A canonical FtsZ protein in <Emphasis Type="Italic">Verrucomicrobium spinosum</Emphasis>, a member of the Bacterial phylum <Emphasis Type="Italic">Verrucomicrobia</Emphasis> that also includes tubulin-producing <Emphasis Type="Italic">Prosthecobacter</Emphasis> species

Background  

The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative.     

The core of the mammalian centriole contains γ-tubulin

Background: The microtubule network, upon which transport occurs in higher cells, is formed by the polymerization of α and β tubulin. The third major tubulin isoform, γ tubulin, is believed to serve a role in organizing this network by nucleating microtubule growth on microtubule-organizing centers, such as the centrosome. Research in vitro has shown that γ tubulin must be restored to stripped centrioles to regenerate the centrosomal functions of duplication and microtubule nucleation.Results We have re-examined the localization of γ tubulin in isolated and in situ mammalian centrosomes using a novel immunocytochemical technique that preserves antigenicity and morphology while allowing increased accessibility. As expected, α tubulin was localized in cytoplasmic and centriolar barrel microtubules and in the associated pericentriolar material. Foci of γ tubulin were observed at the periphery of the organized pericentriolar material, as reported previously, often near the termini of microtubules. A further and major location of γ tubulin was a structure within the proximal end of the centriolar barrel. The distributions were complementary, in that α tubulin was excluded from the core of the centriole, and γ tubulin was excluded from the microtubule barrel.Conclusion We have shown that γ tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that γ tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.     

Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

Rapid and dynamic subcellular reorganization following mechanical stimulation of <Emphasis Type="Italic">Arabidopsis</Emphasis>epidermal cells mimics responses to fungal and oomycete attack

Background  

Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes inArabidopsis plants – after touching the epidermal surface with a microneedle.     

Distribution and Characteristics of βII Tubulin-Enriched Microtubules in Interphase Cells

We have used a polyclonal antibody (Ab196) that specifically recognizes the βII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual β isotypes. Using photoimaging analysis, we observed βII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of βII-enriched microtubules together with the reported increased levels of βII tubulin in taxol-resistant cells (M. Haberet al.,1995,J. Biol. Chem.270, 31269–31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, βII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-βII tubulin antibody but not by anti-acetylated α tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.     

A Change in Tubulin Synthesis in the Process of Tracheary Element Differentiation and Cell Division of Isolated Zinnia Mesophyll Cells

Changes in tubulin synthesis in the process of cytodifferentiationinto tracheary elements and cell division were investigatedusing a culture of single cells isolated from the mesophyllof Zinnia elegans. The tubulin content was measured by a sensitiveimmunoblotting method using a mouse monoclonal antibody to -or ß-tubulin as a probe and mung bean tubulin as astandard. Freshly isolated mesophyll cells had only small amountsof tubulin, but the content increased rapidly between 24 and48 h of culture before morphological differentiation and celldivision. The content rose more than sixfold during 48 h cultureand then decreased slightly. This pattern of increase closelyresembled that of the increase in cortical microtubules (MTs)estimated by electron microscopic analysis. The - and ß-tubulincontents in the cultured cells were almost the same and changedin coordination during culture. The activity of tubulin synthesis was determined by densitometricscanning of spots corresponding to tubulin subunits on an autoradiogramof a two-dimensional polyacrylamide gel of [³⁵S]-methionine-labeledproteins. Tubulin synthesis began as early as between 4 and8 h of culture and its rate increased similarly to the increasein the tubulin content, with the former always preceding thelatter, indicating that the increase in content resulted fromnew tubulin synthesis. (Received December 16, 1986; Accepted February 25, 1987)     

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC₅₀ value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC₅₀ value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10⁻⁷ M⁻¹ s⁻¹ and 4.0 × 10⁻⁹ M⁻¹ s⁻¹ for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin–apigenin binding was 1:1 and binding the binding constant (K_d) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G₂/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α–β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.     

Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant

Background  

EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells.     

Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-Based Analysis of Palmitoylation Sites of In Vitro Palmitoylated Rat Brain Tubulin

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.     

Discrepancy between the <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis

Introduction  

The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-γ (IFN-γ) in these processes.     

The metal tolerance profile of <Emphasis Type="Italic">Thlaspi goesingense</Emphasis> is mimicked in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>heterologously expressing serine acetyl-transferase

Background  

The Ni hyperaccumulator Thlaspi goesingense is tolerant to Ni ≅ Zn, ≅ Co and slightly resistant to > Cd. We previously observed that elevated glutathione, driven by constitutive activation of serine acetyltransferase (SAT), plays a role in the Ni tolerance of T. goesingense.     

