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1
Shu, H. B., and H. C. Joshi. "Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells." Journal of Cell Biology 130, no. 5 (September 1, 1995): 1137–47. http://dx.doi.org/10.1083/jcb.130.5.1137.
Full textAbstract:
alpha-, beta-, and gamma-tubulins are evolutionarily highly conserved members of the tubulin gene superfamily. While the abundant members, alpha- and beta-tubulins, constitute the building blocks of cellular microtubule polymers, gamma-tubulin is a low abundance protein which localized to the pericentriolar material and may play a role in microtubule assembly. To test whether gamma-tubulin mediates the nucleation of microtubule assembly in vivo, and co-assembles with alpha- and beta-tubulins into microtubules or self-assembles into macro-molecular structures, we experimentally elevated the expression of gamma-tubulin in the cell cytoplasm. In most cells, overexpression of gamma-tubulin causes a dramatic reorganization of the cellular microtubule network. Furthermore, we show that when overexpressed, gamma-tubulin causes ectopic nucleation of microtubules which are not associated with the centrosome. In a fraction of cells, gamma-tubulin self-assembles into novel tubular structures with a diameter of approximately 50 nm (named gamma-tubules). Furthermore, unlike microtubules, gamma-tubules are resistant to cold or drug induced depolymerization. These data provide evidence that gamma-tubulin can cause nucleation of microtubule assembly and can self-assemble into novel tubular structures.
2
Inclan, Y. F., and E. Nogales. "Structural models for the self-assembly and microtubule interactions of gamma-, delta- and epsilon-tubulin." Journal of Cell Science 114, no. 2 (January 15, 2001): 413–22. http://dx.doi.org/10.1242/jcs.114.2.413.
Full textAbstract:
alphabeta-tubulin heterodimers self-assemble to form microtubules nucleated by gamma-tubulin in the cell. Gamma-tubulin is believed to recruit the alphabeta-tubulin dimers that form the minus ends of microtubules, but the molecular mechanism of this action remains a matter of heated controversy. Still less is known about the function and molecular interactions of delta-tubulin and epsilon-tubulin. delta-tubulin may seed the formation of the C triplet tubules in the basal bodies of Chlamydomonas and epsilon-tubulin is known to localize to the centrosome in a cell cycle-dependent manner. Using the structure of alphabeta tubulin as a model, we have analyzed the sequences of gamma-, delta- and epsilon-tubulin in regions corresponding to different polymerization interfaces in the tubulin alphabeta dimer. The sequence comparisons sometimes show clear conservation, pointing to similar types of contacts being functionally important for the new tubulin considered. Conversely, certain surfaces show marked differences that rule out equivalent interactions for non-microtubular tubulins. This sequence/structure analysis has led us to structural models of how these special tubulins may be involved in protein-protein contacts that affect microtubule self-assembly. delta-tubulin most likely interacts longitudinally with alpha-tubulin at the minus ends of microtubules, while epsilon-tubulin most likely binds to the plus end of beta-tubulin. Conservation of key residues in gamma-tubulin suggests that it is capable of longitudinal self-assembly. The implications for the protofilament and template models of nucleation are considered.
3
Burke, D., P. Gasdaska, and L. Hartwell. "Dominant effects of tubulin overexpression in Saccharomyces cerevisiae." Molecular and Cellular Biology 9, no. 3 (March 1989): 1049–59. http://dx.doi.org/10.1128/mcb.9.3.1049-1059.1989.
Full textAbstract:
The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.
4
Burke, D., P. Gasdaska, and L. Hartwell. "Dominant effects of tubulin overexpression in Saccharomyces cerevisiae." Molecular and Cellular Biology 9, no. 3 (March 1989): 1049–59. http://dx.doi.org/10.1128/mcb.9.3.1049.
Full textAbstract:
The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.
5
Burland, T. G., E. C. Paul, M. Oetliker, and W. F. Dove. "A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia." Molecular and Cellular Biology 8, no. 3 (March 1988): 1275–81. http://dx.doi.org/10.1128/mcb.8.3.1275-1281.1988.
Full textAbstract:
The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a beta-tubulin cDNA clone, beta 105, which is shown to correspond to the transcript of the betC beta-tubulin locus and to encode beta 2 tubulin, the beta tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that beta 2 tubulin is only 83% identical to the two beta tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum beta 2 tubulin and the beta tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum beta 2 tubulin is no more similar to, for example, Aspergillus beta tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged beta tubulin as well as one or more beta tubulins that conform more closely to a consensus beta-tubulin sequence. We suggest that beta-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among beta tubulins could have resulted through neutral drift. For example, exclusive use of Physarum beta 2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the beta tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of beta tubulins may allow positive selection to operate more freely to refine beta-tubulin function.
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Burland, T. G., E. C. Paul, M. Oetliker, and W. F. Dove. "A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia." Molecular and Cellular Biology 8, no. 3 (March 1988): 1275–81. http://dx.doi.org/10.1128/mcb.8.3.1275.
Full textAbstract:
The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a beta-tubulin cDNA clone, beta 105, which is shown to correspond to the transcript of the betC beta-tubulin locus and to encode beta 2 tubulin, the beta tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that beta 2 tubulin is only 83% identical to the two beta tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum beta 2 tubulin and the beta tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum beta 2 tubulin is no more similar to, for example, Aspergillus beta tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged beta tubulin as well as one or more beta tubulins that conform more closely to a consensus beta-tubulin sequence. We suggest that beta-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among beta tubulins could have resulted through neutral drift. For example, exclusive use of Physarum beta 2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the beta tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of beta tubulins may allow positive selection to operate more freely to refine beta-tubulin function.
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Zhou, Yujun, Jianqiang Xu, Yuanye Zhu, Yabing Duan, and Mingguo Zhou. "Mechanism of Action of the Benzimidazole Fungicide on Fusarium graminearum: Interfering with Polymerization of Monomeric Tubulin But Not Polymerized Microtubule." Phytopathology® 106, no. 8 (August 2016): 807–13. http://dx.doi.org/10.1094/phyto-08-15-0186-r.
Full textAbstract:
Tubulins are the proposed target of clinically relevant anticancer drugs, anthelmintic, and fungicide. β2-tubulin of the plant pathogen Fusarium graminearum was considered as the target of benzimidazole compounds by homology modeling in our previous work. In this study, α1-, α2-, and β2-tubulin of F. graminearum were produced in Escherichia coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant β2-tubulin and reduced the maximum fluorescence intensity of 2 μM β2-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibited the polymerization of α1-/β2-tubulins and α2-/β2-tubulins 90.9 ± 0.4 and 93.5 ± 0.05%, respectively, in vitro. A similar result appeared with benomyl on the polymerization of α1-/β2-tubulins and α2-/β2-tubulins at 89.9 ± 0.1% and 92.6 ± 1.2% inhibition ratios, respectively. In addition, thiabendazole inhibited 81.6 ± 1% polymerization of α1-/β2-tubulins, whereas it had less effect on α2-/β2-tubulin polymerization, with 20.1 ± 1.9% inhibition ratio. However, the three compounds cannot destabilize the polymerized microtubule. To illuminate the issue, mapping the carbendazim binding sites and β/α subunit interface on β/α-tubulin complexes by homology modeling showed that the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin-specific anti-F. graminearum compounds.
