Class V β-tubulin alters dynamic instability and stimulates microtubule detachment from centrosomes

A multigene family produces tubulin isotypes that are expressed in a tissue-specific manner, but the role of these isotypes in microtubule assembly and function is unclear. Recently we showed that overexpression or depletion of β5-tubulin, a minor isotype with wide tissue distribution, inhibits cell division. We now report that elevated β5-tubulin causes uninterrupted episodes of microtubule shortening and increased shortening rates. Conversely, depletion of β5-tubulin reduces shortening rates and causes very short excursions of growth and shortening. A tubulin conformation-sensitive antibody indicated that the uninterrupted shortening can be explained by a relative absence of stabilized patches along the microtubules that contain tubulin in an assembly-competent conformation and normally act to restore microtubule growth. In addition to these changes in dynamic instability, overexpression of β5-tubulin causes fragmentation that results from microtubule detachment from centrosomes, and it is this activity that best explains the effects of β5 on cell division. Paclitaxel inhibits microtubule detachment, increases the number of assembly-competent tubulin patches, and inhibits microtubule shortening, thus providing an explanation for why the drug can counteract the phenotypic effects of β5 overexpression. On the basis of these observations, we propose that cells can use β5-tubulin expression to adjust the behavior of the microtubule cytoskeleton.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo

A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken β-tubulin cDNA and cloned. The fragment was shown to be unique in the yeast genome and to contain the gene for yeast β-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation. A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the β-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single β-tubulin gene of yeast has an essential function. Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain β-tubulins.     

Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7?μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1?μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin.     

Clostridium difficile Toxin A Decreases Acetylation of Tubulin,Leading to Microtubule Depolymerization through Activation of Histone Deacetylase 6, and This Mediates Acute Inflammation

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2′,3′-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.     

Chromosome Breakage and Ultrasound

Human lymphocyte cultures were examined for chromosome damage after exposure to ultrasound. Control and treated slides were scored “blind” and showed no evidence of damage due to ultrasound. Neither was there evidence of chromosome damage in blood cultures from six infants whose mothers had ultrasound during pregnancy when compared with that from six infants whose mothers had not. Our results suggest that if diagnostic ultrasound causes chromosome damage it does so with less frequency than acceptable levels of diagnostic x-irradiation.     

20-Hydroxyecdysone induces the expression of one β-tubulin gene in Drosophila Kc cells

The expression of 56D and 60C β-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with β-tubulin subclones show that the 56D β-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3′-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a β-tubulin subunit (P4); this subunit is the so-called β3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced β3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.     

Analysis of Claviceps africana and C. sorghi from India using AFLPs,EF-1α gene intron 4, and β-tubulin gene intron 3

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1α gene intron 4 and β-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1α gene intron 4, the β-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.     

Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

Microtubules are composed of polymerized α/β-tubulin heterodimers. Biogenesis of assembly-competent tubulin dimers is a complex multistep process that requires sequential actions of distinct molecular chaperones and cofactors. Tubulin folding cofactor A (TFCA), which captures β-tubulin during the folding pathway, has been identified in many organisms. Here, we report the crystal structure of Arabidopsis thaliana TFC A (KIESEL, KIS), which forms a monomeric three-helix bundle. The functional binding analysis demonstrated that KIS interacts with β-tubulin in plant. Furthermore, mutagenesis studies indicated that the α-helical regions of KIS participate in β-tubulin binding. Unlike the budding yeast TFC A, the two loop regions of KIS are not required for this interaction suggesting a distinct binding mechanism of TFC A to β-tubulin in plants.

