Causes and Consequences of Microtubule Acetylation

International audienceAmong the different types of cytoskeletal components, microtubules arguably accumulate the greatest diversity of post-translational modifications (PTMs). Acetylation of lysine 40 (K40) of atubulin has received a particular attention because it is the only tubulin PTM to be found in the lumen of microtubules - most other tubulin PTMs are found at their outer surface. As a consequence, the enzyme catalyzing K40 acetylation needs to penetrate the narrow microtubules lumen to find its substrate. Acetylated microtubules have been considered as stable, long-lived microtubules, however until recently there was little information about whether the longevity of these microtubules is the cause or the consequence of acetylation. Current advances suggest that this PTM helps the microtubule lattice to cope with mechanical stress, thus facilitating microtubule self-repair. These observations now shed a new light on the structural integrity of microtubules, as well as on mechanisms and biological functions of tubulin acetylation. Here we discuss the recentunderstanding on how acetylation is generated in the lumen of microtubules, and how this ‘hidden’PTM can control microtubule properties and functions

A coupled implicit-explicit time integration method for compressible unsteady flows

This paper addresses how two time integration schemes, the Heun's scheme for explicit time integration and the second-order Crank-Nicolson scheme for implicit time integration, can be coupled spatially. This coupling is the prerequisite to perform a coupled Large Eddy Simulation / Reynolds Averaged Navier-Stokes computation in an industrial context, using the implicit time procedure for the boundary layer (RANS) and the explicit time integration procedure in the LES region. The coupling procedure is designed in order to switch from explicit to implicit time integrations as fast as possible, while maintaining stability. After introducing the different schemes, the paper presents the initial coupling procedure adapted from a published reference and shows that it can amplify some numerical waves. An alternative procedure, studied in a coupled time/space framework, is shown to be stable and with spectral properties in agreement with the requirements of industrial applications. The coupling technique is validated with standard test cases, ranging from one-dimensional to three-dimensional flows

Class III PI3K regulates organismal glucose homeostasis by providing negative feedback on hepatic insulin signalling.

Defective hepatic insulin receptor (IR) signalling is a pathogenic manifestation of metabolic disorders including obesity and diabetes. The endo/lysosomal trafficking system may coordinate insulin action and nutrient homeostasis by endocytosis of IR and the autophagic control of intracellular nutrient levels. Here we show that class III PI3K--a master regulator of endocytosis, endosomal sorting and autophagy--provides negative feedback on hepatic insulin signalling. The ultraviolet radiation resistance-associated gene protein (UVRAG)-associated class III PI3K complex interacts with IR and is stimulated by insulin treatment. Acute and chronic depletion of hepatic Vps15, the regulatory subunit of class III PI3K, increases insulin sensitivity and Akt signalling, an effect that requires functional IR. This is reflected by FoxO1-dependent transcriptional defects and blunted gluconeogenesis in Vps15 mutant cells. On depletion of Vps15, the metabolic syndrome in genetic and diet-induced models of insulin resistance and diabetes is alleviated. Thus, feedback regulation of IR trafficking and function by class III PI3K may be a therapeutic target in metabolic conditions of insulin resistance

Clathrin-coated structures support 3D directed migration through local force transmission

Migrating cells navigate in complex environments through sensing and interpreting biochemical and/or mechanical cues. Here, we report that recently identified tubular clathrin/AP-2 lattices (TCALs), a subset of clathrin-coated structures (CCSs) that pinch collagen fibers, mechanically control directed migration along fibers decorated with ligands of CCS cargoes in three-dimensional (3D) environments. We observed that epidermal growth factor or low-density lipoprotein bound to collagen fibers leads to increased local nucleation and accumulation of TCALs. By using engineered, mixed collagen networks, we demonstrate that this mechanism selectively increases local forces applied on ligand-decorated fibers. We show that these effects depend on the ligand’s receptors but do not rely on their ability to trigger signaling events. We propose that the preferential accumulation of TCALs along ligand-decorated fibers steers migration in 3D environments. We conclude that ligand-regulated, local TCAL accumulation results in asymmetric force distribution that orients cell migration in 3D environments