Peptide ligand screening of α-synuclein aggregation modulators by <Emphasis Type="Italic">in silico</Emphasis> panning

Background  

α-Synuclein is a Parkinson's-disease-related protein. It forms aggregates in vivo, and these aggregates cause cell cytotoxicity. Aggregation inhibitors are expected to reduce α-synuclein cytotoxicity, and an aggregation accelerator has recently been reported to reduce α-synuclein cytotoxicity. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.     

Diffusion inside microtubules

Recent high-resolution analysis of tubulin's structure has led to the prediction that the taxol binding site and a tubulin acetylation site are on the interior of microtubules, suggesting that diffusion inside microtubules is potentially a biologically and clinically important process. To assess the rates of transport inside microtubules, predictions of diffusion time scales and concentration profiles were made using a model for diffusion with parameters estimated from experiments reported in the literature. Three specific cases were considered: 1) diffusion of αβ-tubulin dimer, 2) diffusion/binding of taxol, and 3) diffusion/binding of an antibody specific for an epitope on the microtubule's interior surface. In the first case tubulin is predicted to require only ∼1 min to reach half the equilibrium concentration in the center of a 40 μm microtubule open at both ends. This relatively rapid transport occurs because of a lack of appreciable affinity between tubulin and the microtubule inner surface and occurs in spite of a three-fold reduction in diffusivity due to hindrance. By contrast the transport of taxol is much slower, requiring days (at nm concentrations) to reach half the equilibrium concentration in the center of a 40 μm microtubule having both ends open. This slow transport is the result of fast, reversible taxol binding to the microtubule's interior surface and the large capacity for taxol (∼12 mm based on interior volume of the microtubule). An antibody directed toward an epitope in the microtubule's interior is predicted to require years to approach equilibrium. These results are difficult to reconcile with previous experimental results where substantial taxol and antibody binding is achieved in minutes, suggesting that these binding sites are on the microtubule exterior. The slow transport rates also suggest that microtubules might be able to serve as vehicles for controlled-release of drugs. Received: 2 March 1998 / Revised version: 3 May 1998 / Accepted: 3 May 1998     

Osmoconditioning prevents the onset of microtubular cytoskeleton and activation of cell cycle and is detrimental for germination of Jatropha curcas L. seeds

• Jatropha curcas is an oilseed crop renowned for its tolerance to a diverse range of environmental stresses. In Brazil, this species is grown in semiarid regions where crop establishment requires a better understanding of the mechanisms underlying appropriate seed, seedling and plant behaviour under water restriction conditions. In this context, the objective of this study was to investigate the physiological and cytological profiles of J. curcas seeds in response to imbibition in water (control) and in polyethylene glycol solution (osmoticum).
• Seed germinability and reactivation of cell cycle events were assessed by means of different germination parameters and immunohistochemical detection of tubulin and microtubules, i.e. tubulin accumulation and microtubular cytoskeleton configurations in water imbibed seeds (control) and in seeds imbibed in the osmoticum.
• Immunohistochemical analysis revealed increasing accumulation of tubulin and appearance of microtubular cytoskeleton in seed embryo radicles imbibed in water from 48 h onwards. Mitotic microtubules were only visible in seeds imbibed in water, after radicle protrusion, as an indication of cell cycle reactivation and cell proliferation, with subsequent root development. Imbibition in osmoticum prevented accumulation of microtubules, i.e. activation of cell cycle, therefore germination could not be resumed.
• Osmoconditioned seeds were able to survive re‐drying and could resume germination after re‐imbibition in water, however, with lower germination performance, possibly due to acquisition of secondary dormancy. This study provides important insights into understanding of the physiological aspects of J. curcas seed germination in response to water restriction conditions.

     

c-Jun N-terminal kinase (JNK) cooperates with Gsk3β to regulate Dishevelled-mediated microtubule stability

Background  

Wnt factors are a large family of signaling molecules that play important roles in the regulation of cell fate specification, tissue polarity and cell movement. In the nervous system, Wnts also regulates the formation of neuronal connection acting as retrograde signals that regulate the remodeling of the axons prior to the assembly of the presynaptic apparatus. The scaffold protein Dishevelled (Dvl) mimics the effect of Wnt on the neuronal cytoskeleton by increasing the number of stable microtubule along the axon shaft and inducing the formation of looped microtubules (MT) at enlarged growth cones. A divergent Wnt-Dvl canonical pathway which bifurcates downstream of Gsk3β regulates MT dynamics.     

Microtubules regulate disassembly of epithelial apical junctions

Background  

Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

Tyrosine phosphorylation of plant tubulin

Phosphorylation of αβ-tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and differentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modification are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.     

<Emphasis Type="Italic">Gdt</Emphasis>2 regulates the transition of <Emphasis Type="Italic">Dictyostelium</Emphasis>cells from growth to differentiation

Background  

Dictyostelium life cycle consists of two distinct phases – growth and development. The control of growth-differentiation transition in Dictyostelium is not completely understood, and only few genes involved in this process are known.