Mécanismes moléculaires du transport intracellulaire

Publications de l’équipe

Année de publication : 2011

Daphné Dambournet, Mickael Machicoane, Laurent Chesneau, Martin Sachse, Murielle Rocancourt, Ahmed El Marjou, Etienne Formstecher, Rémi Salomon, Bruno Goud, Arnaud Echard (2011 Jun 28)

Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.

Nature cell biology : 981-8 : DOI : 10.1038/ncb2279 En savoir plus
Résumé

Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as lipid and cytoskeleton remodelling. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages. Here, we report that the phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) 5-phosphatase OCRL, which is mutated in Lowe syndrome patients, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PtdIns(4,5)P2 and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patients and can be corrected by the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P2 hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease.

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Patricia Bassereau, Bruno Goud (2011 Jun 10)

Physics, biology and the right chemistry.

F1000 biology reports : 7 : DOI : 10.3410/B3-7 En savoir plus
Résumé

Joint studies that involve biologists and physicists are becoming more frequent and have contributed to the identification and understanding of physical parameters underlying key biological processes. Here, we illustrate the main findings resulting from a 10-year collaboration between a cell biologist and an experimental physicist, both interested in the mechanisms of intracellular transport and membrane dynamics in eukaryotic cells.

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Année de publication : 2010

Mathieu Pinot, Bruno Goud, Jean-Baptiste Manneville (2010 Nov 12)

Physical aspects of COPI vesicle formation.

Molecular membrane biology : 428-42 : DOI : 10.3109/09687688.2010.510485 En savoir plus
Résumé

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.

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Ole Vielemeyer, Clément Nizak, Ana Joaquina Jimenez, Arnaud Echard, Bruno Goud, Jacques Camonis, Jean-Christophe Rain, Franck Perez (2010 Aug 24)

Characterization of single chain antibody targets through yeast two hybrid.

BMC biotechnology : 59 : DOI : 10.1186/1472-6750-10-59 En savoir plus
Résumé

Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody’s binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.

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Bruno Goud, Paul A Gleeson (2010 Mar 16)

TGN golgins, Rabs and cytoskeleton: regulating the Golgi trafficking highways.

Trends in cell biology : 329-36 : DOI : 10.1016/j.tcb.2010.02.006 En savoir plus
Résumé

The architecture of the Golgi apparatus is intimately linked to its role in regulating membrane trafficking. The recruitment of peripheral membrane proteins, in particular golgins and small G proteins has emerged as a key to the understanding of the organization and the dynamics of this organelle. There have been considerable recent advances in defining the structures and binding partners of golgins, and their contribution to membrane-mediated biological processes. In this paper, we review the proposed roles for golgins with a focus on the golgins of the trans-Golgi network (TGN). We explore the potential for TGN golgins, acting as scaffold molecules, to co-ordinate the regulation of TGN structure and function.

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S Miserey-Lenkei, G Chalancon, S Bardin, E Formstecher, B Goud, A Echard (2010 Mar 12)

Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex.

Nature cell biology : 645-54 : DOI : 10.1038/ncb2067 En savoir plus
Résumé

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.

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Kristine Schauer, Tarn Duong, Kevin Bleakley, Sabine Bardin, Michel Bornens, Bruno Goud (2010 Feb 8)

Probabilistic density maps to study global endomembrane organization.

Nature methods : 560-6 : DOI : 10.1038/nmeth.1462 En savoir plus
Résumé

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.

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Joanne Young, Julie Ménétrey, Bruno Goud (2010 Jan 13)

RAB6C is a retrogene that encodes a centrosomal protein involved in cell cycle progression.

Journal of molecular biology : 69-88 : DOI : 10.1016/j.jmb.2010.01.009 En savoir plus
Résumé

Rab-GTPases are key regulators of membrane transport, and growing evidence indicates that their expression levels are altered in certain human malignancies, including cancer. Rab6C, a newly identified Rab6 subfamily member, has attracted recent attention because its reduced expression might confer a selective advantage to drug-resistant breast cancer cells. Here, we report that RAB6C is a primate-specific retrogene derived from a RAB6A’ transcript. RAB6C is transcribed in a limited number of human tissues including brain, testis, prostate, and breast. Endogenous Rab6C is considerably less abundant and has a much shorter half-life than Rab6A’. Comparison of the GTP-binding motifs of Rab6C and Rab6A’, homology modeling, and GTP-blot overlay assays indicate that amino acid changes in Rab6C have greatly reduced its GTP-binding affinity. Instead, the noncanonical GTP-binding domain of Rab6C mediates localization of the protein to the centrosome. Overexpression of Rab6C results in G1 arrest, and its specific depletion generates tetraploid cells with supernumerary centrosomes, revealing a role of Rab6C in events related to the centrosome and cell cycle progression. Thus, RAB6C is a rare example of a recently emerged retrogene that has acquired the status of a new gene, encoding a functional protein with altered characteristics compared to Rab6A’.

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Année de publication : 2009

Ernesto Ambroggio, Benoît Sorre, Patricia Bassereau, Bruno Goud, Jean-Baptiste Manneville, Bruno Antonny (2009 Nov 21)

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions.

The EMBO journal : 292-303 : DOI : 10.1038/emboj.2009.341 En savoir plus
Résumé

ArfGAP1, qui Promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, preferentially Interacts with Positively curved membranes through ict amphipathic lipid packing sensor (ALPS) motifs. This influences the distribution of shoulds Arf1-GTP When flat and curved regions coexist membrane was continuous, notably During COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 Exclusively bound to the tubes. Decreasing the tube radius R Revealed a threshold of Approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing water equivalent of catalytic ArfGAP1 with Arf1 GTP-induced Arf1 a smooth gradient along the tube. This Arf1 That Reflects molecules leaving the tube on GTP hydrolysis are Replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, Suggesting That Arf1-GTP diffusion can Readily Compensate for the loss of localized Arf1 During budding and contribuer to the stability of the coat up to fission.

