Mécanismes moléculaires du transport intracellulaire

Publications de l’équipe

Année de publication : 2013

Andrea Pelikan, James Sillibourne, Stephanie Miserey-Lenkei, Frederique Carlier-Grynkorn, Bruno Goud, Phong T Tran (2013 Aug 27)

Studying mitochondria and microtubule localization and dynamics in standardized cell shapes.

Methods in cell biology : 97-108 : DOI : 10.1016/B978-0-12-407757-7.00007-4 En savoir plus
Résumé

Mammalian cells show a large diversity in shape and are both shape-changing and mobile when cultured on conventional uniform substrates. The use of micropatterning techniques limits the number of variable parameters, by imposing shape and standardized adhesive areas on the cells, which facilitates analysis. By changing size or shape of the micropattern, for example, forcing a polar axis on the cell, it is possible to study how these parameters impact organelle organization, distribution, and dynamics inside the cell. To study the mitochondrial network, which is composed of dynamic tubular organelles dependent on the microtubule cytoskeleton for its distribution, it is important to be able to distinguish between distinct mitochondria. Here, we present a practical method with which we spread the cells on large patterns created with deep UV technique, which not only makes the cells uniform in size and shape as well as immobile, and therefore easier to compare and analyze, but also expands the mitochondrial network and allows for an easier tracking of appropriately labeled individual mitochondria.

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Kristine Schauer, Jean-Philippe Grossier, Tarn Duong, Violaine Chapuis, Sébastien Degot, Aurianne Lescure, Elaine Del Nery, Bruno Goud (2013 Aug 16)

A novel organelle map framework for high-content cell morphology analysis in high throughput.

Journal of biomolecular screening : 317-24 : DOI : 10.1177/1087057113497399 En savoir plus
Résumé

A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and its performance for different cell shapes has been assessed. Density-based analysis detected changes in cell morphology due to compound treatment in a small-scale proof-of-principle screen demonstrating its compatibility with high-throughput screening. This novel tool for high-content and high-throughput cellular phenotyping can potentially be used for a wide range of applications from drug screening to careful characterization of cellular processes.

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James E Sillibourne, Ilse Hurbain, Thierry Grand-Perret, Bruno Goud, Phong Tran, Michel Bornens (2013 Jun 22)

Primary ciliogenesis requires the distal appendage component Cep123.

Biology open : 535-45 : DOI : 10.1242/bio.20134457 En savoir plus
Résumé

Primary cilium formation is initiated at the distal end of the mother centriole in a highly co-ordinated manner. This requires the capping of the distal end of the mother centriole with a ciliary vesicle and the anchoring of the basal body (mother centriole) to the cell cortex, both of which are mediated by the distal appendages. Here, we show that the distal appendage protein Cep123 (Cep89/CCDC123) is required for the assembly, but not the maintenance, of a primary cilium. In the absence of Cep123 ciliary vesicle formation fails, suggesting that it functions in the early stages of primary ciliogenesis. Consistent with such a role, Cep123 interacts with the centriolar satellite proteins PCM-1, Cep290 and OFD1, all of which play a role in primary ciliogenesis. These interactions are mediated by a domain in the C-terminus of Cep123 (400-783) that overlaps the distal appendage-targeting domain (500-600). Together, the data implicate Cep123 as a new player in the primary ciliogenesis pathway and expand upon the role of the distal appendages in this process.

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Anika Thyrock, Edith Ossendorf, Martin Stehling, Mark Kail, Tanja Kurtz, Gottfried Pohlentz, Dieter Waschbüsch, Simone Eggert, Etienne Formstecher, Johannes Müthing, Klaus Dreisewerd, Stefan Kins, Bruno Goud, Angelika Barnekow (2013 Jun 6)

A new Mint1 isoform, but not the conventional Mint1, interacts with the small GTPase Rab6.

PloS one : e64149 : DOI : 10.1371/journal.pone.0064149 En savoir plus
Résumé

Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495-505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.

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Pascale Vonaesch, Steven Cardini, Mikael E Sellin, Bruno Goud, Wolf-Dietrich Hardt, Kristine Schauer (2013 May 8)

Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells.