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Rudolph, J. E., M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff. "Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function." Molecular and Cellular Biology 7, no. 6 (June 1987): 2231–42. http://dx.doi.org/10.1128/mcb.7.6.2231-2242.1987.
Full textAbstract:
The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.
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Rudolph, J. E., M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff. "Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function." Molecular and Cellular Biology 7, no. 6 (June 1987): 2231–42. http://dx.doi.org/10.1128/mcb.7.6.2231.
Full textAbstract:
The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.
10
Chu, Chih-Wen, Fajian Hou, Junmei Zhang, Lilian Phu, Alex V. Loktev, Donald S. Kirkpatrick, Peter K. Jackson, Yingming Zhao, and Hui Zou. "A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation." Molecular Biology of the Cell 22, no. 4 (February 15, 2011): 448–56. http://dx.doi.org/10.1091/mbc.e10-03-0203.
Full textAbstract:
Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.
11
Schneider, A., U. Plessmann, and K. Weber. "Subpellicular and flagellar microtubules of Trypanosoma brucei are extensively glutamylated." Journal of Cell Science 110, no. 4 (February 15, 1997): 431–37. http://dx.doi.org/10.1242/jcs.110.4.431.
Full textAbstract:
To determine the spectrum of tubulin variants in cytoskeletons of Trypanosoma brucei carboxy-terminal fragments of alpha- and beta-tubulin were isolated and characterized by sequencing and mass spectrometry. All variants arise by posttranslational modifications. We confirm the presence of tyrosinated and detyrosinated alpha-tubulin. Unexpectedly, but in line with its sequence, beta-tubulin also occurs with and without its carboxy-terminal tyrosine. Both tyrosinated and detyrosinated alpha- and beta-tubulins are extensively glutamylated. Unglutamylated tubulins are only trace components of the cytoskeletal microtubules. The maximal numbers of glutamyl residues in the lateral chain are 15 and 6 for alpha- and beta-tubulin, respectively. The oligoglutamyl side chain is linked via an isopeptide bond to glutamic acid residues 445 of alpha- and 435 of beta-tubulin. The same sites are used in glutamylated tubulins of mammalian brain. No tubulin variants based on polyglycylation are detected in cytoskeletal preparations or in isolated flagella. Tubulin specific incorporation of radioactive glutamate but not of glycine is observed when protein biosynthesis is completely inhibited in Trypanosoma cells. Possible reasons for the absence of polyglycylated tubulins from the trypanosomal axoneme are discussed. Finally we show that lysine 40 of the flagellar alpha-tubulin is completely acetylated.
12
Chumová, Jana, Hana Kourová, Lucie Trögelová, Petr Halada, and Pavla Binarová. "Microtubular and Nuclear Functions of γ-Tubulin: Are They LINCed?" Cells 8, no. 3 (March 19, 2019): 259. http://dx.doi.org/10.3390/cells8030259.
Full textAbstract:
γ-Tubulin is a conserved member of the tubulin superfamily with a function in microtubule nucleation. Proteins of γ-tubulin complexes serve as nucleation templates as well as a majority of other proteins contributing to centrosomal and non-centrosomal nucleation, conserved across eukaryotes. There is a growing amount of evidence of γ-tubulin functions besides microtubule nucleation in transcription, DNA damage response, chromatin remodeling, and on its interactions with tumor suppressors. However, the molecular mechanisms are not well understood. Furthermore, interactions with lamin and SUN proteins of the LINC complex suggest the role of γ-tubulin in the coupling of nuclear organization with cytoskeletons. γ-Tubulin that belongs to the clade of eukaryotic tubulins shows characteristics of both prokaryotic and eukaryotic tubulins. Both human and plant γ-tubulins preserve the ability of prokaryotic tubulins to assemble filaments and higher-order fibrillar networks. γ-Tubulin filaments, with bundling and aggregating capacity, are suggested to perform complex scaffolding and sequestration functions. In this review, we discuss a plethora of γ-tubulin molecular interactions and cellular functions, as well as recent advances in understanding the molecular mechanisms behind them.
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Yu, Nuo, and Niels Galjart. "TAPping into the treasures of tubulin using novel protein production methods." Essays in Biochemistry 62, no. 6 (November 14, 2018): 781–92. http://dx.doi.org/10.1042/ebc20180033.
Full textAbstract:
Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the ‘tubulome’ of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types.
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SULIMENKO, Vadym, Tetyana SULIMENKO, Slobodan POZNANOVIC, Volodymyr NECHIPORUK-ZLOY, Konrad J. BÖHM, Libor MACUREK, Eberhard UNGER, and Pavel DRÁBER. "Association of brain γ-tubulins with αβ-tubulin dimers." Biochemical Journal 365, no. 3 (August 1, 2002): 889–95. http://dx.doi.org/10.1042/bj20020175.
Full textAbstract:
γ-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by γ-tubulin and the regulation of this process are not fully understood. Here we show that there are two γ-tubulin forms in the brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of a high salt concentration. Both γ-tubulins co-polymerized with tubulin dimers, and multiple γ-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against γ-tubulin and α-tubulin revealed interactions of both γ-tubulin forms with tubulin dimers, irrespective of the size of complexes. We suggest that, besides small and large γ-tubulin complexes, other molecular γ-tubulin form(s) exist in brain extracts. Two-dimensional electrophoresis revealed multiple charge variants of γ-tubulin in both brain extracts and microtubule protein preparations. Post-translational modification(s) of γ-tubulins might therefore have an important role in the regulation of microtubule nucleation in neuronal cells.
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Hoyle, H. D., and E. C. Raff. "Two Drosophila beta tubulin isoforms are not functionally equivalent." Journal of Cell Biology 111, no. 3 (September 1, 1990): 1009–26. http://dx.doi.org/10.1083/jcb.111.3.1009.
Full textAbstract:
We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.
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Lin, Zhewang, Ivana Gasic, Viswanathan Chandrasekaran, Niklas Peters, Sichen Shao, Timothy J. Mitchison, and Ramanujan S. Hegde. "TTC5 mediates autoregulation of tubulin via mRNA degradation." Science 367, no. 6473 (November 14, 2019): 100–104. http://dx.doi.org/10.1126/science.aaz4352.
