A β-Tubulin Leucine Cluster Involved in Microtubule Assembly and Paclitaxel Resistance

ABSTRACTTubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP–β-tubulin by altering the IFT74N–IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP–β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP–β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP–β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

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The Roles of β-Tubulin Mutations and Isotype Expression in Acquired Drug Resistance

Cancer Informatics ◽

10.1177/117693510700300028 ◽

2007 ◽

Vol 3 ◽

pp. 117693510700300 ◽

Cited By ~ 35

Author(s):

J. Torin Huzil ◽

Ke Chen ◽

Lukasz Kurgan ◽

Jack A. Tuszynski

Keyword(s):

Drug Resistance ◽

Rational Design ◽

Resistant Cell ◽

Microtubule Assembly ◽

Acquired Drug Resistance ◽

Tubulin Isotypes ◽

Microtubule Disruption ◽

Tubulin Isotype ◽

Β Tubulin ◽

Isotype Expression

The antitumor drug paclitaxel stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and eventually apoptosis. Upon assembly of the α/β-tubulin heterodimer, GTP becomes bound to both the α and β-tubulin monomers. During microtubule assembly, the GTP bound to β-tubulin is hydrolyzed to GDP, eventually reaching steady-state equilibrium between free tubulin dimers and those polymerized into microtubules. Tubulin-binding drugs such as paclitaxel interact with β-tubulin, resulting in the disruption of this equilibrium. In spite of several crystal structures of tubulin, there is little biochemical insight into the mechanism by which anti-tubulin drugs target microtubules and alter their normal behavior. The mechanism of drug action is further complicated, as the description of altered β-tubulin isotype expression and/or mutations in tubulin genes may lead to drug resistance as has been described in the literature. Because of the relationship between β-tubulin isotype expression and mutations within β-tubulin, both leading to resistance, we examined the properties of altered residues within the taxane, colchicine and Vinca binding sites. The amount of data now available, allows us to investigate common patterns that lead to microtubule disruption and may provide a guide to the rational design of novel compounds that can inhibit microtubule dynamics for specific tubulin isotypes or, indeed resistant cell lines. Because of the vast amount of data published to date, we will only provide a broad overview of the mutational results and how these correlate with differences between tubulin isotypes. We also note that clinical studies describe a number of predictive factors for the response to anti-tubulin drugs and attempt to develop an understanding of the features within tubulin that may help explain how they may affect both microtubule assembly and stability.

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The Seco-Taxane IDN5390 Is Able to Target Class III β-Tubulin and to Overcome Paclitaxel Resistance

Cancer Research ◽

10.1158/0008-5472.can-04-3065 ◽

2005 ◽

Vol 65 (6) ◽

pp. 2397-2405 ◽

Cited By ~ 77

Author(s):

Cristiano Ferlini ◽

Giuseppina Raspaglio ◽

Simona Mozzetti ◽

Lucia Cicchillitti ◽

Flavia Filippetti ◽

...

Keyword(s):

Class Iii ◽

Paclitaxel Resistance ◽

Target Class ◽

Β Tubulin

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Microtubule assembly protects the region 28-38 of the β- tubulin subunit

Cell Motility and the Cytoskeleton ◽

10.1002/cm.970220104 ◽

1992 ◽

Vol 22 (1) ◽

pp. 25-37 ◽

Cited By ~ 5

Author(s):

Patrick Chène ◽

Honoré Mazarguiland ◽

Michel Wright

Keyword(s):

Microtubule Assembly ◽

Tubulin Subunit ◽

Β Tubulin

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Plant β-tubulin phosphorylation on Ser172 as canonical suppressing factor of microtubule growth

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v24.1123 ◽

2019 ◽

Vol 24 ◽

pp. 321-326

Author(s):

P. A. Karpov ◽

Ya. B. Blume

Keyword(s):

