Régulation de la Dynamique des Microtubules par code Tubuline

Publications de l’équipe

Année de publication : 2013

Montserrat Bosch Grau, Gloria Gonzalez Curto, Cecilia Rocha, Maria M Magiera, Patricia Marques Sousa, Tiziana Giordano, Nathalie Spassky, Carsten Janke (2013 Aug 5)

Tubulin glycylases and glutamylases have distinct functions in stabilization and motility of ependymal cilia.

The Journal of cell biology : 441-51 : DOI : 10.1083/jcb.201305041 En savoir plus
Résumé

Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease.

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Andrea E Prota, Maria M Magiera, Marijn Kuijpers, Katja Bargsten, Daniel Frey, Mara Wieser, Rolf Jaussi, Casper C Hoogenraad, Richard A Kammerer, Carsten Janke, Michel O Steinmetz (2013 Feb 4)

Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

The Journal of cell biology : 259-70 : DOI : 10.1083/jcb.201211017 En savoir plus
Résumé

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton.

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Année de publication : 2012

Antonio Castro-Castro, Carsten Janke, Guillaume Montagnac, Perrine Paul-Gilloteaux, Philippe Chavrier (2012 Dec 9)

ATAT1/MEC-17 acetyltransferase and HDAC6 deacetylase control a balance of acetylation of alpha-tubulin and cortactin and regulate MT1-MMP trafficking and breast tumor cell invasion.

European journal of cell biology : 950-60 : DOI : 10.1016/j.ejcb.2012.07.001 En savoir plus
Résumé

Invasive tumor cells use proteases to degrade and migrate through the stromal environment consisting of a 3D network of extracellular matrix macromolecules. In particular, MT1-MMP, a membrane-anchored metalloproteinase, is critical during cancer cell invasion. MT1-MMP is stored in endosomal compartments and then delivered to invadopodia, the specialized plasma membrane domains of invasive cancer cells endowed with extracellular matrix-degradation capacity. In macrophages, traffic of MT1-MMP vesicles to invadopodia-related podosomes requires microtubules. We previously found that in breast tumor MDA-MB-231 cells an increase of microtubule and cortactin acetylation upon inhibition of HDAC6 correlates with a decrease of matrix degradation and invasion in three-dimensional collagen I gel. Here, we investigated the role of the recently identified α-tubulin N-acetyltransferase 1 ATAT1 in invasive MDA-MB-231 cells. We found that the dynamics and distribution of MT1-MMP-positive endosomes require regulation of acetylation levels. We observed that ATAT1 tubulin acetyltransferase binds and regulates cortactin acetylation levels. In addition, ATAT1 colocalizes with cortactin at the adherent surface of the cells and it is required for 2D migration and invasive migration of MDA-MB-231 cells in collagen matrix. All together, our data indicate that a balance of acetylation and deaceylation by ATAT1/HDAC6 enzymes with opposite activities regulates the migratory and invasive capacities of breast tumor cells.

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Année de publication : 2011

Carsten Janke, Jeannette Chloë Bulinski (2011 Nov 16)

Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions.

Nature reviews. Molecular cell biology : 773-86 : DOI : 10.1038/nrm3227 En savoir plus
Résumé

Half a century of biochemical and biophysical experiments has provided attractive models that may explain the diverse functions of microtubules within cells and organisms. However, the notion of functionally distinct microtubule types has not been explored with similar intensity, mostly because mechanisms for generating divergent microtubule species were not yet known. Cells generate distinct microtubule subtypes through expression of different tubulin isotypes and through post-translational modifications, such as detyrosination and further cleavage to Δ2-tubulin, acetylation, polyglutamylation and polyglycylation. The recent discovery of enzymes responsible for many tubulin post-translational modifications has enabled functional studies demonstrating that these post-translational modifications may regulate microtubule functions through an amazing range of mechanisms.

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Benjamin Lacroix, Carsten Janke (2011 Jun 25)

Generation of differentially polyglutamylated microtubules.

Methods in molecular biology (Clifton, N.J.) : 57-69 : DOI : 10.1007/978-1-61779-252-6_4 En savoir plus
Résumé

Microtubules are cytoskeletal structures built of alpha- and beta-tubulins. Although tubulins are highly conserved throughout evolution, microtubules can be adapted to a range of different functions. A powerful mechanism that could regulate the functional specialization of microtubules is the posttranslational modification of tubulin molecules. Two tubulin modifications, polyglutamylation and polyglycylation, generate amino acid side chains of different length on tubulin. These modifications are thought to regulate interactions between microtubules and their associated proteins; however, detailed studies of this potential mechanism have not been performed. The investigation of the potential regulatory role of polyglutamylation requires in vitro tools to visualize the molecular events that could be affected by this modification. Classically, in vitro work with microtubules is performed with tubulin from brain tissue; however, this tubulin is highly posttranslationally modified. Here, we describe a method for the purification of tubulin carrying controlled levels of polyglutamylation, which can be used in basic in vitro assays.

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Marie-Jo Moutin, Annie Andrieux, Carsten Janke (2011 May 25)

[Microtubule polyglutamylation and neurodegeneration].

Médecine sciences : M/S : 464-7 : DOI : 10.1051/medsci/2011275006 En savoir plus
Résumé

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Année de publication : 2010

Krzysztof Rogowski, Juliette van Dijk, Maria M Magiera, Christophe Bosc, Jean-Christophe Deloulme, Anouk Bosson, Leticia Peris, Nicholas D Gold, Benjamin Lacroix, Montserrat Bosch Grau, Nicole Bec, Christian Larroque, Solange Desagher, Max Holzer, Annie Andrieux, Marie-Jo Moutin, Carsten Janke (2010 Nov 12)

A family of protein-deglutamylating enzymes associated with neurodegeneration.