Full textAbstract:
Tubulins play crucial roles in cell division, intracellular traffic, and cell shape. Tubulin concentration is autoregulated by feedback control of messenger RNA (mRNA) degradation via an unknown mechanism. We identified tetratricopeptide protein 5 (TTC5) as a tubulin-specific ribosome-associating factor that triggers cotranslational degradation of tubulin mRNAs in response to excess soluble tubulin. Structural analysis revealed that TTC5 binds near the ribosome exit tunnel and engages the amino terminus of nascent tubulins. TTC5 mutants incapable of ribosome or nascent tubulin interaction abolished tubulin autoregulation and showed chromosome segregation defects during mitosis. Our findings show how a subset of mRNAs can be targeted for coordinated degradation by a specificity factor that recognizes the nascent polypeptides they encode.
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Pamula, Melissa C., Shih-Chieh Ti, and Tarun M. Kapoor. "The structured core of human β tubulin confers isotype-specific polymerization properties." Journal of Cell Biology 213, no. 4 (May 16, 2016): 425–33. http://dx.doi.org/10.1083/jcb.201603050.
Full textAbstract:
Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a “tubulin code” in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human β isotype IIB and characterize polymerization dynamics. Microtubules with βIIB have catastrophe frequencies approximately threefold lower than those with isotype βIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric β tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved β tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin’s polymerization properties across a wide range.
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Liu, Congshan, Jiaqing Yao, Jianhai Yin, Jian Xue, and Haobing Zhang. "Recombinant α- and β-tubulin from Echinococcus granulosus: expression, purification and polymerization." Parasite 25 (2018): 62. http://dx.doi.org/10.1051/parasite/2018063.
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Echinococcosis, which causes a high disease burden and is of great public health significance, is caused by the larval stage of Echinococcus species. It has been suggested that tubulin is the target of benzimidazoles, the only drugs for the treatment of echinococcosis. This study evaluated the characteristics of tubulins from Echinococcus granulosus. The full-length cDNAs of E. granulosus α- and β-tubulin isoforms were cloned by reverse transcription PCR from protoscolex RNA. Then, these two tubulin isoforms (α9 and β4) were recombinantly expressed as insoluble inclusion bodies in Escherichia coli. Nickel affinity chromatography was used to purify and refold the contents of these inclusion bodies as active proteins. The polymerization of tubulins was monitored by UV spectrophotometry (A350) and confirmed by confocal microscopy and transmission electron microscopy (TEM). Nucleotide sequence analysis revealed that E. granulosus 1356 bp α9-tubulin and 1332 bp β4-tubulin encode corresponding proteins of 451 and 443 amino acids. The average yields of α9- and β4-tubulin were 2.0–3.0 mg/L and 3.5–5.0 mg/L of culture, respectively. Moreover, recombinant α9- and β4-tubulin were capable of polymerizing into microtubule-like structures under appropriate conditions in vitro. These recombinant tubulins could be helpful for screening anti-Echinococcus compounds targeting the tubulins of E. granulosus.
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Trivinos-Lagos, L., T. Ohmachi, C. Albrightson, R. G. Burns, H. L. Ennis, and R. L. Chisholm. "The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes." Journal of Cell Science 105, no. 4 (August 1, 1993): 903–11. http://dx.doi.org/10.1242/jcs.105.4.903.
Full textAbstract:
As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.
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Kristensson, Maria Alvarado. "The Game of Tubulins." Cells 10, no. 4 (March 28, 2021): 745. http://dx.doi.org/10.3390/cells10040745.
Full textAbstract:
Members of the tubulin superfamily are GTPases; the activities of GTPases are necessary for life. The members of the tubulin superfamily are the constituents of the microtubules and the γ-tubulin meshwork. Mutations in members of the tubulin superfamily are involved in developmental brain disorders, and tubulin activities are the target for various chemotherapies. The intricate functions (game) of tubulins depend on the activities of the GTP-binding domain of α-, β-, and γ-tubulin. This review compares the GTP-binding domains of γ-tubulin, α-tubulin, and β-tubulin and, based on their similarities, recapitulates the known functions and the impact of the γ-tubulin GTP-binding domain in the regulation of the γ-tubulin meshwork and cellular homeostasis.
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Gong, Z. Y., and B. P. Brandhorst. "Stimulation of tubulin gene transcription by deciliation of sea urchin embryos." Molecular and Cellular Biology 7, no. 12 (December 1987): 4238–46. http://dx.doi.org/10.1128/mcb.7.12.4238-4246.1987.
Full textAbstract:
Deciliation by hypertonic shock of embryos of the sea urchin Lytechinus pictus resulted in an increase in synthesis of alpha- and beta-tubulins, the consequence of an increased concentration of RNA encoding the tubulins. RNA run-on assays in isolated nuclei indicated that this response is due to a transient increase in the rate of synthesis of tubulin RNA beginning within 5 min of deciliation. This enhancement of tubulin gene transcription also occurred in deciliated embryos treated with the microtubule-depolymerizing agent colcemid; thus the reaction to deciliation is not a response to a reduction in concentration of unpolymerized tubulin utilized for ciliogenesis. In deciliated embryos treated with colcemid, the elevated level of tubulin RNA declined rapidly, due to its destabilization by the elevated concentration of unpolymerized tubulin. The increased transcription of tubulin genes is a response to the loss of cilia, not to the hypertonic shock, and occurs even when cilium regeneration is prevented. Inhibition of protein synthesis with puromycin or emetine did not prevent the transcriptional enhancement but stabilized tubulin mRNA, resulting in increased accumulation of tubulin mRNA after deciliation.
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Gong, Z. Y., and B. P. Brandhorst. "Stimulation of tubulin gene transcription by deciliation of sea urchin embryos." Molecular and Cellular Biology 7, no. 12 (December 1987): 4238–46. http://dx.doi.org/10.1128/mcb.7.12.4238.
Full textAbstract:
Deciliation by hypertonic shock of embryos of the sea urchin Lytechinus pictus resulted in an increase in synthesis of alpha- and beta-tubulins, the consequence of an increased concentration of RNA encoding the tubulins. RNA run-on assays in isolated nuclei indicated that this response is due to a transient increase in the rate of synthesis of tubulin RNA beginning within 5 min of deciliation. This enhancement of tubulin gene transcription also occurred in deciliated embryos treated with the microtubule-depolymerizing agent colcemid; thus the reaction to deciliation is not a response to a reduction in concentration of unpolymerized tubulin utilized for ciliogenesis. In deciliated embryos treated with colcemid, the elevated level of tubulin RNA declined rapidly, due to its destabilization by the elevated concentration of unpolymerized tubulin. The increased transcription of tubulin genes is a response to the loss of cilia, not to the hypertonic shock, and occurs even when cilium regeneration is prevented. Inhibition of protein synthesis with puromycin or emetine did not prevent the transcriptional enhancement but stabilized tubulin mRNA, resulting in increased accumulation of tubulin mRNA after deciliation.
