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Ritsu Kamiya - One of the best experts on this subject based on the ideXlab platform.
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Reduced tubulin Polyglutamylation suppresses flagellar shortness in Chlamydomonas
Molecular Biology of the Cell, 2015Co-Authors: Tomohiro Kubo, Masafumi Hirono, Takumi Aikawa, Ritsu Kamiya, George B. WitmanAbstract:Tubulin Polyglutamylation is a posttranslational modification known to affect ciliary/flagellar motility and assembly. Investigation of Chlamydomonas mutants deficient in axonemal Polyglutamylation...
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A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9
Molecular Biology of the Cell, 2014Co-Authors: Tomohiro Kubo, Masafumi Hirono, Haruaki Yanagisawa, Zhongmei Liu, Rie Shibuya, Ritsu KamiyaAbstract:Tubulin undergoes various posttranslational modifications, including Polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased Polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a Polyglutamylation complex specialized for regulation of ciliary motility.
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Tubulin Polyglutamylation regulates flagellar motility by controlling a specific inner-arm dynein that interacts with the dynein regulatory complex.
Cytoskeleton, 2012Co-Authors: Tomohiro Kubo, Toshiki Yagi, Ritsu KamiyaAbstract:The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin Polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin Polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin Polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin Polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin Polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.
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Tubulin Polyglutamylation regulates axonemal motility by modulating activities of inner-arm dyneins.
Current Biology, 2010Co-Authors: Tomohiro Kubo, Masafumi Hirono, Toshiki Yagi, Haruaki Yanagisawa, Ritsu KamiyaAbstract:Tubulin Polyglutamylation is a modification that adds multiple glutamates to the gamma-carboxyl group of a glutamate residue in the C-terminal tails of alpha- and beta-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in alpha-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that Polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins.
Christian Erck - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of Peptides from α- and β-Tubulin Containing Glutamic Acid Side-Chain Linked Oligo-Glu with Defined Length
International Journal of Peptides, 2010Co-Authors: Werner Tegge, Carlos Francisco Sampaio Bonafe, Aileen Teichmann, Christian ErckAbstract:Side-chain oligo- and Polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(E(x))-EEGEEY and CQDATADEQG-E(E(x))-FEEEEGEDEA from the C-termini of mammalian α1- and β1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of Polyglutamylation processes.
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Research Article Synthesis of Peptides from α-andβ-Tubulin Containing Glutamic Acid Side-Chain Linked Oligo-Glu with Defined Length
2010Co-Authors: Werner Tegge, Carlos Francisco Sampaio Bonafe, Aileen Teichmann, Christian ErckAbstract:Copyright © 2010 Werner Tegge et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Side-chain oligo- and Polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(Ex)-EEGEEY and CQDATADEQG-E(Ex)-FEEEEGEDEA from the C-termini of mammalian α1- and β1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of Polyglutamylation processes. 1
Tomohiro Kubo - One of the best experts on this subject based on the ideXlab platform.
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Chlamydomonas as a tool to study tubulin Polyglutamylation.
Microscopy, 2018Co-Authors: Tomohiro Kubo, Toshiyuki OdaAbstract:The diversity of α- and β-tubulin is facilitated by various post-translational modifications (PTMs), such as acetylation, tyrosination, glycylation, glutamylation, phosphorylation and methylation. These PTMs affect the stability and structure of microtubules as well as the interaction between microtubules and microtubule-associated proteins, including molecular motors. Therefore, it is extremely important to investigate the roles of tubulin PTMs for understanding the cell cycle, cell motility and intracellular trafficking. Tubulin PTMs were first studied in the 1980s, and considerable progress has been made since then; it is likely that additional mechanisms remain yet to be elucidated. Here, we discuss one such modification, tubulin glutamylation, and introduce our research on the eukaryotic flagellum of the unicellular green alga Chlamydomonas reinhardtii.
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Reduced tubulin Polyglutamylation suppresses flagellar shortness in Chlamydomonas
Molecular Biology of the Cell, 2015Co-Authors: Tomohiro Kubo, Masafumi Hirono, Takumi Aikawa, Ritsu Kamiya, George B. WitmanAbstract:Tubulin Polyglutamylation is a posttranslational modification known to affect ciliary/flagellar motility and assembly. Investigation of Chlamydomonas mutants deficient in axonemal Polyglutamylation...
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A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9
Molecular Biology of the Cell, 2014Co-Authors: Tomohiro Kubo, Masafumi Hirono, Haruaki Yanagisawa, Zhongmei Liu, Rie Shibuya, Ritsu KamiyaAbstract:Tubulin undergoes various posttranslational modifications, including Polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased Polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a Polyglutamylation complex specialized for regulation of ciliary motility.
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Tubulin Polyglutamylation regulates flagellar motility by controlling a specific inner-arm dynein that interacts with the dynein regulatory complex.
Cytoskeleton, 2012Co-Authors: Tomohiro Kubo, Toshiki Yagi, Ritsu KamiyaAbstract:The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin Polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin Polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin Polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin Polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin Polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.
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Tubulin Polyglutamylation regulates axonemal motility by modulating activities of inner-arm dyneins.