Structured summary

MINT-7968902, MINT-7968915, MINT-7968951, MINT-7968966: KIS (uniprotkb:O04350) physically interacts (MI:0915) with Tub9 (uniprotkb:P29517) by anti tag coimmunoprecipitation (MI:0007)MINT-7968928: KIS (uniprotkb:O04350) and Tub9 (uniprotkb:P29517) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)     

Darwinism and human affairs: By R. D. Alexander. Seattle: University of Washington Press. 317 pp. $14.95

We have examined nuclear transport in Aspergillus nidulans to determine whether microtubles function in the movement of this organelle. Nuclear movement was found to be inhibited in germinating conidia (uninucleate asexual spores) by the microtubule inhibitor benomyl. To show that the benomyl inhibition was due to its effect on microtubules, the test was repeated with mutants which have genetic lesions in β-tubulin which produce resistance to benomyl in one case (benA15) and super-sensitivity in another (benA16). Nuclear movement was resistant to benomyl in the strain carrying benA15 and super-sensitive in the strain carrying benAl6. Since altered sensitivity to benomyl in these strains is specifically due to alterations in β-tubulin, these results show that β-tubulin is involved in nuclear movement. To eliminate the possibility that nuclear movement blockage was a secondary consequence of nuclear division blockage, this experiment was repeated with temperature-sensitive nuclear division mutants. At restrictive temperature, nuclear division was blocked in these mutants but nuclear movement was not. In the presence of benomyl, nuclear division and migration were blocked at permissive and restrictive temperatures. Thus nuclear division blockage alone is not sufficient to block nuclear movement. These experiments were corroborated by similar experiments on a temperature-sensitive nuclear movement mutant. Five previously isolated nonallelic temperature-sensitive nuclear movement mutants, nudA-E, were analyzed genetically and found not to be allelic to the benA (β-tubulin) tubA α-tubulin genes.     

Purification and evaluation of 2, 4-di-tert butylphenol (DTBP) as a biocontrol agent against phyto-pathogenic fungi

A fungal strain, Marasmiellus sp (PUK64), isolated from the mangrove forests in Muthupet, Tamil Nadu, East coast of India, along with others were screened for the search of potent bioactive compounds. A phenolic compound, 2,4-di-tert-butylphenol (DTBP), was isolated from the most promising strain PUK64 and its chemical structure was ascertained. DTBP demonstrated remarkable antifungal activity against the phytopathogenic fungi Aspergillus oryzae, Curvularia lunata and Fusarium verticillioides. In an in-vitro experimental setup, DTBP suppressed the growth of all three fungi, among which F. verticillioides was found to be highly susceptible. This effect relates with the inhibition of spore germination and hyphal growth that we observed. DTBP showed high affinity with the F. verticillioides's β-tubulin protein (determined by ligand-protein docking) as compared to the standard fungicide carbendazim (CBZ). Molecular docking and simulation studies of DTBP with target β-tubulin further confirmed the potential of β-tubulin binding in F. verticillioides. To our knowledge, this is the first report on DTBP-mediated biocontrol of phytopathogenic fungi, produced by Marasmiellus sp. PUK64 that can be potent inhibitor of β-tubulin protein of F. verticillioides.     

Successful Posaconazole Therapy of Disseminated Alternariosis due to <Emphasis Type="Italic">Alternaria infectoria</Emphasis> in a Heart Transplant Recipient

We report a case of phaeohyphomycosis caused by Alternaria infectoria in a 61-year-old heart transplant recipient with multiple skin lesions and pulmonary infiltrates. The infection spread via the haematogenous route from the primary cutaneous lesions into the lungs. The diagnosis was based on the histopathological examination, direct microscopy, skin lesion cultures and detection of Alternaria DNA in the bronchoalveolar lavage fluid using molecular methods. The treatment consisted of a combination of surgical excision and systemic antifungal therapy. Voriconazole was the first agent used but had a weak effect. Posaconazole was subsequently used to achieve a successful response. The isolate was identified as A. infectoria by sequencing of the rDNA ITS region and the partial β-tubulin gene.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

TGF-β-stimulated aberrant expression of class III β-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

The class III β-tubulin isotype (β_III) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III β-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-β (TGF-β) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-β on the aberrant expression of class III β-tubulin and the intracellular signaling pathway mediating these changes. TGF-β-induced aberrant expression and O-linked-β-N-acetylglucosamine (O-GlcNac) modification of class III β-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-β also stimulated phosphorylation of ERK. TGF-β-induced aberrant expression of class III β-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-β stimulated aberrant expression of class III β-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-β stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.     