Protein Kinases ◽

Catalytic Domain ◽

Homo Sapiens ◽

Fruit Fly ◽

Microtubule Assembly ◽

Multiple Sequence ◽

Significant Similarity ◽

Domain Structures ◽

Phylogenetic Profiling ◽

Β Tubulin

Aim. The estimation of potential role of plant β-tubulin Ser172 phosphorylation for correct function of microtubules and cell division due to selection of protein kinases, most probable associated with phosphorylation of Ser172 in Arabidopsis thaliana (L.) Heynh. Methods. Literature and database search. Comparison of protein sequences and structures: multiple sequence alignment, phylogenetic profiling, protein structure modeling, etc. Results. Comparison of Ser172 site region from all known β-tubulins from Homo sapiens, Sus scrofa, Saccharomyces cerevisiae, Drosophila melanogaster and A. thaliana confirms its significant similarity. Joint clusterization of all Ser172 site regions (in S±10 a.a. format) reveals that plant site is most similar to Ser172±10 fragment of β-tubulin from S. cerevisiae. At the same time, sequences and catalytic domain structures of cyclin-dependent kinases 1 and YAK1-related kinases (MNB/DYRK1a/YAK1) associated with Ser172 phosphorylation, found maximal similarity in A. thaliana and S. cerevisiae. Сonclusions. The results confirm similarity of amino acid environment of Ser172 in β-tubulin isotypes in human, pig, fruit fly, yeast and arabidopsis. This suggests similar effect of β-tubulin phosphorylation at Ser172 for inhibition of microtubule assembly onto their protofilaments and its association with CDK1 and YAK1-related protein kinases. Similarity of Ser172 sites and associated protein kinases, allows us to expect similar effect of this modification on structure of microtubules in A. thaliana and S. cerevisiae. Keywords: β-tubulin, Ser172, phosphorylation, CDK1, DYRK1, MNB, YAK1.

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Convergent Evolution of C239S Mutation in Pythium spp. β-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes

Phytopathology ◽

10.1094/phyto-01-19-0022-r ◽

2019 ◽

Vol 109 (12) ◽

pp. 2087-2095 ◽

Cited By ~ 2

Author(s):

Zachary A. Noel ◽

Hyunkyu Sang ◽

Mitchell G. Roth ◽

Martin I. Chilvers

Keyword(s):

Convergent Evolution ◽

Evolutionary History ◽

Amino Acid Change ◽

Soybean Seed ◽

Effective Concentration ◽

Microtubule Assembly ◽

Virulence Assay ◽

Treated Seed ◽

Repeated Evolution ◽

Β Tubulin

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to β-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 μg ml−1), whereas 16% were insensitive (EC50 > 11 μg ml−1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in β-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available β-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

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Functional roles of α 1 -, α 2 -, β 1 -, β 2 -tubulin in vegetative growth, microtubule assembly and sexual reproduction of Fusarium graminearum .

Applied and Environmental Microbiology ◽

10.1128/aem.00967-21 ◽

2021 ◽

Author(s):

Yuanye Zhu ◽

Yuanshuai Zhang ◽

Yabing Duan ◽

Dongya Shi ◽

Yi ping Hou ◽

...

Keyword(s):

Sexual Reproduction ◽

Fusarium Graminearum ◽

Vegetative Growth ◽

Microtubule Assembly ◽

Mitochondrial Outer Membrane ◽

Deletion Mutants ◽

Voltage Dependent Anion Channel ◽

Microtubule Cytoskeleton ◽

Functional Roles ◽

Β Tubulin

The plant pathogen Fusarium graminearum contains two α-tubulin (α 1 and α 2 ) isotypes and two β-tubulin isotypes (β 1 and β 2 ). The functional roles of these tubulins in microtubule assembly are not clear. Previous studies showed that α 1 - and β 2 -tubulin deletion mutants showed severe growth defects and hypersensitivity to carbendazim, which have not been well explained. Here, we investigated the interaction between α- and β-tubulin of F. graminearum . Co-localization experiments demonstrated that β 1 - and β 2 -tubulin are co-localized. Co-immunoprecipitation experiment suggested that β 1 -tubulin binds to both α 1 - and α 2 -tubulin and β 2 -tubulin can also bind to α 1 - or α 2 -tubulin. Interestingly, deletion of α 1 -tubulin increased the interaction between β 2 -tubulin and α 2 -tubulin. Microtubule observation assays showed that deletion of α 1 -tubulin completely disrupted β 1 -tubulin-containing microtubules and significantly decreased β 2 -tubulin-containing microtubules. Deletion of α 2 -, β 1 - or β 2 -tubulin respectively had no obvious effect on the microtubule cytoskeleton. However, microtubules in α 1 - and β 2 -tubulin deletion mutants were easily depolymerized in the presence of carbendazim. The sexual reproduction assay indicates that α 1 - and β 1 -tubulin deletion mutants could not produce asci and ascospores. These results implied that α 1 -tubulin may be essential for the microtubule cytoskeleton. However, our Δα 1 -2×α 2 mutant (α 1 -tubulin deletion mutant containing two copies of α 2 -tubulin) exhibited a normal microtubule network, growth and sexual reproduction. Interestingly, the Δα 1 -2×α 2 mutant was still hypersensitive to carbendazim. In addition, both β 1 -tubulin and β 2 -tubulin were found to bind the mitochondrial outer membrane voltage-dependent anion channel (VDAC), indicating they could regulate the function of VDAC. Importance: In this study, we found that F. graminearum contains four different α-/β-tubulin heterodimers (α 1 -β 1 , α 1 -β 2 , α 2 -β 1 and α 2 -β 2 ) and they assemble together into a single microtubule. Moreover, α 1 -, α 2 -tubulins are functionally interchangeable in microtubule assembly, vegetative growth and sexual reproduction. These results provide more insights into functional roles of different tubulins of F. graminearum which could be helpful for purification of tubulin heterodimers and developing new tubulin-binding agents.

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