scholarly journals The Caenorhabditis elegans Microtubule-severing Complex MEI-1/MEI-2 Katanin Interacts Differently with Two Superficially Redundant β-Tubulin Isotypes

2004 ◽  
Vol 15 (1) ◽  
pp. 142-150 ◽  
Author(s):  
Chenggang Lu ◽  
Martin Srayko ◽  
Paul E. Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Background ◽  
Morphological Changes ◽  
Meiotic Spindle ◽  
Tubulin Isotypes ◽  
Microtubule Severing ◽  
Tubulin Isotype ◽  
Embryonic Viability ◽  
Β Tubulin

The microtubule-severing protein complex katanin is required for a variety of important microtubule-base morphological changes in both animals and plants. Caenorhabditis elegans katanin is encoded by the mei-1 and mei-2 genes and is required for oocyte meiotic spindle formation and must be inactivated before the first mitotic cleavage. We identified a mutation, sb26, in the tbb-2 β-tubulin gene that partially inhibits MEI-1/MEI-2 activity: sb26 rescues lethality caused by ectopic MEI-1/MEI-2 expression during mitosis, and sb26 increases meiotic defects in a genetic background where MEI-1/MEI-2 activity is lower than normal. sb26 does not interfere with MEI-1/MEI-2 microtubule localization, suggesting that this mutation likely interferes with severing. Tubulin deletion alleles and RNA-mediated interference revealed that TBB-2 and the other germline enriched β-tubulin isotype, TBB-1, are redundant for embryonic viability. However, limiting MEI-1/MEI-2 activity in these experiments revealed that MEI-1/MEI-2 preferentially interacts with TBB-2–containing microtubules. Our results demonstrate that these two superficially redundant β-tubulin isotypes have functionally distinct roles in vivo.

scholarly journals Mutation of the α-tubulin Tuba1a leads to straighter microtubules and perturbs neuronal migration

2017 ◽  
Vol 216 (8) ◽  
pp. 2443-2461 ◽  
Author(s):  
Richard Belvindrah ◽  
Kathiresan Natarajan ◽  
Preety Shabajee ◽  
Elodie Bruel-Jungerman ◽  
Jennifer Bernard ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Glial Cells ◽  
Neuronal Migration ◽  
Cortical Malformations ◽  
Tubulin Isotype ◽  
Modeling Data ◽  
Migratory Stream ◽  
Β Tubulin

Brain development involves extensive migration of neurons. Microtubules (MTs) are key cellular effectors of neuronal displacement that are assembled from α/β-tubulin heterodimers. Mutation of the α-tubulin isotype TUBA1A is associated with cortical malformations in humans. In this study, we provide detailed in vivo and in vitro analyses of Tuba1a mutants. In mice carrying a Tuba1a missense mutation (S140G), neurons accumulate, and glial cells are dispersed along the rostral migratory stream in postnatal and adult brains. Live imaging of Tuba1a-mutant neurons revealed slowed migration and increased neuronal branching, which correlated with directionality alterations and perturbed nucleus–centrosome (N–C) coupling. Tuba1a mutation led to increased straightness of newly polymerized MTs, and structural modeling data suggest a conformational change in the α/β-tubulin heterodimer. We show that Tuba8, another α-tubulin isotype previously associated with cortical malformations, has altered function compared with Tuba1a. Our work shows that Tuba1a plays an essential, noncompensated role in neuronal saltatory migration in vivo and highlights the importance of MT flexibility in N–C coupling and neuronal-branching regulation during neuronal migration.

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

2007 ◽  
Vol 3 ◽  
pp. 117693510700300 ◽  
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.

scholarly journals MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis

2000 ◽  
Vol 14 (9) ◽  
pp. 1072-1084
Author(s):  
Martin Srayko ◽  
Dan W. Buster ◽  
Omar A. Bazirgan ◽  
Francis J. McNally ◽  
Paul E. Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Subcellular Location ◽  
Meiotic Spindle ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Acid Protein ◽  
Spindle Function

The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm ∼20 minutes apart. Themei-1 and mei-2 genes of C. elegans are required for the establishment of the oocyte meiotic spindle but are not required for mitotic spindle function. mei-1 encodes an AAA ATPase family member with similarity to the p60 catalytic subunit of the heterodimeric sea urchin microtubule-severing protein, katanin. We report that mei-2 encodes a 280-amino acid protein containing a region similar to the p80-targeting subunit of katanin. MEI-1 and MEI-2 antibodies decorate the polar ends of meiotic spindle microtubules and meiotic chromatin. We find that the subcellular location of MEI-2 depends on wild-type mei-1 activity and vice versa. These experiments, combined with MEI-1 and MEI-2's similarity to p60 and p80 katanin, suggest that the C. elegans proteins function as a complex. In support of this idea, MEI-1 and MEI-2 physically associate in HeLa cells. Furthermore, co-expression of MEI-1 and MEI-2 in HeLa cells results in the disassembly of microtubules. These data lead us to conclude that MEI-1/MEI-2 microtubule-severing activity is required for meiotic spindle organization in C. elegans.