Cdk1 and BRCA1 target γ-tubulin to microtubule domains

DNA damage is a critical event that requires an appropriate cellular response. This is mediated by checkpoint proteins such as Cdk1 that controls S/G2 and G2/M transition. Cdk1 is required for BRCA1 transport to DNA damage sites inside the nucleus where BRCA1 functions as a scaffold to initiate a signaling cascade. BRCA1 is a multifunctional protein that also ubiquitinates γ-tubulin and, consequently, inhibits microtubule nucleation at the centrosome. Here, we report that γ-tubulin also localizes at confined areas in the microtubule network. Nocodazole-mediated microtubule depolymeration results in disappearance of this γ-tubulin fraction, while microtubule stabilization by taxol preserves this structure. Surprisingly, overexpression of Cdk1 or BRCA1 greatly expands the γ-tubulin coating of microtubules, suggesting that the microtubule-bound γ-tubulin is involved in DNA damage response. This is in accordance with numerous reports of microtubule-associated DNA damage proteins, such as p53, that are transported to the nucleus when DNA damage occurs. γ-Tubulin itself has been reported to form complexes with DNA repair proteins in the nucleus.     

Organization and expression of a-tubulin genes in Drosophila melanogaster: One member of the a-tubulin multigene family is transcribed in both oogenesis and later embryonic development

Genomic clones containing α-tubulin sequences were isolated from a library of Drosophila melanogaster DNA and identified by a hybridization-selection and in vitro-translation procedure. The in vitro translation products were identical to the two electrophoretic variants of α-tubulin present in Drosophila embryos. They co-assembled with an embryonic tubulin fraction to form microtubules in vitro and generated the same partial proteolytic fragments as Drosophila α-tubulins. Hybridization in situ to polytene chromosomes revealed that the α-tubulin sequences constitute a multigene family localized on the right arm of chromosome 3 at sites 84 B3–6, 84 D4–8 and 85 E6–10. Clones hybridizing to these sites corresponded to the three major α-tubulin sequences in genomic DNA. The α-tubulin sequences at 84 B3–6 were present twice per haploid genome, embedded in a large duplicated DNA segment. The sequences of the three genomic α-tubulin genes showed considerable divergence, making it possible to conclude that both of the α-tubulin variants present in embryos are encoded by the genes at 84 B3–6. Furthermore, the abundance of this α-tubulin messenger RNA changes with the requirements for microtubule synthesis during embryo development. The genes at 84 B3–6 encoded both the stored maternal mRNA of the oocyte and the major mRNA transcribed during embryonic development.     

The marine red alga Chondrus crispus has a highly divergent β-tubulin gene with a characteristic 5′ intron: functional and evolutionary implications

We characterized a nuclear gene and its corresponding cDNA encoding β-tubulin (gene TubB1) of the marine red alga Chondrus crispus. The deduced TubB1 protein is the most divergent β-tubulin so far reported with only 64 to 69% amino acid identity relative to other β-tubulins from higher and lower eukaryotes. Our analysis reveals that TubB1 has an accelerated evolutionary rate probably due to a release of functional constraints in connexion with a specialization of microtubular structures in rhodophytes. It further indicates that isoform diversity and functional differentiation of tubulins in eukaryotic cells may be controlled by independent selective constraints. TubB1 has a short spliceosomal intron at its 5′ end which seems to be a characteristic feature of nuclear protein-coding genes from rhodophytes. The splice junctions of the four known rhodophyte introns comply well with the corresponding consensus sequences of higher plants in agreement with previous suggestions from phylogenetic inference that red algae and green plants may be sister groups. The paucity and asymmetrical location of introns in rhodophyte genes can be explained by differential intron loss due to conversion of genes by homologous recombination with cDNAs corresponding to reverse transcribed mRNAs or partially spliced pre-mRNAs, respectively. The identification of an intron containing TubB1 cDNA in C. crispus confirms that pre-mRNAs can escape both splicing and degradation in the nucleus prior to transport into the cytoplasm. Differential Southern hybridizations under non-stringent conditions with homologous and heterologous probes suggest that C. crispus contains a second degenerate β-tubulin gene (or pseudogene?) which, however, is only distantly related to TubB1 as it is to the more conserved homologues of other organisms.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.