Mécanismes moléculaires du transport intracellulaire

Publications de l’équipe

Année de publication : 2015

Cédric Delevoye, Bruno Goud (2015 Sep 12)

Rab GTPases and kinesin motors in endosomal trafficking.

Methods in cell biology : 235-46 : DOI : 10.1016/bs.mcb.2015.05.004 En savoir plus
Résumé

The endocytic pathway is composed of distinct types of endosomes that vary in shape, function, and molecular composition. In addition, endosomes are highly dynamic structures that continuously receive, sort, and deliver molecules to other organelles. Among organizing machineries that contribute to endosomal functions, Rab GTPases and kinesin motors play critical roles. Rab proteins define the identity of endosomal subdomains by recruiting set of effectors among which kinesins shape and transport membranous carriers along the microtubule network. In this review, we provide detailed protocols from live cell imaging to electron microscopy and biochemical approaches to address how Rab and kinesin proteins cooperate molecularly and functionally within the endocytic pathway.

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Sabine Bardin, Stéphanie Miserey-Lenkei, Ilse Hurbain, Daniela Garcia-Castillo, Graça Raposo, Bruno Goud (2015 Aug 26)

Phenotypic characterisation of RAB6A knockout mouse embryonic fibroblasts.

Biology of the cell / under the auspices of the European Cell Biology Organization : 427-39 : DOI : 10.1111/boc.201400083 En savoir plus
Résumé

Rab6 is one of the most conserved Rab GTPaes throughout evolution and the most abundant Rab protein associated with the Golgi complex. The two ubiquitous Rab isoforms, Rab6A and Rab6A’, that are generated by alternative splicing of the RAB6A gene, regulate several transport steps at the Golgi level, including retrograde transport between endosomes and Golgi, anterograde transport between Golgi and the plasma membrane, and intra-Golgi and Golgi to endoplasmic reticulum transport.

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Andrew J Lindsay, Stéphanie Miserey-Lenkei, Bruno Goud (2015 Mar 25)

Analysis of the interactions between Rab GTPases and class V myosins.

Methods in molecular biology (Clifton, N.J.) : 73-83 : DOI : 10.1007/978-1-4939-2569-8_6 En savoir plus
Résumé

Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli’s Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid « living chip » assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation.

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Maria P Gavilan, Marina Arjona, Angel Zurbano, Etienne Formstecher, Juan R Martinez-Morales, Michel Bornens, Rosa M Rios (2015 Mar 13)

Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

PLoS biology : e1002087 : DOI : 10.1371/journal.pbio.1002087 En savoir plus
Résumé

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

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Année de publication : 2014

Matteo Fossati, Bruno Goud, Nica Borgese, Jean-Baptiste Manneville (2014 Sep 12)

An investigation of the effect of membrane curvature on transmembrane-domain dependent protein sorting in lipid bilayers.

Cellular logistics : e29087 En savoir plus
Résumé

Sorting of membrane proteins within the secretory pathway of eukaryotic cells is a complex process involving discrete sorting signals as well as physico-chemical properties of the transmembrane domain (TMD). Previous work demonstrated that tail-anchored (TA) protein sorting at the interface between the Endoplasmic Reticulum (ER) and the Golgi complex is exquisitely dependent on the length and hydrophobicity of the transmembrane domain, and suggested that an imbalance between TMD length and bilayer thickness (hydrophobic mismatch) could drive long TMD-containing proteins into curved membrane domains, including ER exit sites, with consequent export of the mismatched protein out of the ER. Here, we tested a possible role of curvature in TMD-dependent sorting in a model system consisting of Giant Unilamellar Vesicles (GUVs) from which narrow membrane tubes were pulled by micromanipulation. Fluorescent TA proteins differing in TMD length were incorporated into GUVs of uniform lipid composition or made of total ER lipids, and TMD-dependent sorting and diffusion, as well as the bending rigidity of bilayers made of microsomal lipids, were investigated. Long and short TMD-containing constructs were inserted with similar orientation, diffused equally rapidly in GUVs and in tubes pulled from GUVs, and no difference in their final distribution between planar and curved regions was detected. These results indicate that curvature alone is not sufficient to drive TMD-dependent sorting at the ER-Golgi interface, and set the basis for the investigation of the additional factors that must be required.