Cell : 564-78 : DOI : 10.1016/j.cell.2010.10.014 En savoir plus
Résumé

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.

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Carsten Janke, Matthias Kneussel (2010 Aug 1)

Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton.

Trends in neurosciences : 362-72 : DOI : 10.1016/j.tins.2010.05.001 En savoir plus
Résumé

In the past decades, a range of post-translational modifications has been discovered on tubulins, the major constituents of microtubules. Pioneering studies have described the occurrence and dynamics of these modifications and provided first insights into their potential functions in regulating the microtubule cytoskeleton. By contrast, several tubulin-modifying enzymes were only discovered in the last few years, and studies on molecular mechanisms and cellular functions of tubulin modifications are just beginning to emerge. This review highlights the roles of tubulin modifications in neurons. Recent studies are also discussed in relation to how the combinatorial use of tubulin modifications could generate a dynamic microtubule code, and how such a code might regulate basic as well as higher-order neuronal functions.

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Benjamin Lacroix, Juliette van Dijk, Nicholas D Gold, Julien Guizetti, Gudrun Aldrian-Herrada, Krzysztof Rogowski, Daniel W Gerlich, Carsten Janke (2010 Jun 14)

Tubulin polyglutamylation stimulates spastin-mediated microtubule severing.

The Journal of cell biology : 945-54 : DOI : 10.1083/jcb.201001024 En savoir plus
Résumé

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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Kotoe Kashiwaya, Hidewaki Nakagawa, Masayo Hosokawa, Yasuo Mochizuki, Koji Ueda, Lianhua Piao, Suyoun Chung, Ryuji Hamamoto, Hidetoshi Eguchi, Hiroaki Ohigashi, Osamu Ishikawa, Carsten Janke, Yasuhisa Shinomura, Yusuke Nakamura (2010 May 15)

Involvement of the tubulin tyrosine ligase-like family member 4 polyglutamylase in PELP1 polyglutamylation and chromatin remodeling in pancreatic cancer cells.

Cancer research : 4024-33 : DOI : 10.1158/0008-5472.CAN-09-4444 En savoir plus
Résumé

Polyglutamylation is a new class of posttranslational modification in which glutamate side chains are formed in proteins, although its biological significance is not well known. Through our genome-wide gene expression profile analyses of pancreatic ductal adenocarcinoma (PDAC) cells, we identified the overexpression of tubulin tyrosine ligase-like family member 4 (TTLL4) in PDAC cells. Subsequent reverse transcription-PCR and Northern blot analyses confirmed its upregulation in several PDACs. TTLL4 belongs to the TTLL family which was reported to have polyglutamylase activity. Knockdown of TTLL4 by short hairpin RNA in PDAC cells attenuated the growth of PDAC cells and exogenous introduction of TTLL4 enhanced cell growth. We also found that TTLL4 expression was correlated with polyglutamylation levels of a glutamate stretch region of the proline, glutamate, and leucine-rich protein 1 (PELP1) that was shown to interact with various proteins such as histone H3, and was involved in several signaling pathways through its function as a scaffold protein. PELP1 polyglutamylation could influence its interaction with histone H3 and affect histone H3 acetylation. We also identified the interaction of PELP1 with LAS1L and SENP3, components of the MLL1-WDR5 supercomplex involving chromatin remodeling. Our findings imply that TTLL4 could play important roles in pancreatic carcinogenesis through its polyglutamylase activity and subsequent coordination of chromatin remodeling, and might be a good molecular candidate for the development of new therapeutic strategies for pancreatic cancer.

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Année de publication : 2009

Dorota Wloga, Danielle M Webster, Krzysztof Rogowski, Marie-Hélène Bré, Nicolette Levilliers, Maria Jerka-Dziadosz, Carsten Janke, Scott T Dougan, Jacek Gaertig (2009 Jun 15)

TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia.

Developmental cell : 867-76 : DOI : 10.1016/j.devcel.2009.04.008 En savoir plus
Résumé

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer’s vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

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Krzysztof Rogowski, François Juge, Juliette van Dijk, Dorota Wloga, Jean-Marc Strub, Nicolette Levilliers, Daniel Thomas, Marie-Hélène Bré, Alain Van Dorsselaer, Jacek Gaertig, Carsten Janke (2009 Jun 12)

Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation.

Cell : 1076-87 : DOI : 10.1016/j.cell.2009.05.020 En savoir plus
Résumé

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

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Christoph Maas, Dorthe Belgardt, Han Kyu Lee, Frank F Heisler, Corinna Lappe-Siefke, Maria M Magiera, Juliette van Dijk, Torben J Hausrat, Carsten Janke, Matthias Kneussel (2009 May 26)

Synaptic activation modifies microtubules underlying transport of postsynaptic cargo.

Proceedings of the National Academy of Sciences of the United States of America : 8731-6 : DOI : 10.1073/pnas.0812391106 En savoir plus
Résumé

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.

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Année de publication : 2008

Peter Bieling, Stefanie Kandels-Lewis, Ivo A Telley, Juliette van Dijk, Carsten Janke, Thomas Surrey (2008 Dec 29)

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites.

The Journal of cell biology : 1223-33 : DOI : 10.1083/jcb.200809190 En savoir plus
Résumé

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end-binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated alpha-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

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Carsten Janke, Krzysztof Rogowski, Juliette van Dijk (2008 Jul 1)

Polyglutamylation: a fine-regulator of protein function? ‘Protein Modifications: beyond the usual suspects’ review series.

EMBO reports : 636-41 : DOI : 10.1038/embor.2008.114 En savoir plus
Résumé

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.

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