The Experts below are selected from a list of 2052 Experts worldwide ranked by ideXlab platform
Peter L Graumann - One of the best experts on this subject based on the ideXlab platform.
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Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation.
BMC molecular and cell biology, 2020Co-Authors: Simon Dersch, Herve Joel Defeu Soufo, Christian Reimold, Joshua Stoll, Hannes Breddermann, Thomas Heimerl, Peter L GraumannAbstract:BACKGROUND MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles. RESULTS Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent magnesium and calcium ions, all three proteins formed highly disordered sheets of filaments that could split up or merge, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs. CONCLUSIONS Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerise. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently split up filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament splitting in vivo.
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translation elongation factor ef tu modulates filament formation of actin like MreB protein in vitro
Journal of Molecular Biology, 2015Co-Authors: Herve Joel Defeu Soufo, Christian Reimold, Hannes Breddermann, Hans Georg Mannherz, Peter L GraumannAbstract: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 co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching 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.
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Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology
Molecular biology of the cell, 2013Co-Authors: Christian Reimold, Herve Joel Defeu Soufo, Felix Dempwolff, Peter L GraumannAbstract:The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.
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Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.
PloS one, 2011Co-Authors: Felix Dempwolff, Christian Reimold, Michael Reth, Peter L GraumannAbstract:Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.
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bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system
Molecular Microbiology, 2010Co-Authors: Herve Joel Defeu Soufo, Peter L GraumannAbstract:Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria.
Herve Joel Defeu Soufo - One of the best experts on this subject based on the ideXlab platform.
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Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation.
BMC molecular and cell biology, 2020Co-Authors: Simon Dersch, Herve Joel Defeu Soufo, Christian Reimold, Joshua Stoll, Hannes Breddermann, Thomas Heimerl, Peter L GraumannAbstract:BACKGROUND MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles. RESULTS Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent magnesium and calcium ions, all three proteins formed highly disordered sheets of filaments that could split up or merge, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs. CONCLUSIONS Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerise. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently split up filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament splitting in vivo.
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Polymerization of Bacillus Subtilis MreB on A Lipid Membrane Reveals Lateral Co-Polymerization of MreB Paralogs and Strong Effects of Cations on Filament Formation
2020Co-Authors: Simon Dersch, Herve Joel Defeu Soufo, Christian Reimold, Hannes Breddermann, Thomas Heimerl, Peter GraumannAbstract:Abstract Background MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles. Results Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent ions, all three proteins formed highly disordered filaments that appeared to branch and fuse, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs. Conslusions Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerase. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently branched filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament branching in vivo.
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translation elongation factor ef tu modulates filament formation of actin like MreB protein in vitro
Journal of Molecular Biology, 2015Co-Authors: Herve Joel Defeu Soufo, Christian Reimold, Hannes Breddermann, Hans Georg Mannherz, Peter L GraumannAbstract: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 co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching 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.
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Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology
Molecular biology of the cell, 2013Co-Authors: Christian Reimold, Herve Joel Defeu Soufo, Felix Dempwolff, Peter L GraumannAbstract:The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.
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bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system
Molecular Microbiology, 2010Co-Authors: Herve Joel Defeu Soufo, Peter L GraumannAbstract:Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria.
Zemer Gitai - One of the best experts on this subject based on the ideXlab platform.
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AimB Is a Small Protein Regulator of Cell Size and MreB Assembly.
Biophysical journal, 2020Co-Authors: John N. Werner, Zemer Gitai, Handuo Shi, Jen Hsin, Kerwyn Casey Huang, Eric A. KleinAbstract:Abstract The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeleton systems, few MreB-interacting proteins have been well characterized. Here, we identify a small protein from Caulobacter crescentus, an assembly inhibitor of MreB (AimB). AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Furthermore, aimB appears to be essential, and its depletion results in decreased cell width and increased resistance to A22, a small-molecule inhibitor of MreB assembly. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft and that this interaction is favored for C. crescentus MreB over Escherichia coli MreB because of a closer match in the degree of opening with AimB size, suggesting coevolution of AimB with MreB conformational dynamics in C. crescentus. We support this model through functional analysis of point mutants in both AimB and MreB, photo-cross-linking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings are consistent with AimB promoting MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor. Because AimB has only 104 amino acids and small proteins are often poorly characterized, our work suggests the possibility of more bacterial cytoskeletal regulators to be found in this class. Thus, like FtsZ and eukaryotic actin, MreB may have a rich repertoire of regulators to tune its precise assembly and dynamics.