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Mathieu Pinot, Stefano Vanni, Sophie Pagnotta, Sandra Lacas-Gervais, Laurie-Anne Payet, Thierry Ferreira, Romain Gautier, Bruno Goud, Bruno Antonny, Hélène Barelli (2014 Aug 9)

Lipid cell biology. Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins.

Science (New York, N.Y.) : 693-7 : DOI : 10.1126/science.1255288 En savoir plus
Résumé

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.

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Jean-Philippe Grossier, Bruno Goud, Kristine Schauer (2014 Jun 21)

Probabilistic density maps to study the spatial organization of endocytosis.

Methods in molecular biology (Clifton, N.J.) : 117-38 : DOI : 10.1007/978-1-4939-0944-5_8 En savoir plus
Résumé

Despite a large body of publications on endocytosis, only a few studies have focused on its spatial organization. To study how endocytosis is related to distinct cellular sites, we combine cell normalization by the « micropatterning technique » with the quantification of spatial organization by « probabilistic density mapping. » Micropatterns of extracellular matrix proteins impose adhesive and non-adhesive areas to cultured cells and allow the control of adhesion geometry, shape, and cell organization. Probabilistic density maps provide a visual summary for 3D localization of the structures of interest and enable the extraction of robust statistics for quantification of cellular organization. Here, we provide a method to analyze and compare the spatial distribution of endocytosed ligands in micropatterned cells. This approach permits to establish the role of cellular adhesion on uptake mechanisms and to address the potential function of predefined sites of endocytosis.

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Laura Picas, Julien Viaud, Kristine Schauer, Stefano Vanni, Karim Hnia, Vincent Fraisier, Aurélien Roux, Patricia Bassereau, Frédérique Gaits-Iacovoni, Bernard Payrastre, Jocelyn Laporte, Jean-Baptiste Manneville, Bruno Goud (2014 May 19)

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin.

Nature communications : 5647 : DOI : 10.1038/ncomms6647 En savoir plus
Résumé

Phosphoinositide play a central role in physiological processes by Many Assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How is this recruitment 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 ict downstream partner dynamin. By using Several mutants associated with centronuclear myopathies, we find that N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin we membranes, respectively. We that show phosphoinositide Clustering is a mechanism shared by –other That proteins interact with PtdIns (4,5) P2, do not aim to contain a BAR domain. Our numerical simulations Point Out That clustering is a diffusion-driven process in which molecules are not sequestered phosphoinositide. We propose That this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

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Kristine Schauer, Bruno Goud (2014 Apr 11)

Why does endocytosis in single cells care which side up?

Bioarchitecture : 62-7 : DOI : 10.4161/bioa.28809 En savoir plus
Résumé

Eukaryotic cells display an asymmetric distribution of cellular compartments relying on their adhesion and the underlying anisotropy of the actin and microtubule cytoskeleton. Studies using a minimal cell culture system based on confined adhesion on micropatterns have illustrated that trafficking compartments are well organized at the single cell level in response to the geometry of cellular adhesion cues. Expanding our analysis on cellular uptake processes, we have found that cellular adhesion additionally defines the topology of endocytosis and signaling. During endocytosis, transferrin (Tfn) and epidermal growth factor (EGF) concentrate at distinct cellular sites in micropatterned cells. Tfn is enriched in adhesive sites during uptake, whereas EGF endocytosis is restricted to the dorsal cellular surface. This unexpected dorsal/ventral asymmetry is regulated by uptake mechanisms and actin dynamics. Interestingly, restricted EGF uptake leads to asymmetry of EGF receptor activation that is required to sustain downstream signaling. Based on our results, we propose that differential sorting begins at the plasma membrane leading to spatially distinct intracellular trafficking routes that are well defined in space. We speculate that the intracellular positioning of trafficking compartments sustains an important coupling between the endocytic and signaling systems that allows cells to sense their environment.

