scholarly journals ZapA stabilizes FtsZ filament bundles without slowing down treadmilling dynamics

2019 ◽  
Author(s):  
Paulo Caldas ◽  
Mar López-Pelegrín ◽  
Daniel J.G. Pearce ◽  
Nazmi B. Budanur ◽  
Jan Brugués ◽  
...  
Keyword(s):  
Large Scale ◽  
Strong Influence ◽  
Bacterial Cell Division ◽  
Supported Membrane ◽  
Slowing Down ◽  
Associated Proteins ◽  
Filament Bundles ◽  
Z Ring ◽  
Filament Network

AbstractFor bacterial cell division, treadmilling filaments of FtsZ organize into a ring-like structure at the center of the cell. What governs the architecture and stability of this dynamic Z-ring is currently unknown, but FtsZ-associated proteins have been suggested to play an important role. Here, we used anin vitroreconstitution approach combined with fluorescence microscopy to study the influence of the well-conserved protein ZapA on the organization and dynamics of FtsZ filaments recruited to a supported membrane. We found that ZapA increases the spatial order and stabilizes the steady-state architecture of the FtsZ filament network in a highly cooperative manner. Despite its strong influence on their large-scale organization, ZapA binds only transiently to FtsZ filaments and has no effect on their treadmilling velocity. Together, our data explains how FtsZ-associated proteins can contribute to the precision and stability of the Z-ring without compromising treadmilling dynamics.

scholarly journals Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Paulo Caldas ◽  
Mar López-Pelegrín ◽  
Daniel J. G. Pearce ◽  
Nazmi Burak Budanur ◽  
Jan Brugués ◽  
...  
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Large Scale ◽  
Bacterial Cell Division ◽  
Division Site ◽  
Associated Proteins ◽  
Z Ring ◽  
Cytoskeletal Networks ◽  
Cooperative Ordering

AbstractDuring bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.

scholarly journals Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranesin vitro

2018 ◽  
Author(s):  
Kousik Sundararajan ◽  
Anthony Vecchiarelli ◽  
Kiyoshi Mizuuchi ◽  
Erin D. Goley
Keyword(s):  
Cell Wall ◽  
Lipid Bilayers ◽  
Caulobacter Crescentus ◽  
Dynamic Structure ◽  
Bacterial Cell Division ◽  
Filament Bundles ◽  
Z Ring ◽  
Local Cell

SummaryBacterial cell division requires the assembly of FtsZ protofilaments into a dynamic structure called the ‘Z-ring’. The Z-ring recruits the division machinery and directs local cell wall remodeling for constriction. The organization and dynamics of protofilaments within the Z-ring coordinate local cell wall synthesis during cell constriction, but their regulation is largely unknown. The disordered C-terminal linker (CTL) region ofCaulobacter crescentusFtsZ (CcFtsZ) regulates polymer structure and turnover in solutionin vitro, and regulates Z-ring structure and activity of cell wall enzymesin vivo. To investigate the contributions of the CTL to the polymerization properties of FtsZ on its physiological platform, the cell membrane, we reconstitutedCcFtsZ polymerization on supported lipid bilayers (SLB) and visualized polymer dynamics and structure using total internal reflection fluorescence microscopy. UnlikeE. coliFtsZ protofilaments that organized into large, bundled patterns,CcFtsZ protofilaments assembled into small, dynamic clusters on SLBs. Moreover,CcFtsZ lacking its CTL formed large networks of straight filament bundles that underwent slower turnover than the dynamic clusters of wildtype FtsZ. Ourin vitrocharacterization provides novel insights into species- and CTL-dependent differences between FtsZ assembly properties that are relevant to Z-ring assembly and function on membranesin vivo.

Tetrins: polypeptides that form bundled filaments in Tetrahymena

1990 ◽  
Vol 96 (2) ◽  
pp. 293-302
Author(s):  
J.E. Honts ◽  
N.E. Williams
Keyword(s):  
Intermediate Filaments ◽  
Tetrahymena Pyriformis ◽  
Ciliated Protozoan ◽  
Microtubule Associated Proteins ◽  
Fine Filament ◽  
Helical Content ◽  
In Vitro Assembly ◽  
Associated Proteins ◽  
Filament Bundles

