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Mécanismes moléculaires du transport intracellulaire

Mots-clés : appareil de Golgi, GTPases Rab, transport intracellulaire, membranes modèles, cycle cellulaire, cytocinèse

Chef d'équipe : Bruno Goud

Des défauts dans les processus de transport intracellulaire sont fréquemment associés à de nombreuses pathologies, y compris le cancer. Notre équipe s’intéresse aux mécanismes régulant le transport intracellulaire entre les compartiments de la cellule eucaryote, en particulier au niveau de l’appareil de Golgi. Pour cet objectif, nous utilisons une variété d’approches méthodologiques. Quatre thèmes principaux sont développés :


- Fonction des GTPases de la famille Rab dans le trafic membranaire et la biogénèse des membranes (Bruno Goud)


Les GTPases de la famille Rab (une soixantaine identifiées chez l’Homme) sont des régulateurs majeurs du transport intracellulaire. Nous étudions plus particulièrement la protéine Rab6, présente sur les membranes de l’appareil de Golgi (Fig. 1), et les protéines qui partagent des effecteurs communs avec Rab6, comme Rab8, Rab11 et Rab39. Nos travaux récents ont mis en particulier en évidence un rôle de la myosine II dans la fission des vésicules de transport à partir des membranes de l’appareil de Golgi et d’identifier les domaines de la myosine Va liant plusieurs protéines Rab. Nos projets en cours portent principalement sur la reconstitution in vitro du processus de fission dépendant de la myosine II, le rôle de Rab6, Rab8 et de la myosine V dans le transport post-golgien, le rôle des protéines Rab dans le transport d’APP (« amyloid precursor protein ») et de BACE-1, et la dérégulation de la fonction des protéines Rab dans le cancer de la vessie.

Rôle de Rab6 dans la régulation du transport au niveau de l’appareil de Golgi
Fig. 1: Role de Rab6 dans la régulation du transport au niveau de l’appareil de Golgi


- Paramètres physiques qui sous-tendent les processus de transport (Jean-Baptiste Manneville)

Pour comprendre le rôle des paramètres physiques tels que la tension ou la courbure membranaires dans les processus de transport, nous développons depuis plusieurs années des systèmes biomimétiques basés sur des vésicules géantes unilamellaires (GUVs) à partir desquelles des tubes de membrane sont tirés avec des moteurs moléculaire ou une pince optique. Nos travaux récents ont porté sur les changements des propriétés membranaires après incorporation d'acides gras polyinsaturés et sur ​​le rôle de BIN1/M-amphiphysin2 dans l’agrégation des lipides. Nous avons également développé une nouvelle approche pour sonder la mécanique de l'appareil de Golgi dans les cellules vivantes en utilisant des billes de polystyrène internalisées dans les cellules et piégées avec une pince optique (Fig. 2).

Montage experimental pour mesurer la mécanique du Golgi dans les cellules
Fig. 2: Montage experimental pour mesurer la mécanique du Golgi dans les cellules


- Comprendre l'organisation globale des endomembranes (Kristine Schauer)

Nous avons développé une nouvelle approche d’imagerie basée sur des cartes de densité permettant de quantifier la distribution en 2D et 3D des compartiments cellulaires dans des cellules « micropatternées ». Cette approche est utilisée pour identifier les facteurs cellulaires et extra-cellulaires sous-tendant l’organisation des compartiments de la cellule. Nos travaux récents ont mis en évidence le rôle de l'adhésion cellulaire dans la topologie de l'endocytose et de la signalisation (Fig. 3). Les études en cours visent à identifier les moteurs moléculaires de la famille des myosines et des kinésines participant à l’organisation des endomembranes et à détecter les changements de cette organisation dans les cellules cancéreuses.

Comparison de l’endocytose de la transferrine et de l’EGF (« Epidermal Growth Factor ») dans des cellules micro-patternées
Fig. 3: Comparison de l’endocytose de la transferrine et de l’EGF (« Epidermal Growth Factor ») dans des cellules micro-patternées

- Fonctions des myosines dans le trafic membranaire (Evelyne Coudrier)

En plus de déplacer les vésicules de transport le long des filaments d'actine, les moteurs moléculaires de la famille des myosines régulent la morphologie des organites et facilitent le bourgeonnement des vésicules de transport. Elles peuvent également ancrer les vésicules de transport sur les filaments d'actine et faciliter leur fusion avec les organites. Nos travaux récents ont montré qu'une myosine non processive, la myosine 1b, régule la formation de tubules membranaires émanant du réseau trans-Golgien (TGN) et peut extraire des tubes membranaires le long des faisceaux de filaments d'actine à partir de liposomes géants. Nos études actuelles portent sur le rôle de la myosine 1b à la membrane plasmique et sur l'interaction des myosines avec le réseau d'actine au niveau du TGN.