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Al-Bassam, Jawdat. "Revisiting the tubulin cofactors and Arl2 in the regulation of soluble αβ-tubulin pools and their effect on microtubule dynamics." Molecular Biology of the Cell 28, no. 3 (February 2017): 359–63. http://dx.doi.org/10.1091/mbc.e15-10-0694.
Full textAbstract:
Soluble αβ-tubulin heterodimers are maintained at high concentration inside eukaryotic cells, forming pools that fundamentally drive microtubule dynamics. Five conserved tubulin cofactors and ADP ribosylation factor–like 2 regulate the biogenesis and degradation of αβ-tubulins to maintain concentrated soluble pools. Here I describe a revised model for the function of three tubulin cofactors and Arl2 as a multisubunit GTP-hydrolyzing catalytic chaperone that cycles to promote αβ-tubulin biogenesis and degradation. This model helps explain old and new data indicating these activities enhance microtubule dynamics in vivo via repair or removal of αβ-tubulins from the soluble pools
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Xie, Yixin, and Lin Li. "Computational Study on E-Hooks of Tubulins in the Binding Process with Kinesin." International Journal of Molecular Sciences 23, no. 4 (February 12, 2022): 2035. http://dx.doi.org/10.3390/ijms23042035.
Full textAbstract:
Cargo transport within cells is essential to healthy cells, which requires microtubules-based motors, including kinesin. The C-terminal tails (E-hooks) of alpha and beta tubulins of microtubules have been proven to play important roles in interactions between the kinesins and tubulins. Here, we implemented multi-scale computational methods in E-hook-related analyses, including flexibility investigations of E-hooks, binding force calculations at binding interfaces between kinesin and tubulins, electrostatic potential calculations on the surface of kinesin and tubulins. Our results show that E-hooks have several functions during the binding process: E-hooks utilize their own high flexibilities to increase the chances of reaching a kinesin; E-hooks help tubulins to be more attractive to kinesin. Besides, we also observed the differences between alpha and beta tubulins: beta tubulin shows a higher flexibility than alpha tubulin; beta tubulin generates stronger attractive forces (about twice the strengths) to kinesin at different distances, no matter with E-hooks in the structure or not. Those facts may indicate that compared to alpha tubulin, beta tubulin contributes more to attracting and catching a kinesin to microtubule. Overall, this work sheds the light on microtubule studies, which will also benefit the treatments of neurodegenerative diseases, cancer treatments, and preventions in the future.
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Saoudi, Y., I. Paintrand, L. Multigner, and D. Job. "Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins." Journal of Cell Science 108, no. 1 (January 1, 1995): 357–67. http://dx.doi.org/10.1242/jcs.108.1.357.
Full textAbstract:
The acidic carboxy-terminal regions of alpha- and beta-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of alpha- and beta-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.
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Weatherbee, J. A., G. S. May, J. Gambino, and N. R. Morris. "Involvement of a particular species of beta-tubulin (beta 3) in conidial development in Aspergillus nidulans." Journal of Cell Biology 101, no. 3 (September 1, 1985): 706–11. http://dx.doi.org/10.1083/jcb.101.3.706.
Full textAbstract:
Strains of Aspergillus containing the benA22 mutation are resistant to benomyl for vegetative growth but do not produce conidia. To test whether conidiation involved an additional benomyl-sensitive tubulin (i.e., was mediated by a tubulin other than the tubulins coded for by the benA locus), a collection of mutants was produced that formed conidia in the presence of benomyl, i.e., were conidiation-resistant (CR-) mutants. We analyzed the tubulins of these CR- mutants using two-dimensional gel electrophoresis and found that the mutants lacked one species of beta-tubulin (designated beta 3). We have examined two of these mutants in detail. In crosses with strains containing wild-type tubulins, we found that the absence of the beta 3-tubulin co-segregated perfectly with the CR- phenotype. In diploids containing both the benA22 and CR- mutations, we found that the CR- phenotype was recessive and that beta 3-tubulin was present on two-dimensional gels of tubulins prepared from these diploids. In another set of crosses, these two CR- strains and seven others were first made auxotrophic for uridine and then crossed against strains that had homologously integrated a plasmid containing an incomplete internal fragment of the beta 3-tubulin gene and the pyr4 gene of Neurospora crassa (which confers uridine prototrophy on transformants). If the CR- phenotype were produced by a mutation in a gene distinct from the structural gene for beta 3-tubulin (designated the tubC gene), then crossing over should have produced some CR+ segregants among the uridine auxotrophic progeny of the second cross. All of the uridine auxotrophs from this type of cross, however, showed the CR- phenotype, suggesting that the mutation in these strains is at or closely linked to the tubC locus. The most obvious explanation of these results is that beta 3-tubulin is ordinarily used during conidiation and the presence of this species of beta-tubulin renders conidiation sensitive to benomyl. In the CR- mutants, beta 3-tubulin is absent, and in the presence of the benA22 mutation the benomyl-resistant beta 1-and/or beta 2-tubulin substitutes for beta 3 to make conidiation benomyl resistant. We discuss these results and give two models to explain the interactions between these beta-tubulin species.
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Khabudaev, Kirill V., Darya P. Petrova, Yekaterina D. Bedoshvili, Yelena V. Likhoshway, and Mikhail A. Grachev. "Molecular Evolution of Tubulins in Diatoms." International Journal of Molecular Sciences 23, no. 2 (January 6, 2022): 618. http://dx.doi.org/10.3390/ijms23020618.
Full textAbstract:
Microtubules are formed by α- and β-tubulin heterodimers nucleated with γ-tubulin. Tubulins are conserved eukaryotic proteins. Previously, it was shown that microtubules are involved in diatom silica frustule morphogenesis. Diatom frustules are varied, and their morphology is species-specific. Despite the attractiveness of the problem of elucidating the molecular mechanisms of genetically programmed morphogenesis, the structure and evolution of diatom tubulins have not been studied previously. Based on available genomic and transcriptome data, we analyzed the phylogeny of the predicted amino acid sequences of diatom α-, β- and γ-tubulins and identified five groups for α-tubulins, six for β-tubulins and four for γ-tubulins. We identified characteristic amino acids of each of these groups and also analyzed possible posttranslational modification sites of diatom tubulins. According to our results, we assumed what changes occurred in the diatom tubulin structures during their evolution. We also identified which tubulin groups are inherent in large diatom taxa. The similarity between the evolution of diatom tubulins and the evolution of diatoms suggests that molecular changes in α-, β- and γ-tubulins could be one of the factors in the formation of a high morphological diversity of diatoms.