scholarly journals Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

2014 ◽  
Vol 25 (7) ◽  
pp. 1037-1049 ◽  
Author(s):  
Karen McNally ◽  
Evan Berg ◽  
Daniel B. Cortes ◽  
Veronica Hernandez ◽  
Paul E. Mains ◽  
...  
Keyword(s):  
Point Mutations ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Microtubule Organization ◽  
Bipolar Structure ◽  
Microtubule Severing ◽  
Meiotic Spindles

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.

scholarly journals Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations

2021 ◽  
Author(s):  
Ben Paul Jones ◽  
Arnoud H. M. van Vliet ◽  
E. James La Course ◽  
Martha Betson
Keyword(s):  
Amino Acid ◽  
Obvious Effect ◽  
Middle Income ◽  
Docking Simulations ◽  
In Silico Docking ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Amino Acid Mutations ◽  
Dynamics Simulations ◽  
Β Tubulin

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes.

scholarly journals Identification of Key Interactions of Benzimidazole Resistance-Associated Amino Acid Mutations in Ascaris β-Tubulins By Molecular Docking Simulations

2021 ◽  
Author(s):  
Ben P. Jones ◽  
Arnoud H.M. Vliet ◽  
E. James LaCourse ◽  
Martha Betson
Keyword(s):  
Amino Acid ◽  
Obvious Effect ◽  
Middle Income ◽  
Docking Simulations ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Amino Acid Mutations ◽  
Dynamics Simulations ◽  
Tubulin Protein ◽  
Β Tubulin

Abstract Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes.

scholarly journals In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.

1987 ◽  
Vol 105 (4) ◽  
pp. 1707-1720 ◽  
Author(s):  
M A Lopata ◽  
D W Cleveland
Keyword(s):  
Polyclonal Antibodies ◽  
Cell Types ◽  
Amino Acid Residues ◽  
Beta Tubulin ◽  
Tubulin Isotypes ◽  
Peptide Antigens ◽  
Tubulin Isotype ◽  
Carboxy Terminal ◽  
Different Cell Types

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta-tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

scholarly journals In vivo coassembly of a divergent beta-tubulin subunit (c beta 6) into microtubules of different function.

1987 ◽  
Vol 105 (5) ◽  
pp. 2179-2190 ◽  
Author(s):  
H C Joshi ◽  
T J Yen ◽  
D W Cleveland
Keyword(s):  
Random Copolymers ◽  
Resolution Limit ◽  
Beta Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Tubulin Subunit ◽  
Cytoplasmic Microtubules ◽  
Chicken Erythrocytes

alpha- and beta-Tubulin are encoded in vertebrate genomes by a family of approximately 6-7 functional genes whose polypeptide products differ in amino acid sequence. In the chicken, one beta-tubulin isotype (c beta 6) has previously been found to be expressed only in thrombocytes and erythroid cells, where it is assembled into a circumferential ring of marginal band microtubules. In light of its unique in vivo utilization and its divergent assembly properties in vitro, we used DNA transfection to test whether this isotype could be assembled in vivo into microtubules of divergent functions. Using an antibody specific to c beta 6, we have found that upon transfection this polypeptide is freely coassembled into an extensive array of interphase cytoplasmic microtubules and into astral and pole-to-chromosome or pole-to-pole microtubules during mitosis. Further, examination of developing chicken erythrocytes reveals that both beta-tubulins that are expressed in these cells (c beta 6 and c beta 3) are found as co-polymers of the two isoforms. These results, in conjunction with efforts that have localized various other beta-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo microtubules in vertebrates are random copolymers of available isotypes. Although these findings are consistent with functional interchangeability of beta-tubulin isotypes, we have also found that in vivo microtubules enriched in c beta 3 polypeptides are more sensitive to cold depolymerization than those enriched in c beta 6. This differential quantitative utilization of the two endogenous isotypes documents that some in vivo functional differences between isotypes do exist.

scholarly journals Katanin regulates dynamics of microtubules and biogenesis of motile cilia

2007 ◽  
Vol 178 (6) ◽  
pp. 1065-1079 ◽  
Author(s):  
Neeraj Sharma ◽  
Jessica Bryant ◽  
Dorota Wloga ◽  
Rachel Donaldson ◽  
Richard C. Davis ◽  
...  
Keyword(s):  
Cortical Microtubules ◽  
Cell Type ◽  
Post Translational Modifications ◽  
Microtubule Severing ◽  
Spatial Regulation ◽  
Internal Network ◽  
A Cell ◽  
Motile Cilia ◽  
Β Tubulin

The in vivo significance of microtubule severing and the mechanisms governing its spatial regulation are not well understood. In Tetrahymena, a cell type with elaborate microtubule arrays, we engineered null mutations in subunits of the microtubule-severing complex, katanin. We show that katanin activity is essential. The net effect of katanin on the polymer mass depends on the microtubule type and location. Although katanin reduces the polymer mass and destabilizes the internal network of microtubules, its activity increases the mass of ciliary microtubules. We also show that katanin reduces the levels of several types of post-translational modifications on tubulin of internal and cortical microtubules. Furthermore, katanin deficiencies phenocopy a mutation of β-tubulin that prevents deposition of polymodifications (glutamylation and glycylation) on microtubules. We propose that katanin preferentially severs older, post-translationally modified segments of microtubules.

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