Régulation de la Dynamique des Microtubules par code Tubuline

Publications de l’équipe

Année de publication : 2016

Jeffrey J Nirschl, Maria M Magiera, Jacob E Lazarus, Carsten Janke, Erika L F Holzbaur (2016 Mar 22)

α-Tubulin Tyrosination and CLIP-170 Phosphorylation Regulate the Initiation of Dynein-Driven Transport in Neurons.

Cell reports : 2637-52 : DOI : 10.1016/j.celrep.2016.02.046 En savoir plus
Résumé

Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single-molecule resolution, live-cell transport assays in primary neurons, computational image analysis, and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron.

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Chrystelle Aillaud, Christophe Bosc, Yasmina Saoudi, Eric Denarier, Leticia Peris, Laila Sago, Nicolas Taulet, Adeline Cieren, Olivia Tort, Maria M Magiera, Carsten Janke, Virginie Redeker, Annie Andrieux, Marie-Jo Moutin (2016 Feb 15)

Evidence for new C-terminally truncated variants of α- and β-tubulins.

Molecular biology of the cell : 640-53 : DOI : 10.1091/mbc.E15-03-0137 En savoir plus
Résumé

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.

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M Blosa, C Bursch, S Weigel, M Holzer, C Jäger, C Janke, R T Matthews, T Arendt, M Morawski (2016 Jan 1)

Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice.

Neural plasticity : 2828536 : DOI : 10.1155/2016/2828536 En savoir plus
Résumé

The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis.

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Année de publication : 2015

Carsten Janke, Michel O Steinmetz (2015 Aug 13)

Optochemistry to control the microtubule cytoskeleton.

The EMBO journal : 2114-6 : DOI : 10.15252/embj.201592415 En savoir plus
Résumé

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Marin Barisic, Ricardo Silva e Sousa, Suvranta K Tripathy, Maria M Magiera, Anatoly V Zaytsev, Ana L Pereira, Carsten Janke, Ekaterina L Grishchuk, Helder Maiato (2015 May 15)

Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

Science (New York, N.Y.) : 799-803 : DOI : 10.1126/science.aaa5175 En savoir plus
Résumé

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

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Nathalie Delgehyr, Alice Meunier, Marion Faucourt, Montserrat Bosch Grau, Laetitia Strehl, Carsten Janke, Nathalie Spassky (2015 Apr 4)

Ependymal cell differentiation, from monociliated to multiciliated cells.

Methods in cell biology : 19-35 : DOI : 10.1016/bs.mcb.2015.01.004 En savoir plus
Résumé

Primary and motile cilia differ in their structure, composition, and function. In the brain, primary cilia are immotile signalling organelles present on neural stem cells and neurons. Multiple motile cilia are found on the surface of ependymal cells in all brain ventricles, where they contribute to the flow of cerebrospinal fluid. During development, monociliated ependymal progenitor cells differentiate into multiciliated ependymal cells, thus providing a simple system for studying the transition between these two stages. In this chapter, we provide protocols for immunofluorescence staining of developing ependymal cells in vivo, on whole mounts of lateral ventricle walls, and in vitro, on cultured ependymal cells. We also provide a list of markers we currently use to stain both types of cilia, including proteins at the ciliary membrane and tubulin posttranslational modifications of the axoneme.

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Magali Casanova, Frédérique de Monbrison, Juliette van Dijk, Carsten Janke, Michel Pagès, Patrick Bastien (2015 Feb 1)

Characterisation of polyglutamylases in trypanosomatids.

International journal for parasitology : 121-32 : DOI : 10.1016/j.ijpara.2014.09.005 En savoir plus
Résumé

Microtubules are subject to post-translational modifications, which are thought to have crucial roles in the function of complex microtubule-based organelles. Among these, polyglutamylation was relatively recently discovered, and was related to centrosome stability, axonemal maintenance and mobility, and neurite outgrowth. In trypanosomatids, parasitic protozoa where microtubules constitute the essential component of the cytoskeleton, the function of polyglutamylated microtubules is unknown. Here, in order to better understand the role of this conserved but highly divergent post-translational modification, we characterised glutamylation and putative polyglutamylases in these parasites. We showed that microtubules are intensely glutamylated in all stages of the cell cycle, including interphase. Moreover, a cell cycle-dependent gradient of glutamylation was observed along the cell anteroposterior axis, which might be related to active growth of the microtubule ‘corset’ during the cell cycle. We also identified two putative polyglutamylase proteins (among seven analysed here) which appeared to be clearly and directly involved in microtubule polyglutamylation in in vitro activity assays. Paradoxically, in view of the importance of tubulins and of their extensive glutamylation in these organisms, RNA interference-based knockdown of all these proteins had no effect on cell growth, suggesting either functional redundancy or, more likely, subtle roles such as function modulation or interaction with protein partners.