The cortex of the ciliated protozoan Tetrahymena contains a number of fibrous elements, including a network of filaments that pervades the feeding organelle of this organism. The cluster of polypeptides (79–89K; K = 10(3) Mr) in Tetrahymena pyriformis GL-C that constitute these filaments has been purified by in vitro assembly after solubilization in 1.0 M KI. Four distinct sets of these polypeptides, designated ‘tetrins’, have been shown to be distinguishable from each other by immunochemical and biochemical criteria. The smallest filaments reassembled in vitro were 3–4 nm in diameter and these fine filaments were seen to be bundled together into thicker strands of varying diameters, similar to those within the cell. The thicker filament bundles were clearly distinguishable from intermediate filaments, but fine filaments in these bundles were superficially similar to the 2–5 nm filaments described as microtubule-associated proteins in other organisms. The ultrastructure of the tetrin filaments localized within the feeding organelle reveals a substantial presence of these filaments apart from microtubules. In addition, circular dichroism measurements indicate a relatively low alpha-helical content for these filaments and suggest that the tetrins may be substantially different from other fine filament proteins such as the tektins and giardins.

scholarly journals Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Piotr Szwedziak ◽  
Qing Wang ◽  
Tanmay A M Bharat ◽  
Matthew Tsim ◽  
Jan Löwe
Keyword(s):  
Cell Division ◽  
Molecular Model ◽  
Three Dimensional ◽  
Bacterial Cell Division ◽  
Electron Cryomicroscopy ◽  
E Coli ◽  
Ftsz Ring ◽  
Z Ring

Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.

scholarly journals ZapA tetramerization is required for midcell localization and ZapB interaction in Escherichia coli

2019 ◽  
Author(s):  
Nils Y. Meiresonne ◽  
Tanneke den Blaauwen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bacterial Cell Division ◽  
Cellular Processes ◽  
Interaction Partners ◽  
Associated Proteins

AbstractBacterial cell division is guided by FtsZ treadmilling precisely at midcell. FtsZ itself is regulated by FtsZ associated proteins (Zaps) that couple it to different cellular processes. ZapA is known to enhance FtsZ bundling but also forms the synchronizing link with chromosome segregation through ZapB and matS bound MatP. ZapA exists as dimers and tetramers in the cell. Using the ZapAI83E mutant that only forms dimers, this paper investigates the effects of ZapA multimerization state on its interaction partners and cell division. By employing (fluorescence) microscopy and Förster Resonance Energy Transfer in vivo it is shown that; dimeric ZapA is unable to complement a zapA deletion strain and localizes diffusely through the cell but still interacts with FtsZ that is not part of the cell division machinery. Dimeric ZapA is unable to recruit ZapB, which localizes in its presence unipolarly in the cell. Interestingly, the localization profiles of the chromosome and unipolar ZapB anticorrelate. The work presented here confirms previously reported in vitro effects of ZapA multimerization in vivo and further places it in a broader context by revealing the strong implications for ZapB localization and ter linkage.

scholarly journals Assembly properties of bacterial tubulin homolog FtsZ regulated by the positive regulator protein ZipA and ZapA from Pseudomonas aeruginosa

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mujeeb ur Rahman ◽  
Zhe Li ◽  
Tingting Zhang ◽  
Shuheng Du ◽  
Xueqin Ma ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Species ◽  
Biochemical Properties ◽  
Gtpase Activity ◽  
Bacterial Cell Division ◽  
Contractile Ring ◽  
Positive Regulator ◽  
Regulator Protein ◽  
Z Ring

AbstractBacterial tubulin homolog FtsZ self-assembles into dynamic protofilaments, which forms the scaffold for the contractile ring (Z-ring) to achieve bacterial cell division. Here, we study the biochemical properties of FtsZ from Pseudomonas aeruginosa (PaFtsZ) and the effects of its two positive regulator proteins, ZipA and ZapA. Similar to Escherichia coli FtsZ, PaFtsZ had a strong GTPase activity, ~ 7.8 GTP min-1 FtsZ-1 at pH 7.5, and assembled into mainly short single filaments in vitro. However, PaFtsZ protofilaments were mixtures of straight and “intermediate-curved” (100–300 nm diameter) in pH 7.5 solution and formed some bundles in pH 6.5 solution. The effects of ZipA on PaFtsZ assembly varied with pH. In pH 6.5 buffer ZipA induced PaFtsZ to form large bundles. In pH 7.5 buffer PaFtsZ-ZipA protofilaments were not bundled, but ZipA enhanced PaFtsZ assembly and promoted more curved filaments. Comparable to ZapA from other bacterial species, ZapA from P. aeruginosa induced PaFtsZ protofilaments to associate into long straight loose bundles and/or sheets at both pH 6.5 and pH 7.5, which had little effect on the GTPase activity of PaFtsZ. These results provide us further information that ZipA functions as an enhancer of FtsZ curved filaments, while ZapA works as a stabilizer of FtsZ straight filaments.