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David Guet, Kalpana Mandal, Mathieu Pinot, Jessica Hoffmann, Yara Abidine, Walter Sigaut, Sabine Bardin, Kristine Schauer, Bruno Goud, Jean-Baptiste Manneville (2014 Apr 8)

Mechanical role of actin dynamics in the rheology of the Golgi complex and in Golgi-associated trafficking events.

Current biology : CB : 1700-11 : DOI : 10.1016/j.cub.2014.06.048 En savoir plus
Résumé

In vitro studies have shown that physical parameters, such as membrane curvature, tension, and composition, influence the budding and fission of transport intermediates. Endocytosis in living cells also appears to be regulated by the mechanical load experienced by the plasma membrane. In contrast, how these parameters affect intracellular membrane trafficking in living cells is not known. To address this question, we investigate here the impact of a mechanical stress on the organization of the Golgi complex and on the formation of transport intermediates from the Golgi complex.

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Pin-I Chen, Kristine Schauer, Chen Kong, Andrew R Harding, Bruno Goud, Philip D Stahl (2014 Mar 4)

Rab5 isoforms orchestrate a « division of labor » in the endocytic network; Rab5C modulates Rac-mediated cell motility.

PloS one : e90384 : DOI : 10.1371/journal.pone.0090384 En savoir plus
Résumé

Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.

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Année de publication : 2013

Jean-Philippe Grossier, Georgia Xouri, Bruno Goud, Kristine Schauer (2013 Dec 23)

Cell adhesion defines the topology of endocytosis and signaling.

The EMBO journal : 35-45 : DOI : 10.1002/embj.201385284 En savoir plus
Résumé

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.

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Vincent Fraisier, Amal Kasri, Stéphanie Miserey-Lenkei, Jean-Baptiste Sibarita, Deepak Nair, Adeline Mayeux, Sabine Bardin, Yusuke Toyoda, Ina Poser, Andrei Poznyakovskiy, Bruno Goud, Anthony A Hyman, Ariane Dimitrov (2013 Dec 7)

C11ORF24 is a novel type I membrane protein that cycles between the Golgi apparatus and the plasma membrane in Rab6-positive vesicles.

PloS one : e82223 : DOI : 10.1371/journal.pone.0082223 En savoir plus
Résumé

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

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Kristine Schauer, Tarn Duong, Carina S Gomes-Santos, Bruno Goud (2013 Dec 4)

Studying intracellular trafficking pathways with probabilistic density maps.

Methods in cell biology : 325-43 : DOI : 10.1016/B978-0-12-417164-0.00020-3 En savoir plus
Résumé

The compartmentalization of cellular functions in complex membranous organelles is a key feature of eukaryotic cells. To cope with the enormous complexity of trafficking pathways that connect these compartments, new approaches need to be considered and introduced into the field of cell biology. We exploit the advantages of the « micropatterning technique, » which is to bring cells to adopt a highly reproducible shape, and probabilistic density mapping, which quantifies spatial organization of trafficking compartments, to study regulatory mechanisms of intracellular trafficking. Here, we provide a protocol to analyze and quantify alterations in trafficking compartments upon cellular manipulation. We demonstrate how this approach can be employed to study the regulation of Rab6-labeled transport carriers by the cytoskeleton.

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Andrew J Lindsay, Florence Jollivet, Conor P Horgan, Amir R Khan, Graça Raposo, Mary W McCaffrey, Bruno Goud (2013 Sep 6)

Identification and characterization of multiple novel Rab-myosin Va interactions.

Molecular biology of the cell : 3420-34 : DOI : 10.1091/mbc.E13-05-0236 En savoir plus
Résumé

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.

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