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Jesus Cardenas, Sabrina Rivero, Bruno Goud, Michel Bornens, Rosa M Rios (2009 Sep 1)

Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms.

BMC biology : 56 : DOI : 10.1186/1741-7007-7-56 En savoir plus
Résumé

The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated.

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Fiona J Houghton, Pau Ling Chew, Sylvain Lodeho, Bruno Goud, Paul A Gleeson (2009 Aug 26)

The localization of the Golgin GCC185 is independent of Rab6A/A’ and Arl1.

Cell : 787-94 : DOI : 10.1016/j.cell.2009.05.048 En savoir plus
Résumé

Mammalian golgins of the trans-Golgi network (TGN) are small G protein effectors that are required for membrane transport and contain a Golgi targeting C-terminal GRIP domain. The localization of two TGN golgins, p230/golgin-245 and golgin-97, is mediated by the small GTPase Arl1, whereas recruitment of the TGN golgin GCC185 is controversial. Recently, GCC185 was proposed to localize to the Golgi by the co-operation of two small GTPases, Rab6A/A’ and Arl1 (Burguete et al., 2008), a model based predominantly on in vitro interactions. Here we demonstrate that Golgi recruitment of endogenous GCC185 does not involve Rab6A/A’ and Arl1. We find minimal colocalization between Rab6A/A’ and endogenous GCC185 on Golgi membranes and failed to detect an interaction between Rab6A/A’ and C-terminal domains of GCC185 by yeast two-hybrid analyses. Moreover, depletion of both Rab6A/A’ and Arl1 also had no effect on the localization of endogenous GCC185 or the isolated GRIP domain of GCC185.

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Matias Ostrowski, Nuno B Carmo, Sophie Krumeich, Isabelle Fanget, Graça Raposo, Ariel Savina, Catarina F Moita, Kristine Schauer, Alistair N Hume, Rui P Freitas, Bruno Goud, Philippe Benaroch, Nir Hacohen, Mitsunori Fukuda, Claire Desnos, Miguel C Seabra, François Darchen, Sebastian Amigorena, Luis F Moita, Clotilde Thery (2009 Aug 14)

Rab27a and Rab27b control different steps of the exosome secretion pathway.

Nature cell biology : 19-30; sup pp 1-13 : DOI : 10.1038/ncb2000 En savoir plus
Résumé

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

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Thomas Wassmer, Naomi Attar, Martin Harterink, Jan R T van Weering, Colin J Traer, Jacqueline Oakley, Bruno Goud, David J Stephens, Paul Verkade, Hendrik C Korswagen, Peter J Cullen (2009 Jul 22)

The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.

Developmental cell : 110-22 : DOI : 10.1016/j.devcel.2009.04.016 En savoir plus
Résumé

Early endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150(glued) component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

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Benoit Sorre, Andrew Callan-Jones, Jean-Baptiste Manneville, Pierre Nassoy, Jean-François Joanny, Jacques Prost, Bruno Goud, Patricia Bassereau (2009 Mar 24)

Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins.

Proceedings of the National Academy of Sciences of the United States of America : 5622-6 : DOI : 10.1073/pnas.0811243106 En savoir plus
Résumé

Sorting of lipids and proteins is a key process ALLOWING eukaryotic cells to execute accurate and efficient transportation and intracellular membrane to Maintain homeostasis. It OCCURS DURING THE formation of highly curved transportation intermediates That shuttle entre cell compartments. Protein sorting is reasonably well described, goal lipid sorting is much less Understood. Lipid sorting has-been Proposed to be mediated by a mechanism based on the physical coupling entre membrane composition and high curvature of the transportation intermediates. To test this hypothesis, we-have Performed a combination of strength and fluorescence measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory HAS aussi beens Developed to explain our results. We show quantitatively, using 2 independent Approaches, That a difference in lipid composition can build up entre is curved and has noncurved membrane. Importantly, and consistent with our theory, lipid sorting OCCURS only if the system is close to a point of demixing. Remarkably, this process is amplified When low-even a fraction of lipids is clustered upon cholera toxin binding. This Can Be Explained by the reduction of the entropic penalty of lipid sorting When Some lipids are bound together by the toxin. Our results show That curvature-induced lipid sorting results from the collective behavior of lipids and is-even amplified in the presence of lipid-clustering proteins. In addition, They suggest a generic mechanism by qui proteins can Facilitate lipid segregation in vivo.

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Année de publication : 2008

Thierry Pécot, Charles Kervrann, Sabine Bardin, Bruno Goud, Jean Salamero (2008 Nov 6)

Patch-based Markov models for event detection in fluorescence bioimaging.

Medical image computing and computer-assisted intervention : MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention : 95-103 En savoir plus
Résumé

The study of protein dynamics is essential for understanding the multi-molecular complexes at subcellular levels. Fluorescent Protein (XFP)-tagging and time-lapse fluorescence microscopy enable to observe molecular dynamics and interactions in live cells, unraveling the live states of the matter. Original image analysis methods are then required to process challenging 2D or 3D image sequences. Recently, tracking methods that estimate the whole trajectories of moving objects have been successfully developed. In this paper, we address rather the detection of meaningful events in spatio-temporal fluorescence image sequences, such as apparent stable « stocking areas » involved in membrane transport. We propose an original patch-based Markov modeling to detect spatial irregularities in fluorescence images with low false alarm rates. This approach has been developed for real image sequences of cells expressing XFP-tagged Rab proteins, known to regulate membrane trafficking.

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