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Guo, Wenhan, Tolulope Ayodeji Ale, Shengjie Sun, Jason E. Sanchez, and Lin Li. "A Comprehensive Study on the Electrostatic Properties of Tubulin-Tubulin Complexes in Microtubules." Cells 12, no. 2 (January 5, 2023): 238. http://dx.doi.org/10.3390/cells12020238.
Full textAbstract:
Microtubules are key players in several stages of the cell cycle and are also involved in the transportation of cellular organelles. Microtubules are polymerized by α/β tubulin dimers with a highly dynamic feature, especially at the plus ends of the microtubules. Therefore, understanding the interactions among tubulins is crucial for characterizing microtubule dynamics. Studying microtubule dynamics can help researchers make advances in the treatment of neurodegenerative diseases and cancer. In this study, we utilize a series of computational approaches to study the electrostatic interactions at the binding interfaces of tubulin monomers. Our study revealed that among all the four types of tubulin-tubulin binding modes, the electrostatic attractive interactions in the α/β tubulin binding are the strongest while the interactions of α/α tubulin binding in the longitudinal direction are the weakest. Our calculations explained that due to the electrostatic interactions, the tubulins always preferred to form α/β tubulin dimers. The interactions between two protofilaments are the weakest. Thus, the protofilaments are easily separated from each other. Furthermore, the important residues involved in the salt bridges at the binding interfaces of the tubulins are identified, which illustrates the details of the interactions in the microtubule. This study elucidates some mechanistic details of microtubule dynamics and also identifies important residues at the binding interfaces as potential drug targets for the inhibition of cancer cells.
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Hoyle, Henry D., F. Rudolf Turner, Linda Brunick, and Elizabeth C. Raff. "Tubulin Sorting during Dimerization In Vivo." Molecular Biology of the Cell 12, no. 7 (July 2001): 2185–94. http://dx.doi.org/10.1091/mbc.12.7.2185.
Full textAbstract:
We demonstrate sorting of β-tubulins during dimerization in theDrosophila male germ line. Different β-tubulin isoforms exhibit distinct affinities for α-tubulin during dimerization. Our data suggest that differences in dimerization properties are important in determining isoform-specific microtubule functions. The differential use of β-tubulin during dimerization reveals structural parameters of the tubulin heterodimer not discernible in the resolved three-dimensional structure. We show that the variable β-tubulin carboxyl terminus, a surface feature in the heterodimer and in microtubules, and which is disordered in the crystallographic structure, is of key importance in forming a stable α-β heterodimer. If the availability of α-tubulin is limiting, α-β dimers preferentially incorporate intact β-tubulins rather than a β-tubulin missing the carboxyl terminus (β2ΔC). When α-tubulin is not limiting, β2ΔC forms stable α-β heterodimers. Once dimers are formed, no further sorting occurs during microtubule assembly: α-β2ΔC dimers are incorporated into axonemes in proportion to their contribution to the total dimer pool. Co-incorporation of β2ΔC and wild-type β2-tubulin results in nonmotile axonemes because of a disruption of the periodicity of nontubulin axonemal elements. Our data show that the β-tubulin carboxyl terminus has two distinct roles: 1) forming the α-β heterodimer, important for all microtubules and 2) providing contacts for nontubulin components required for specific microtubule structures, such as axonemes.
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Lajoie-Mazenc, I., C. Detraves, V. Rotaru, M. Gares, Y. Tollon, C. Jean, M. Julian, M. Wright, and B. Raynaud-Messina. "A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres." Journal of Cell Science 109, no. 10 (October 1, 1996): 2483–92. http://dx.doi.org/10.1242/jcs.109.10.2483.
Full textAbstract:
Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of alpha- and beta-tubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for alpha- and beta-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of gamma-tubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for gamma-tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64–76% identity with other gamma-tubulins. However, immunological studies identified two gamma-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two gamma-tubulins, called gamma s- and gamma f-tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of gamma-tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two gamma-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, gamma s-tubulin was tightly bound to microtubule organizing centers (MTOCs) while gamma f-tubulin was loosely associated with these structures. This first demonstration of the presence of two gamma-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed.
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Johnson, K. A. "The axonemal microtubules of the Chlamydomonas flagellum differ in tubulin isoform content." Journal of Cell Science 111, no. 3 (February 1, 1998): 313–20. http://dx.doi.org/10.1242/jcs.111.3.313.
Full textAbstract:
Little is known of the molecular basis for the diversity of microtubule structure and function found within the eukaryotic flagellum. Antibodies that discriminate between tyrosinated alpha tubulin and post-translationally detyrosinated alpha tubulin were used to localize these complementary tubulin isoforms in flagella of the single-celled green alga Chlamydomonas reinhardtii. Immunofluorescence analysis of intact axonemes detected both isoforms along most of the lengths of flagella; however, each had a short distal zone rich in tyrosinated tubulin. Localizations on splayed axonemes revealed that the microtubules of the central-pair apparatus were rich in tyrosinated tubulin, while outer doublets contained a mixture of both isoforms. Immunoelectron analysis of individual outer doublets revealed that while tyrosinated tubulin was present in both A and B tubules, detyrosinated tubulin was largely confined to the wall of the B hemi-tubules. The absence of detyrosinated tubulin from the A tubules of the outer doublets and the microtubules of the central pair, both of which extend past the B hemi-tubules of the outer doublets in the flagellar tip, explained the appearance of a tyrosinated tubulin-rich distal zone on intact axonemes. Localizations performed on cells regenerating flagella revealed that flagellar assembly used tyrosinated tubulin; detyrosination of the B tubule occurred during later stages of regeneration, well after microtubule polymerization. The developmental timing of detyrosination, which occurs over a period during which the regrowing flagella begin to beat more effectively, suggests that post-translational modification of the B tubule surface may enhance dynein/B tubule interactions that power flagellar beating.
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Hecht, N. B., R. J. Distel, P. C. Yelick, S. M. Tanhauser, C. E. Driscoll, E. Goldberg, and K. S. Tung. "Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain." Molecular and Cellular Biology 8, no. 2 (February 1988): 996–1000. http://dx.doi.org/10.1128/mcb.8.2.996-1000.1988.
Full textAbstract:
Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.
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Hecht, N. B., R. J. Distel, P. C. Yelick, S. M. Tanhauser, C. E. Driscoll, E. Goldberg, and K. S. Tung. "Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain." Molecular and Cellular Biology 8, no. 2 (February 1988): 996–1000. http://dx.doi.org/10.1128/mcb.8.2.996.
Full textAbstract:
Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.
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Ruiz, F., P. Dupuis-Williams, C. Klotz, F. Forquignon, M. Bergdoll, J. Beisson, and F. Koll. "Genetic Evidence for Interaction between η- and β-Tubulins." Eukaryotic Cell 3, no. 1 (February 2004): 212–20. http://dx.doi.org/10.1128/ec.3.1.212-220.2004.
