confinement domains in the cell across corrals or interactions with specialized domains. [62][63][64][65] Bayesian infer ence quantifies the present state of knowledge and refines it on the basis of new data.…”

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

“…For example, Kim et al observed the dynamics of the single cytoskeletal protein MreB in living bacterial cells using a total internal reflection fluorescence microscope. 5 This method visualizes fluorescence signals of labeled target molecules, and therefore only indirect molecular movement can be detected as a trajectory. Moreover, the spatial resolution of optical microscopy is not sufficient for observing the dynamic structural change of intracellular molecules.…”