Current Biology, 2010Co-Authors: Tomohiro Kubo, Masafumi Hirono, Toshiki Yagi, Haruaki Yanagisawa, Ritsu KamiyaAbstract:Tubulin Polyglutamylation is a modification that adds multiple glutamates to the gamma-carboxyl group of a glutamate residue in the C-terminal tails of alpha- and beta-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in alpha-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that Polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins.
Rocío I. Díaz De La Garza - One of the best experts on this subject based on the ideXlab platform.
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Folate levels and Polyglutamylation profiles of papaya (Carica papaya cv. Maradol) during fruit development and ripening.
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Perla A. Ramos-parra, Carolina García-salinas, Carmen Hernández-brenes, Rocío I. Díaz De La GarzaAbstract:Folates are essential micronutrients for humans, and their deficiency causes several detrimental effects on human health. Papaya fruit is an important natural source of some micronutrients. This paper presents a first complete characterization of folate derivatives accumulated in cv. Maradol papaya during fruit development and ripening processes. During postharvest ripening, the fruit accumulated up to 24.5% of the daily folate recommended dietary allowance (RDA) for an adult in a 1 cup (145 g) portion. Tetrahydrofolate (THF) and 5-methyl-THF were the predominant folate classes observed. Surprisingly, an unusually long Polyglutamylation profile of tentatively up to 17 glutamates linked to 5-methyl-THF was detected; to the authors’ knowledge, this very long polyglutamyl tail has not been reported for any organism, and it is probably characteristic of this plant species. This Polyglutamylation degree changed throughout fruit development and ripening, showing the largest differences at the onset of ripening....
Carsten Janke - One of the best experts on this subject based on the ideXlab platform.
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Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules
The Company of Biologists, 2014Co-Authors: Teresa Mendes Maia, Carsten Janke, Delphine Gogendeau, Carole Pennetier, Renata BastoAbstract:Summary Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, Polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal Polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme
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post translational regulation of the microtubule cytoskeleton mechanisms and functions
Nature Reviews Molecular Cell Biology, 2011Co-Authors: Carsten Janke, Jeannette Chloe BulinskiAbstract:Cells generate distinct microtubule subtypes by expressing different tubulin isotypes and through tubulin post-translational modifications, such as detyrosination, acetylation, Polyglutamylation and polyglycylation. The recent discovery of enzymes responsible for many of these modifications has shown how they may regulate microtubule functions.
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Polyglutamylation des microtubules et neurodégénérescence
médecine sciences, 2011Co-Authors: Marie-jo Moutin, Annie Andrieux, Carsten JankeAbstract:m/s n° 5, vol. 27, mai 2011 DOI : 10.1051/medsci/2011275006 Les microtubules et leur regulation par des modifications posttraductionnelles Les microtubules sont des fibres du cytosquelette cellulaire. Dans les cellules eucaryotes, ils forment un reseau dynamique essentiel a la mobilite et a la morphologie de la cellule ainsi qu’a sa polarite. Dans les cellules nerveuses, par exemple, les microtubules determinent le choix de l’extension neuritique qui deviendra l’axone du neurone differencie. Les microtubules permettent egalement le trafic de nombreux organites (mitochondries, vesicules golgiennes ou synaptiques, etc.) ou molecules (comme les ARN) dans le cytoplasme. Lorsque les cellules entrent en mitose, les microtubules forment le fuseau mitotique qui segrege les chromosomes entre les cellules filles. Les microtubules sont egalement les composants principaux des cils et des flagelles : ils en constituent l’axe central, l’axoneme. Au niveau moleculaire, les microtubules se presentent comme de larges tubes creux dont la paroi est composee de proteines tres conservees dans l’evolution, les tubulines. Plus precisement, ce sont des assemblages orientes et dynamiques de dimeres de tubuline α et β (Figure 1A). Leurs extremites, en particulier celles appelees bouts « plus » (cote β-tubuline) qui se trouvent vers la peripherie cellulaire, polymerisent et depolymerisent en permanence. Parce que Polyglutamylation des microtubules et neurodegenerescence
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Tubulin Polyglutamylation stimulates spastin-mediated microtubule severing.
Journal of Cell Biology, 2010Co-Authors: Benjamin Lacroix, Krzysztof Rogowski, Juliette Van Dijk, Nicholas D Gold, Julien Guizetti, Gudrun Aldrian-herrada, Daniel W Gerlich, Carsten JankeAbstract:Posttranslational glutamylation of tubulin is present on selected subsets of microtubules in cells. Although the modification is expected to contribute to the spatial and temporal organization of the cytoskeleton, hardly anything is known about its functional relevance. Here we demonstrate that glutamylation, and in particular the generation of long glutamate side chains, promotes the severing of microtubules. In human cells, the generation of long side chains induces spastin-dependent microtubule disassembly and, consistently, only microtubules modified by long glutamate side chains are efficiently severed by spastin in vitro. Our study reveals a novel control mechanism for microtubule mass and stability, which is of fundamental importance to cellular physiology and might have implications for diseases related to microtubule severing.
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Polyglutamylation a fine regulator of protein function
EMBO Reports, 2008Co-Authors: Carsten Janke, Krzysztof Rogowski, Juliette Van DijkAbstract:Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various Polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of Polyglutamylation and now allows for direct functional studies of the role of tubulin Polyglutamylation. Moreover, the recent identification of new substrates of Polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.