Full textAbstract:
ABSTRACT The thermosensitive allelic mutations sm19-1 and sm19-2 of Paramecium tetraurelia cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the SM19 gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, α to ε, characterized in P. tetraurelia. In order to analyze η-tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the sm19-1 mutation, the su3-1 mutation was characterized as an E288K substitution in the β-PT2 gene coding for a β-tubulin, while the mutation nocr 1 conferring nocodazole resistance and localized in another β-tubulin gene, β-PT3, was shown to enhance the mutant phenotype. The interaction between η-tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that η-tubulin, like γ- and δ-tubulins, might be a microtubule minus-end capping molecule. The likely function of η-tubulin as part of a complex specifically involved in basal body biogenesis is discussed.
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Nami, Babak, and Zhixiang Wang. "Genetics and Expression Profile of the Tubulin Gene Superfamily in Breast Cancer Subtypes and Its Relation to Taxane Resistance." Cancers 10, no. 8 (August 18, 2018): 274. http://dx.doi.org/10.3390/cancers10080274.
Full textAbstract:
Taxanes are a class of chemotherapeutic agents that inhibit cell division by disrupting the mitotic spindle through the stabilization of microtubules. Most breast cancer (BC) tumors show resistance against taxanes partially due to alterations in tubulin genes. In this project we investigated tubulin isoforms in BC to explore any correlation between tubulin alterations and taxane resistance. Genetic alteration and expression profiling of 28 tubulin isoforms in 6714 BC tumor samples from 4205 BC cases were analyzed. Protein-protein, drug-protein and alterations neighbor genes in tubulin pathways were examined in the tumor samples. To study correlation between promoter activity and expression of the tubulin isoforms in BC, we analyzed the ChIP-seq enrichment of active promoter histone mark H3K4me3 and mRNA expression profile of MCF-7, ZR-75-30, SKBR-3 and MDA-MB-231 cell lines. Potential correlation between tubulin alterations and taxane resistance, were investigated by studying the expression profile of taxane-sensitive and resistant BC tumors also the MDA-MB-231 cells acquired resistance to paclitaxel. All genomic data were obtained from public databases. Results showed that TUBD1 and TUBB3 were the most frequently amplified and deleted tubulin genes in the BC tumors respectively. The interaction analysis showed physical interactions of α-, β- and γ-tubulin isoforms with each other. The most of FDA-approved tubulin inhibitor drugs including taxanes target only β-tubulins. The analysis also revealed sex tubulin-interacting neighbor proteins including ENCCT3, NEK2, PFDN2, PTP4A3, SDCCAG8 and TBCE which were altered in at least 20% of the tumors. Three of them are tubulin-specific chaperons responsible for tubulin protein folding. Expression of tubulin genes in BC cell lines were correlated with H3K4me3 enrichment on their promoter chromatin. Analyzing expression profile of BC tumors and tumor-adjacent normal breast tissues showed upregulation of TUBA1A, TUBA1C, TUBB and TUBB3 and downregulation of TUBB2A, TUBB2B, TUBB6, TUBB7P pseudogene, and TUBGCP2 in the tumor tissues compared to the normal breast tissues. Analyzing taxane-sensitive versus taxane-resistant tumors revealed that expression of TUBB3 and TUBB6 was significantly downregulated in the taxane-resistant tumors. Our results suggest that downregulation of tumor βIII- and βV-tubulins is correlated with taxane resistance in BC. Based on our results, we conclude that aberrant protein folding of tubulins due to mutation and/or dysfunction of tubulin-specific chaperons may be potential mechanisms of taxane resistance. Thus, we propose studying the molecular pathology of tubulin mutations and folding in BC and their impacts on taxane resistance.
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Westermann, S., A. Schneider, E. K. Horn, and K. Weber. "Isolation of tubulin polyglutamylase from Crithidia; binding to microtubules and tubulin, and glutamylation of mammalian brain alpha- and beta-tubulins." Journal of Cell Science 112, no. 13 (July 1, 1999): 2185–93. http://dx.doi.org/10.1242/jcs.112.13.2185.
Full textAbstract:
Trypanosomatids have a striking cage-like arrangement of submembraneous microtubules. We previously showed that alpha- and beta- tubulins of these stable microtubules are extensively modified by polyglutamylation. Cytoskeletal microtubular preparations obtained by Triton extraction of Leishmania tarentolae and Crithidia fasciculata retain an enzymatic activity that incorporates radioactive glutamic acid in a Mg2+-ATP-dependent manner into alpha- and beta-tubulins. The tubulin polyglutamylase is extracted by 0.25 M salt. The Crithidia enzyme can be purified by ATP-affinity chromatography, glycerol-gradient centrifugation and ion-exchange chromatography. After extraction from the microtubular cytoskeleton the glutamylase forms a complex with alphabeta tubulin, but behaves after removal of tubulin as a globular protein with a molecular mass of 38x10(3). In highly enriched fractions a corresponding band is the major polypeptide visible in SDS-PAGE. The enzyme from Crithidia recognises mammalian brain tubulin, where it incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins. Synthetic peptides with an oligoglutamyl side chain, corresponding to the carboxy-terminal end of brain alpha- and beta-tubulins, are accepted by the enzyme, albeit at low efficiency. The polyglutamylase elongates the side chain by up to 3 and 5 residues, respectively. Other properties of the tubulin polyglutamylase are also discussed.
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Makarova, Liubov, and Alena Korshunova. "Abstract P-36: Structural Analysis of Conformational Changes of Bacterial and Eukaryotic Tubulins." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S27—S28. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p36.
Full textAbstract:
Background: Eukaryotic α- and β-tubulin proteins stand out among tubulin-like proteins by their ability to form hollow dynamically unstable microtubules (MT) with 13 protofilaments. Microtubules are part of the cell cytoskeleton and play a key role in chromosome division in mitosis. A considerable amount of anticancer drugs works on microtubules level breaking its dynamic. But the mechanism of dynamic instability and works of these drugs remains unknown. Bacteria of the genus Prostecobacter have unique bacterial tubulins (BtubA/B) capable to form hollow dynamically unstable 5 protofilament MTs (miniMT). Instead of great differences, both tubulins have many common features. Eukaryotic tubulin was known to have structural changes through GTP hydrolysis (compactization for approximately 2 Å and a twist for 0,1˚). «Anchor point» structure in alpha-tubulin was noticed to be a fixed point in this movement. Methods: We performed comparative structural analysis of BtubA/B and α- and β-tubulin proteins using USCF Chimera10 and MEGA X software. This data was obtained due to a comparison of 3 structures of microtubules with different nucleotides [pdb6DPU, 6DPV, 6DPW] and two structures for bacterial tubulins (miniMT [pdb5o09] and BtubA/B-dimer [pdb2BTQ]). Results: We noticed that bacterial tubulins form shorter protofilaments in miniMT than eukaryotic ones. It can be explained as compaction in two sites instead of one site in eukaryotic MT. Also, the most motionless point of min MT turned out the same "anchor point." Phylogenetic analysis showed that this structure is very conservative in these orthologs. Moreover, the final state of both tubulins (GDP) repeats each other. Conclusion: Our results suggest that bacterial tubulin can have movements through GTP hydrolysis similar to eukaryotic one. And it means that despite different amino acid sequences, bacterial and eukaryotic tubulins have similar keys structures for dynamic instability.