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Année de publication : 2014

Olivia Tort, Sebastián Tanco, Cecilia Rocha, Ivan Bièche, Cecilia Seixas, Christophe Bosc, Annie Andrieux, Marie-Jo Moutin, Francesc Xavier Avilés, Julia Lorenzo, Carsten Janke (2014 Oct 1)

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids.

Molecular biology of the cell : 3017-27 : DOI : 10.1091/mbc.E14-06-1072 En savoir plus
Résumé

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.

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Cecilia Rocha, Laura Papon, Wulfran Cacheux, Patricia Marques Sousa, Valeria Lascano, Olivia Tort, Tiziana Giordano, Sophie Vacher, Benedicte Lemmers, Pascale Mariani, Didier Meseure, Jan Paul Medema, Ivan Bièche, Michael Hahne, Carsten Janke (2014 Oct 1)

Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon.

The EMBO journal : 2247-60 : DOI : 10.15252/embj.201488466 En savoir plus
Résumé

TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development.

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Carsten Janke (2014 Aug 18)

The tubulin code: molecular components, readout mechanisms, and functions.

The Journal of cell biology : 461-72 : DOI : 10.1083/jcb.201406055 En savoir plus
Résumé

Microtubules are cytoskeletal filaments that are dynamically assembled from α/β-tubulin heterodimers. The primary sequence and structure of the tubulin proteins and, consequently, the properties and architecture of microtubules are highly conserved in eukaryotes. Despite this conservation, tubulin is subject to heterogeneity that is generated in two ways: by the expression of different tubulin isotypes and by posttranslational modifications (PTMs). Identifying the mechanisms that generate and control tubulin heterogeneity and how this heterogeneity affects microtubule function are long-standing goals in the field. Recent work on tubulin PTMs has shed light on how these modifications could contribute to a « tubulin code » that coordinates the complex functions of microtubules in cells.

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Maria M Magiera, Carsten Janke (2014 May 5)

Post-translational modifications of tubulin.

Current biology : CB : R351-4 : DOI : 10.1016/j.cub.2014.03.032 En savoir plus
Résumé

Microtubules are the largest filamentous components of the eukaryotic cytoskeleton. In spite of their extraordinary level of structural conservation, microtubules fulfill a vast range of different functions in cells. How this functional diversity is achieved remains an open question; however, recent advances point towards post-translational modifications (PTMs) of tubulin as a potent mechanism to generate microtubule identities. As many microtubule functions have direct implications for development and homeostasis of organisms, understanding the molecular functions of tubulin PTMs could provide a more differentiated view on the role of microtubules in both normal and pathological aspects of organism development.

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Annemarie Wehenkel, Carsten Janke (2014 Apr 1)

Towards elucidating the tubulin code.

Nature cell biology : 303-5 : DOI : 10.1038/ncb2938 En savoir plus
Résumé

Genetically encoded and post-translationally generated variations of tubulin C-terminal tails give rise to extensive heterogeneity of the microtubule cytoskeleton. The generation of different tubulin variants in yeast now demonstrates how single amino-acid differences or post-translational modifications can modulate the behaviour of selected molecular motors.

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Teresa Mendes Maia, Delphine Gogendeau, Carole Pennetier, Carsten Janke, Renata Basto (2014 Feb 15)

Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules.

Biology open : 138-51 : DOI : 10.1242/bio.20146577 En savoir plus
Résumé

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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Année de publication : 2013

Carsten Janke (2013 Oct 16)

Mysterious modification of tubulin.

Nature Reviews Molecular Cell BiologyNature Reviews Molecular Cell Biology En savoir plus
Maria M Magiera, Carsten Janke (2013 Aug 27)

Investigating tubulin posttranslational modifications with specific antibodies.

Methods in cell biology : 247-67 : DOI : 10.1016/B978-0-12-407757-7.00016-5 En savoir plus
Résumé

Microtubules play highly diverse and essential roles in every eukaryotic cell. While built from conserved dimers of α- and β-tubulin, microtubules can be diversified by posttranslational modifications in order to fulfill specific functions in cells. The tubulin posttranslational modifications: acetylation, detyrosination, polyglutamylation, and polyglycylation play important roles in microtubule functions; however, only little functional and mechanistic insight has been gained so far. The modification state of microtubules can be visualized with specific antibodies. A drawback is that detailed information about the specificities and limitations of these antibodies are not easily accessible in the literature. We provide here a comprehensive description of the currently available set of antibodies specific to tubulin modifications. Focusing on glutamylation antibodies, we discuss specific protocols that allow using these antibodies to gain semi-quantitative information on the levels and distribution of tubulin modifications in immunocytochemistry and immunoblot.

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