Tubules membranaires formées par la myosine 1b à partir de liposomes géants en présence de câbles d’actine.
Fig. 4: Tubules membranaires formées par la myosine 1b à partir de liposomes géants en présence de câbles d’actine.


Publications clés

  • Year of publication : 2016

  • Integrins have key functions in cell adhesion and migration. How integrins are dynamically relocalized to the leading edge in highly polarized migratory cells has remained unexplored. Here, we demonstrate that β1 integrin (known as PAT-3 in Caenorhabditis elegans), but not β3, is transported from the plasma membrane to the trans-Golgi network, to be resecreted in a polarized manner. This retrograde trafficking is restricted to the non-ligand-bound conformation of β1 integrin. Retrograde trafficking inhibition abrogates several β1-integrin-specific functions such as cell adhesion in early embryonic development of mice, and persistent cell migration in the developing posterior gonad arm of C. elegans. Our results establish a paradigm according to which retrograde trafficking, and not endosomal recycling, is the key driver for β1 integrin function in highly polarized cells. These data more generally suggest that the retrograde route is used to relocalize plasma membrane machinery from previous sites of function to the leading edge of migratory cells.

  • Year of publication : 2015

  • Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis.

  • Year of publication : 2014

  • Preferred sites of endocytosis have been observed in various cell types, but whether they occur randomly or are linked to cellular cues is debated. Here, we quantified the sites of endocytosis of transferrin (Tfn) and epidermal growth factor (EGF) in cells whose adhesion geometry was defined by micropatterns. 3D probabilistic density maps revealed that Tfn was enriched in adhesive sites during uptake, whereas EGF endocytosis was restricted to the dorsal cellular surface. This spatial separation was not due to distributions of corresponding receptors but was regulated by uptake mechanisms. Asymmetric uptake of Tfn resulted from the enrichment of clathrin and adaptor protein 2 at adhesive areas. Asymmetry in EGF uptake was strongly dependent on the actin cytoskeleton and led to asymmetry in EGF receptor activation. Mild alteration of actin dynamics abolished asymmetry in EGF uptake and decreased EGF-induced downstream signaling, suggesting that cellular adhesion cues influence signal propagation. We propose that restriction of endocytosis at distinct sites allows cells to sense their environment in an "outside-in" mechanism.

  • Early endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration, and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but although factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes (SEs) remain unknown. We show that the microtubule motor KIF13A associates with recycling endosome tubules and controls their morphogenesis. Interfering with KIF13A function impairs the formation of endosomal tubules from SEs with consequent defects in endosome homeostasis and cargo recycling. Moreover, KIF13A interacts and cooperates with RAB11 to generate endosomal tubules. Our data illustrate how a microtubule motor couples early endosome morphogenesis to its motility and function.

  • Phospholipids (PLs) with polyunsaturated acyl chains are extremely abundant in a few specialized cellular organelles such as synaptic vesicles and photoreceptor discs, but their effect on membrane properties is poorly understood. Here, we found that polyunsaturated PLs increased the ability of dynamin and endophilin to deform and vesiculate synthetic membranes. When cells incorporated polyunsaturated fatty acids into PLs, the plasma membrane became more amenable to deformation by a pulling force and the rate of endocytosis was accelerated, in particular, under conditions in which cholesterol was limiting. Molecular dynamics simulations and biochemical measurements indicated that polyunsaturated PLs adapted their conformation to membrane curvature. Thus, by reducing the energetic cost of membrane bending and fission, polyunsaturated PLs may help to support rapid endocytosis.

  • Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

  • Year of publication : 2013

  • Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A', 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

  • Year of publication : 2010

  • Trafficking between membrane compartments is a characteristic of eukaryotic cells and relies on transport carriers that bud and fission from a donor membrane, before being transported and fusing with the correct acceptor compartment. Rab GTPases ensure specificity and directionality of trafficking steps by regulating the movement of transport carriers along cytoskeletal tracks, and the recruitment of tethering factors required for the docking and fusion processes. Here we show that Rab6, a Golgi-associated Rab, forms a complex with myosin II, contributes to its localization at the Golgi complex and, unexpectedly, controls the fission of Rab6 vesicles. Inhibition of either Rab6 or myosin II function impairs both the fission of Rab6 transport carriers from Golgi membranes and the trafficking of anterograde and retrograde cargo from the Golgi. These effects are consistent with myosin II being an effector of Rab6 in these processes. Our results provide evidence that the actomyosin system is required in vesicle biogenesis at the Golgi, and uncover a function for Rab GTPases in vesicle fission.

  • We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps. Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells. The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry. Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance observed for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.