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Linhartová, I., P. Dráber, E. Dráberová, and V. Viklický. "Immunological discrimination of β-tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III β-tubulins." Biochemical Journal 288, no. 3 (December 15, 1992): 919–24. http://dx.doi.org/10.1042/bj2880919.
Full textAbstract:
Individual beta-tubulin isoforms in developing mouse brain were characterized using immunoblotting, after preceding high-resolution isoelectric focusing, with monoclonal antibodies against different structural regions of beta-tubulin. Some of the antibodies reacted with a limited number of tubulin isoforms in all stages of brain development and in HeLa cells. The epitope for the TU-14 antibody was located in the isotype-defining domain and was present on the beta-tubulin isotypes of classes I, II and IV, but absent on the neuron-specific class-III isotype. The data suggest that non-class-III beta-tubulins in mouse brain are substrates for developmentally regulated post-translational modifications and that beta-tubulins of non-neuronal cells are also post-translationally modified.
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Anders, Kirk R., and David Botstein. "Dominant-Lethal α-Tubulin Mutants Defective in Microtubule Depolymerization in Yeast." Molecular Biology of the Cell 12, no. 12 (December 2001): 3973–86. http://dx.doi.org/10.1091/mbc.12.12.3973.
Full textAbstract:
The dynamic instability of microtubules has long been understood to depend on the hydrolysis of GTP bound to β-tubulin, an event stimulated by polymerization and necessary for depolymerization. Crystallographic studies of tubulin show that GTP is bound by β-tubulin at the longitudinal dimer-dimer interface and contacts particular α-tubulin residues in the next dimer along the protofilament. This structural arrangement suggests that these contacts could account for assembly-stimulated GTP hydrolysis. As a test of this hypothesis, we examined, in yeast cells, the effect of mutating the α-tubulin residues predicted, on structural grounds, to be involved in GTPase activation. Mutation of these residues to alanine (i.e., D252A and E255A) created poisonous α-tubulins that caused lethality even as minor components of the α-tubulin pool. When the mutant α-tubulins were expressed from the galactose-inducible promoter ofGAL1, cells rapidly acquired aberrant microtubule structures. Cytoplasmic microtubules were largely bundled, spindle assembly was inhibited, preexisting spindles failed to completely elongate, and occasional, stable microtubules were observed unattached to spindle pole bodies. Time-lapse microscopy showed that microtubule dynamics had ceased. Microtubules containing the mutant proteins did not depolymerize, even in the presence of nocodazole. These data support the view that α-tubulin is a GTPase-activating protein that acts, during microtubule polymerization, to stimulate GTP hydrolysis in β-tubulin and thereby account for the dynamic instability of microtubules.
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Gudi, Radhika, Chaozhong Zou, Jun Li, and Qingshen Gao. "Centrobin–tubulin interaction is required for centriole elongation and stability." Journal of Cell Biology 193, no. 4 (May 16, 2011): 711–25. http://dx.doi.org/10.1083/jcb.201006135.
Full textAbstract:
Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin–tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1–associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin–tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin–tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.
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Schneider, A., T. Sherwin, R. Sasse, D. G. Russell, K. Gull, and T. Seebeck. "Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms." Journal of Cell Biology 104, no. 3 (March 1, 1987): 431–38. http://dx.doi.org/10.1083/jcb.104.3.431.
Full textAbstract:
The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha-tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules.
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Gaertig, J., M. A. Cruz, J. Bowen, L. Gu, D. G. Pennock, and M. A. Gorovsky. "Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila." Journal of Cell Biology 129, no. 5 (June 1, 1995): 1301–10. http://dx.doi.org/10.1083/jcb.129.5.1301.
Full textAbstract:
In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha-tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.
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Fees, Colby P., and Jeffrey K. Moore. "Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin." Life Science Alliance 1, no. 2 (April 19, 2018): e201800054. http://dx.doi.org/10.26508/lsa.201800054.
Full textAbstract:
Dynamic instability is an intrinsic property of microtubules; however, we do not understand what domains of αβ-tubulins regulate this activity or how these regulate microtubule networks in cells. Here, we define a role for the negatively charged carboxy-terminal tail (CTT) domain of β-tubulin in regulating dynamic instability. By combining in vitro studies with purified mammalian tubulin and in vivo studies with tubulin mutants in budding yeast, we demonstrate that β-tubulin CTT inhibits microtubule stability and regulates the structure and stability of microtubule plus ends. Tubulin that lacks β-tubulin CTT polymerizes faster and depolymerizes slower in vitro and forms microtubules that are more prone to catastrophe. The ends of these microtubules exhibit a more blunted morphology and rapidly switch to disassembly after tubulin depletion. In addition, we show that β-tubulin CTT is required for magnesium cations to promote depolymerization. We propose that β-tubulin CTT regulates the assembly of stable microtubule ends and provides a tunable mechanism to coordinate dynamic instability with ionic strength in the cell.
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Li, Zhongping, Lingling Ma, Chengyong Wu, Tao Meng, Lanping Ma, Wenyue Zheng, Yamei Yu, Qiang Chen, Jinliang Yang, and Jingkang Shen. "The Structure of MT189-Tubulin Complex Provides Insights into Drug Design." Letters in Drug Design & Discovery 16, no. 9 (September 11, 2019): 1069–73. http://dx.doi.org/10.2174/1570180816666181122122655.
Full textAbstract:
Background: Drugs that interfere with microtubule dynamics are used widely in cancer chemotherapy. Microtubules are composed of αβ-tubulin heterodimers, and the colchicine binding site of tubulin is an important pocket for designing tubulin polymerization inhibitors. We have previously designed and synthesized a series of colchicine binding site inhibitors (CBSIs). However, these compounds showed no anticancer activity in vivo. Then, we have used a deconstruction approach to obtain a new derivative MT189, which showed in vivo anticancer activity. Methods: We crystallized a protein complex including two tubulins, one stathmin-like domain of RB3 and one tubulin tyrosine ligase, and soaked MT189 into the crystals. We collected the diffraction data and determined the tubulin-MT189 structure to 2.8 Å. Results: Here, we report the crystal structure of tubulin complexed with MT189, elucidate how the small-molecular agent binds to tubulin and inhibits microtubule assembly, and explain previous results of the structure-activity-relationship studies. Conclusion: The tubulin-MT189 complex structure reveals the interactions between this agent and tubulin and provides insights into the design of new derivatives targeting the colchicine binding site.