scholarly journals Storage of proliferating gooseberry cultures under slow growth conditions

2021 ◽  
Author(s):  
Danuta Kucharska ◽  
Robert Maciorowski ◽  
Małgorzata Kunka ◽  
Angelika Niewiadomska-Wnuk
Keyword(s):  
Large Scale ◽  
Slow Growth ◽  
Shoot Proliferation ◽  
Growth Conditions ◽  
Storage Conditions ◽  
In Vitro Cultures ◽  
Slowing Down ◽  
Growth Room

Short storage of in vitro cultures under slow-growth conditions is included in the commercial large-scale micropropagation process. It is dictated by the organizational scheme that provides temporary stop multiplication of shoots for some months. To avoid subculturing to fresh media every 4 weeks, which is obligatory for gooseberry, they can be kept in conditions that protect them from ageing, by slowing down their metabolism. To develop a rational schedule of gooseberry micropropagation, two experiments were used to adopt a temperature and length of time for storage. The best results were obtained with storage conditions at 2 °C for two or four months for proliferating cultures. Under these conditions, the percentage of necrotic shoots was low (< 10%), and shoot proliferation in the subsequent passages was at a level similar to proliferation cultures incubated in the growth room and sub-cultured monthly. The rate of shoots > 1 cm was higher than in the control in the growth room. Storage at 4 °C increased the probability of necrotic shoots up to 80% and decreased the number of all shoots and shoots > 1 cm in subsequent passages.

scholarly journals Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro.

1981 ◽  
Vol 90 (2) ◽  
pp. 467-473 ◽  
Author(s):  
R F Sattilaro ◽  
W L Dentler ◽  
E L LeCluyse
Keyword(s):  
Actin Filaments ◽  
Muscle Actin ◽  
Actin Bundles ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Filament Bundles ◽  
Reversible Formation ◽  
Brain Microtubules ◽  
28 Nm

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP-actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene-adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.

scholarly journals ZapE Is a Novel Cell Division Protein Interacting with FtsZ and Modulating the Z-Ring Dynamics

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Benoit S. Marteyn ◽  
Gouzel Karimova ◽  
Andrew K. Fenton ◽  
Anastasia D. Gazi ◽  
Nicholas West ◽  
...  
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Dependent Manner ◽  
Bacterial Cell Division ◽  
Low Oxygen ◽  
Ftsz Ring ◽  
Division Process ◽  
Z Ring ◽  
Cell Division Protein

ABSTRACTBacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss ofzapEresults in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation ofzapEleads to elongation ofEscherichia coliand affects the dynamics of the Z-ring during division.In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner.IMPORTANCEBacterial cell division has mainly been characterizedin vitro. In this report, we could identify ZapE as a novel cell division protein which is not essentialin vitrobut is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. AszapEis not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future.

FtsA G50E mutant suppresses the essential requirement for FtsK during bacterial cell division in Escherichia coli

2020 ◽  
Vol 66 (4) ◽  
pp. 313-327
Author(s):  
Alison M. Berezuk ◽  
Elyse J. Roach ◽  
Laura Seidel ◽  
Reggie Y. Lo ◽  
Cezar M. Khursigara
Keyword(s):  
Escherichia Coli ◽  
Protein Interaction ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Bacterial Cell Division ◽  
Essential Requirement ◽  
Essential Protein ◽  
Z Ring

In Escherichia coli, the N-terminal domain of the essential protein FtsK (FtsKN) is proposed to modulate septum formation through the formation of dynamic and essential protein interactions with both the Z-ring and late-stage division machinery. Using genomic mutagenesis, complementation analysis, and in vitro pull-down assays, we aimed to identify protein interaction partners of FtsK essential to its function during division. Here, we identified the cytoplasmic Z-ring membrane anchoring protein FtsA as a direct protein–protein interaction partner of FtsK. Random genomic mutagenesis of an ftsK temperature-sensitive strain of E. coli revealed an FtsA point mutation (G50E) that is able to fully restore normal cell growth and morphology, and further targeted site-directed mutagenesis of FtsA revealed several other point mutations capable of fully suppressing the essential requirement for functional FtsK. Together, this provides insight into a potential novel co-complex formed between these components during division and suggests FtsA may directly impact FtsK function.

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