Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc Natl Acad Sci USA

Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2006; 103(29):10929-34. DOI: 10.1073/pnas.0604503103
Source: PubMed


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.

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    • "However, this model has not been stringently proven. It is also still unclear how MreB obtains its localization pattern underneath the cell membrane and what determines the turnover of MreB filaments, which is high in B. subtilis and C. crescentus cells [21] [22] but is apparently low in E. coli cells. We have shown that translation elongation factor EF-Tu forms a structure underneath the cell membrane in B. subtilis cells and co-localizes and interacts with MreB [23]. "
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    ABSTRACT: EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro, and was active in dynamic light scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of colocalizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. FRAP analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 02/2015; 230(8). DOI:10.1016/j.jmb.2015.01.025 · 4.33 Impact Factor
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    • "Interestingly, the diffusion coefficient displayed by the unpolymerized MreB form was significantly slower than expected for a free cytoplasmic protein of similar size (Kim et al., 2006). Such slower diffusion indicated that the movement of soluble MreB is restricted by a yet uncharacterized force, and raised the possibility that unpolymerized MreB interacts either with the cell membrane or with a larger protein complex in the cytoplasm (Kim et al., 2006). Notably, DapI foci were still detected using up to 30 ms exposure time in our HILO time-lapses (Fig. S9C). "
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    ABSTRACT: MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intra-cellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intra-cellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries.
    Molecular Microbiology 11/2013; 91(2). DOI:10.1111/mmi.12467 · 4.42 Impact Factor
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    • "−1 ) (Deich et al., 2004; Kim et al., 2006; Leake et al., 2006; Mullineaux et al., 2006 "
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    ABSTRACT: In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in E. coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule based superresolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments.
    Molecular Microbiology 07/2013; 89(6). DOI:10.1111/mmi.12331 · 4.42 Impact Factor
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