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Lyons-Abbott, Sally, Dan L. Sackett, Dorota Wloga, Jacek Gaertig, Rachel E. Morgan, Karl A. Werbovetz, and Naomi S. Morrissette. "α-Tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties To Confer Dinitroaniline Resistance." Eukaryotic Cell 9, no. 12 (September 24, 2010): 1825–34. http://dx.doi.org/10.1128/ec.00140-10.
Full textAbstract:
ABSTRACT Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin, and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii, and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provide additional support for the proposed dinitroaniline binding site on α-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.
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MacRae, Thomas H., and Carrie M. Langdon. "Tubulin synthesis, structure, and function: what are the relationships?" Biochemistry and Cell Biology 67, no. 11-12 (November 1, 1989): 770–90. http://dx.doi.org/10.1139/o89-116.
Full textAbstract:
In most eukaryotes, families of tubulin genes give rise to multiple isoforms of tubulin, which may be modified post-translationally. The synthesis of isotubulins is spatially and temporally regulated, leading to the presence of different tubulins within an organism. The cellular localization of tubulin is also nonrandom with discrete isoforms residing in specific regions of some cells. Much work, dependent upon interrelated molecular and immunological technologies, has gone into determining why cells produce multiple isotubulins. One proposal would have us believe that isotubulins are functionally discrete and that the isotubulin composition of a microtubule determines its function. A second idea is that tubulin multigene families arose by gene duplication and subsequent genetic drift. The duplicated genes survive in modified form, as they provide the cell with the ability to effectively regulate tubulin synthesis at the transcriptional level while yielding multifunctional tubulins. Analysis of mutant tubulin genes is revealing functionally important regions of tubulin, prompting a return to the consideration of those properties of the molecule that are fundamental to microtubule formation rather than function per se. A model for autoregulation of tubulin synthesis has been developed, but tittle is yet known of other molecular signals or mechanisms involved in regulation of tubulin production. Further study of flagella formation promises to change this situation. Complementing the genetic studies is the examination of tubulin posttranslational modifications including detyrosination/tyrosination, acetylation/deacetylation, and phosphorylation. Enzymatic mechanisms mediating tubulin posttranslational changes are partially elucidated, as are the influences of the modifications on microtubule properties. Potential functions of the posttranslational changes are the modulation of microtubule-associated protein binding to microtubules, the provision of a mechanism to indicate microtubule age, or as mediators of cellular morphogenesis, a role which could be dependent upon the first two functions. Despite the progress that has been made, the physiological significance of having several isotubulins within a single cell and the molecular details of microtubule function remain obscure.Key words: tubulin genes, multitubulin hypothesis, autoregulation, posttranslational modifications.
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Zhou, Yujun, Yuanye Zhu, Yanjun Li, Yabing Duan, Rongsheng Zhang, and Mingguo Zhou. "β1 Tubulin Rather Than β2 Tubulin Is the Preferred Binding Target for Carbendazim in Fusarium graminearum." Phytopathology® 106, no. 9 (September 2016): 978–85. http://dx.doi.org/10.1094/phyto-09-15-0235-r.
Full textAbstract:
Tubulins are the proposed target of anticancer drugs, anthelminthics, and fungicides. In Fusarium graminearum, β2 tubulin has been reported to be the binding target of methyl benzimidazole carbamate (MBC) fungicides. However, the function of F. graminearum β1 tubulin, which shares 76% amino acid sequence identity with β2 tubulin, in MBC sensitivity has been unclear. In this study, MBC sensitivity relative to that of a parental strain (2021) was significantly reduced in a β1 tubulin deletion strain but increased in a β2 tubulin deletion strain, suggesting that β1 tubulin was involved in the MBC sensitivity of F. graminearum. When strain 2021 was grown in a medium with a low or high concentration of the MBC fungicide carbendazim (0.5 or 1.4 μg/ml), the protein accumulation levels were reduced by 47 and 87%, respectively, for β1 tubulin but only by 6 and 24%, respectively, for β2 tubulin. This result was consistent with observations that MBC fungicides are more likely to disrupt β1 tubulin microtubules rather than β2 tubulin microtubules in GFP-β tubulin fusion mutants in vivo. Furthermore, sequence analysis indicated that a difference in tubulin amino acid 240 (240L in β1 versus 240F in β2) may explain the difference in MBC binding affinity; this result was consistent with the result that an F240L mutation in β2 tubulin greatly increased sensitivity to carbendazim in F. graminearum. We suggest that β1 tubulin rather than β2 tubulin is the preferred binding target for MBC fungicides in F. graminearum.
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Garant, Katy A., and Thomas H. MacRae. "Cloning and sequencing of tubulin cDNAs from Artemia franciscana: evidence for differential expression of α- and β-tubulin genes." Biochemistry and Cell Biology 87, no. 6 (December 2009): 989–97. http://dx.doi.org/10.1139/o09-050.
Full textAbstract:
Tubulin is a heterodimeric protein composed of α- and β-tubulin. In most organisms, they are encoded by multiple gene families whose members are subject to differential regulation. The objective of the work described herein was to better understand tubulin gene expression in the extremophile Artemia franciscana To this end tubulin cDNAs were cloned and sequenced. αAT2, an α-tubulin cDNA, differed by one nucleotide from αAT1, a previously cloned Artemia cDNA. This change, possibly generated by allelic variation, caused an M313V substitution in α-tubulin. The amino acid sequence of β-tubulin encoded by βAT1, one of only a very limited number of cloned crustacean β-tubulin cDNA sequences yet available, and the first from Artemia, was similar to other β-tubulins. However, βAT1 possessed four degenerate TATA boxes in the 5′ untranslated region, although authentic TATA and CCAAT boxes occurred in the 3′ non-coding sequence. Analyses by quantitative PCR demonstrated that the amount of tubulin mRNA declined relative to total mRNA in progressive life history stages of Artemia and also that the organism contained more αAT2- than βAT1-tubulin mRNA at all developmental phases examined.
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Boggs, B., and F. Cabral. "Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells." Molecular and Cellular Biology 7, no. 8 (August 1987): 2700–2707. http://dx.doi.org/10.1128/mcb.7.8.2700-2707.1987.
Full textAbstract:
Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.
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Boggs, B., and F. Cabral. "Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells." Molecular and Cellular Biology 7, no. 8 (August 1987): 2700–2707. http://dx.doi.org/10.1128/mcb.7.8.2700.
Full textAbstract:
Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.