αTAT1 controls longitudinal spreading of acetylation marks from open microtubules extremities OPEN

International audienceAcetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time. Bias for MTs extremities was even more pronounced when using non-dynamic MTs extracted from HeLa cells. In contrast, K40 acetylation was mostly uniform along the length of MTs reconstituted from purified tubulin in vitro. Quantitative modelling of luminal diffusion of αTAT1 suggested that the uniform acetylation pattern observed in vitro is consistent with defects in the MT lattice providing lateral access to the lumen. Indeed, we observed that in vitro MTs are permeable to macromolecules along their shaft while cellular MTs are not. Our results demonstrate αTAT1 enters the lumen from open extremities and spreads K40 acetylation marks longitudinally along cellular MTs. This mode of tip-directed microtubule acetylation may allow for selective acetylation of subsets of microtubules. Results and Discussion Microtubules (MTs) are dynamic polymers composed of α β-tubulin dimers that assembled into hollow tubes. In most eukaryotic cells, MTs can undergo post-translational modifications (PTMs) that modify their properties and functions 1. Acetylation of the lysine 40 of α-tubulin (K40) is a common PTM that is catalysed by the α-tubulin acetyl-transferase α TAT1 and is associated with stable, long-lived MTs 2-4. Remarkably, K40 acetylation occurs in the lumen of MTs 5,6 and is the only such PTM that we know of ref. 1. Supporting this, Szyk et al. recently used in vitro approaches to demonstrate that α TAT1 enters into and diffuses within the MT lumen 7. However, Szyk et al. also suggested that fast diffusivity of α TAT1 leads to stochastic acetylation that occurs uniformly along the length of MTs. This was in marked contrast with earlier in vivo observations of discrete acetylated segments along MTs 8-10 progressively elongating with time 11. More recently, several groups have reported that the acetylated segments were predominately associated with the ends of MTs in vivo 3,12. Thus, reported observations in vivo do not match the proposed model of uniformly distributed acetylated K40 marks based on experiments performed with in vitro MTs 7. To understand how acetylated K40 marks spreading occurs in vivo, we first analyzed acetylation dynamics in HeLa cells. In order to synchronize acetylation events, HeLa cells were subjected to complete MT depolymeri-sation by a prolonged treatment with Nocodazole before being allowed to reassemble MTs after washing out th

Novel integrin endocytosis motif

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.We gratefully acknowledge the following funding sources: N.d.F. FinPharma Doctoral Program, Instrumentarium Foundation, Orion Research Foundation, Liv och Halsa foundation, Finsk-Norska Medicinska Stiftelsen and the Magnus Ehrnrooth Foundation; J.I. Academy of Finland CoE, European Research Council Consolidator Grant, the Sigrid Juselius Foundation, The Finnish Heart Foundation and Finnish Cancer Organizations. DJO, AGW and TW are funded by Wellcome Trust fellowship 090909 (DJO).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.3161

Etude du rôle du récepteur de faible affinité aux IgE, CD23, dans le transport transépithélial de complexes IgE/allergènes

Le récepteur de faible affinité aux IgE (CD) est impliqué dans le transport transépithélial de complexes IgE / allergène. Au niveau intestinal, ce transport est à l'origine du déclenchement allergique. Mon travail de thèse a visé à étudier le rôle du CD23 dans ce processus. Nous avons établi que les cellules épithéliales intestinales murines expriment la forme CD23b ainsi qu'une nouvelle forme épissée, bDELTA5, dont l'expression est induite par la sensibilisation in vivo. La régulation de l'internalisation du CD23 murin est complexe, faisant intervenir des domaines intra et extracellulaires. Nous avons ensuite déterminé que la forme CD23b peut prendre en charge la transcytose de complexes IgE/allergène tandis que bDELTA5 peut transporter des IgE libres. Les études que nous avons menées en parallèle chez l'homme suggèrent que le CD23 y joue un rôle similaire bien que les modalités régulant l'endocytose et la transcytose soient différentes.PARIS5-BU-Necker : Fermée (751152101) / SudocSudocFranceF