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AimB is a small protein regulator of cell size and MreB assembly
2019Co-Authors: John N. Werner, Zemer Gitai, Handuo Shi, Jen Hsin, Kerwyn Casey Huang, Eric A. KleinAbstract:Abstract The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeletons, few MreB interacting proteins have been well characterized. Here we identify a small protein from Caulobacter crescentus, AimB, as an Assembly Inhibitor of MreB. AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft. We validate this model through functional analysis of point mutants in both AimB and MreB, photo-crosslinking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings indicate that AimB promotes MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor.
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MreB polymers and curvature localization are enhanced by RodZ and predict E. coli's cylindrical uniformity.
Nature communications, 2018Co-Authors: Benjamin P. Bratton, Zemer Gitai, Joshua W. Shaevitz, Randy M. MorgensteinAbstract:The actin-like protein MreB has been proposed to coordinate the synthesis of the cell wall to determine cell shape in bacteria. MreB is preferentially localized to areas of the cell with specific curved geometries, avoiding the cell poles. It remains unclear whether MreB’s curvature preference is regulated by additional factors, and which specific features of MreB promote specific features of rod shape growth. Here, we show that the transmembrane protein RodZ modulates MreB curvature preference and polymer number in E. coli, properties which are regulated independently. An unbiased machine learning analysis shows that MreB polymer number, the total length of MreB polymers, and MreB curvature preference are key correlates of cylindrical uniformity, the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2 = 0.93). Our data thus suggest RodZ promotes the assembly of geometrically-localized MreB polymers that lead to the growth of uniform cylinders.
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rodz promotes MreB polymer formation and curvature localization to determine the cylindrical uniformity of e coli shape
bioRxiv, 2017Co-Authors: Randy M. Morgenstein, Benjamin P. Bratton, Joshua W. Shaevitz, Zemer GitaiAbstract:Cell shape in bacteria is determined by the cell wall, which is synthesized by a variety of proteins whose actions are coordinated by the actin-like MreB protein. MreB uses local geometric cues of envelope curvature to avoid the cell poles and localize to specific regions of the cell body. However, it remains unclear whether MreB9s curvature preference is regulated by additional factors, and which features of MreB are essential for specific aspects of rod shape growth, such as cylindrical uniformity. Here we show that in addition to its previously-described role in mediating MreB motion, RodZ also modulates MreB polymer number and curvature preference. MreB polymer number and curvature localization can be regulated independently. Quantitative 3D measurements and a series of mutant strains show that among various properties of MreB, polymer number, total length of MreB polymers, and MreB curvature preference are the key determinants of cylindrical uniformity, a measure of the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2=0.93). Our data suggest a model for rod shape in which RodZ promotes the assembly of multiple long MreB polymers that ensure the growth of a uniform cylinder
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MreB Orientation Correlates with Cell Diameter in Escherichia coli
Biophysical journal, 2016Co-Authors: Nikolay Ouzounov, Zemer Gitai, Benjamin P. Bratton, Jeffrey Nguyen, David Jacobowitz, Joshua W. ShaevitzAbstract:Bacteria have remarkably robust cell shape control mechanisms. For example, cell diameter only varies by a few percent across a given population. The bacterial actin homolog, MreB, is necessary for establishment and maintenance of rod shape although the detailed properties of MreB that are important for shape control remained unknown. In this study, we perturb MreB in two ways: by treating cells with the polymerization-inhibiting drug A22 and by creating point mutants in MreB. These perturbations modify the steady-state diameter of cells over a wide range, from 790 ± 30 nm to 1700 ± 20 nm. To determine which properties of MreB are important for diameter control, we correlated structural characteristics of fluorescently tagged MreB polymers with cell diameter by simultaneously analyzing three-dimensional images of MreB and cell shape. Our results indicate that the helical pitch angle of MreB inversely correlates with the cell diameter of Escherichia coli. Other correlations between MreB and cell diameter are not found to be significant. These results demonstrate that the physical properties of MreB filaments are important for shape control and support a model in which MreB organizes the cell wall growth machinery to produce a chiral cell wall structure and dictate cell diameter.
