Regulation of microtubule dynamics and function

Keywords : cell cylce, cilia, microtubule, neuron, polyglutamylation, polyglycylation, posttranslational modifications, tubulin

Group leader  : Carsten Janke

             

Posttranslational modifications enable the rapid, reversible and specific modulations of protein functions. The research projects of our group are focussed on the role of tubulin posttranslational modifications in the regulation of the functions of the microtubule cytoskeleton. In the past years, our group has discovered and characterised many of the enzymes that are involved in the catalysis of two complex tubulin modifications, polyglutamylation and polyglycylation. Our first functional studies using these enzymes have revealed important implications of both, polyglutamylation and polyglycylation in different functions of the microtubule cytoskeleton. We are now focussing on dissection of the molecular mechanisms that underlie the potential regulatory roles of these modifications. In parallel, we are focussing on the investigation of the functional roles of tubulin modifications in cells, organism development and disease. The complexity of these modifications might provide a fine-tuning mechanism for interactions between microtubules and their binding partners, the molecular motors and MAPs.


More details can be found on our group webpage at :

http://perso.curie.fr/Carsten.Janke/

Key publications

  • Year of publication : 2015

  • 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.

  • Year of publication : 2014

  • 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.

  • 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.

  • Year of publication : 2013

  • 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.

  • Year of publication : 2011

  • 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.

  • Year of publication : 2010

  • 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.

  • Year of publication : 2009

  • 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.

  • Year of publication : 2007

  • Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level.

  • Year of publication : 2005

  • Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.