James W Gober - One of the best experts on this subject based on the ideXlab platform.
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MreB: pilot or passenger of cell wall synthesis?
Trends in microbiology, 2011Co-Authors: Courtney L White, James W GoberAbstract:The discovery that the bacterial cell shape determinant MreB is related to actin spurred new insights into bacterial morphogenesis and development. The trafficking and mechanical roles of the eukaryotic cytoskeleton were hypothesized to have a functional ancestor in MreB based on evidence implicating MreB as an organizer of cell wall synthesis. Genetic, biochemical and cytological studies implicate MreB as a coordinator of a large multi-protein peptidoglycan (PG) synthesizing holoenzyme. Recent advances in microscopy and new biochemical evidence, however, suggest that MreB may function differently than previously envisioned. This review summarizes our evolving knowledge of MreB and attempts to refine the generalized model of the proteins organizing PG synthesis in bacteria. This is generally thought to be conserved among eubacteria and the majority of the discussion will focus on studies from a few well-studied model organisms.
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positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and mred
Molecular Microbiology, 2010Co-Authors: Courtney L White, Aleksandar Kitich, James W GoberAbstract:In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.
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the cell shape proteins MreB and mrec control cell morphogenesis by positioning cell wall synthetic complexes
Molecular Microbiology, 2007Co-Authors: Arun V Divakaruni, Courtney L White, Cyril Baida, James W GoberAbstract:Summary MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogen- esis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthe- sized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan- synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluores- cent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of pepti- doglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orches- trate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient pepti- doglycan synthetic activity.
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MreB the cell shape determining bacterial actin homologue co ordinates cell wall morphogenesis in caulobacter crescentus
Molecular Microbiology, 2004Co-Authors: Rainer M Figge, Arun V Divakaruni, James W GoberAbstract:Summary The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2–peptidoglycan biosynthesis complex.
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MreB, the cell shape‐determining bacterial actin homologue, co‐ordinates cell wall morphogenesis in Caulobacter crescentus
Molecular microbiology, 2004Co-Authors: Rainer M Figge, Arun V Divakaruni, James W GoberAbstract:Summary The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2–peptidoglycan biosynthesis complex.
Christian Reimold - One of the best experts on this subject based on the ideXlab platform.
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Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation.
BMC molecular and cell biology, 2020Co-Authors: Simon Dersch, Herve Joel Defeu Soufo, Christian Reimold, Joshua Stoll, Hannes Breddermann, Thomas Heimerl, Peter L GraumannAbstract:BACKGROUND MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles. RESULTS Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent magnesium and calcium ions, all three proteins formed highly disordered sheets of filaments that could split up or merge, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs. CONCLUSIONS Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerise. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently split up filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament splitting in vivo.
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Polymerization of Bacillus Subtilis MreB on A Lipid Membrane Reveals Lateral Co-Polymerization of MreB Paralogs and Strong Effects of Cations on Filament Formation
2020Co-Authors: Simon Dersch, Herve Joel Defeu Soufo, Christian Reimold, Hannes Breddermann, Thomas Heimerl, Peter GraumannAbstract:Abstract Background MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles. Results Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent ions, all three proteins formed highly disordered filaments that appeared to branch and fuse, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs. Conslusions Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerase. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently branched filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament branching in vivo.
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translation elongation factor ef tu modulates filament formation of actin like MreB protein in vitro
Journal of Molecular Biology, 2015Co-Authors: Herve Joel Defeu Soufo, Christian Reimold, Hannes Breddermann, Hans Georg Mannherz, Peter L GraumannAbstract: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 co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching 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.
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Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology
Molecular biology of the cell, 2013Co-Authors: Christian Reimold, Herve Joel Defeu Soufo, Felix Dempwolff, Peter L GraumannAbstract:The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.
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Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.
PloS one, 2011Co-Authors: Felix Dempwolff, Christian Reimold, Michael Reth, Peter L GraumannAbstract:Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.