Cellular microbiology : 1851-65 : DOI : 10.1111/cmi.12154 En savoir plus
Résumé

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively study actin rearrangements triggered by Salmonella Typhimurium (S. Tm). Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape avoiding strong cell-to-cell variations, a major limitation in classical cell culture conditions. S. Tm induced F-actin-rich ruffles and invaded micropatterned cells similar to unconstrained cells. Yet, standardized conditions allowed fast and unbiased comparison of cellular changes triggered by the SipA and SopE bacterial effector proteins. Intensity measurements in defined regions revealed that the content of pre-existing F-actin remained unchanged during infection, suggesting that newly polymerized F-actin in bacteria-triggered ruffles originates from the G-actin pool. Analysing bacterial target sites, we found that bacteria did not show any preferences for the local actin cytoskeleton specificities. Rather, invasion was constrained to a specific ‘cell height’, due to flagella-mediated near-surface swimming. We found that invasion sites were similar to bacterial binding sites, indicating that S. Tm can induce a permissive invasion site wherever it binds. As micropatterned cells can be infected by many different pathogens they represent a valuable new tool for quantitative analysis of host-pathogen interactions.

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Ernesto E Ambroggio, James Sillibourne, Bruno Antonny, Jean-Baptiste Manneville, Bruno Goud (2013 May 3)

Arf1 and membrane curvature cooperate to recruit Arfaptin2 to liposomes.

PloS one : e62963 : DOI : 10.1371/journal.pone.0062963 En savoir plus
Résumé

Arfaptin2 contains a Bin/Amphiphysin/Rvs (BAR) domain and directly interacts with proteins of the Arf/Arl family in their active GTP-bound state. It has been proposed that BAR domains are able to sense membrane curvature and to induce membrane tubulation. We report here that active Arf1 is required for the recruitment of Arfaptin2 to artificial liposomes mimicking the Golgi apparatus lipid composition. The Arf1-dependent recruitment of Arfaptin2 increases with membrane curvature, while the recruitment of Arf1 itself is not sensitive to curvature. At high protein concentrations, the binding of Arfaptin2 induces membrane tubulation. Finally, membrane-bound Arfaptin2 is released from the liposome when ArfGAP1 catalyzes the hydrolysis of GTP to GDP in Arf1. These results show that both Arf1 activation and high membrane curvature are required for efficient recruitment of Arfaptin2 to membranes.

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Rodrigo D Militello, Daniela B Munafó, Walter Berón, Luis A López, Solange Monier, Bruno Goud, María I Colombo (2013 Feb 8)

Rab24 is required for normal cell division.

Traffic (Copenhagen, Denmark) : 502-18 : DOI : 10.1111/tra.12057 En savoir plus
Résumé

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.

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Année de publication : 2012

Eoin E Kelly, Conor P Horgan, Bruno Goud, Mary W McCaffrey (2012 Nov 27)

The Rab family of proteins: 25 years on.

Biochemical Society transactions : 1337-47 : DOI : 10.1042/BST20120203 En savoir plus
Résumé

Intracellular membrane trafficking requires the complex interplay of several classes of trafficking proteins. Rab proteins, the largest subfamily of the Ras superfamily of small G-proteins, are central regulators of all aspects of intracellular trafficking processes including vesicle budding and uncoating, motility, tethering and fusion. In the present paper, we discuss the discovery, evolution and characterization of the Rab GTPase family. We examine their basic functional roles, their important structural features and the regulatory proteins which mediate Rab function. We speculate on outstanding issues in the field, such as the mechanisms of Rab membrane association and the co-ordinated interplay between distinct Rab proteins. Finally, we summarize the data implicating Rab proteins in an ever increasing number of diseases.

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Cédric Delevoye, Stéphanie Miserey-Lenkei, Guillaume Montagnac, Floriane Gilles-Marsens, Perrine Paul-Gilloteaux, Francesca Giordano, François Waharte, Michael S Marks, Bruno Goud, Graça Raposo (2012 Oct 3)

Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A.

Cell reports : 445-54 : DOI : 10.1016/j.celrep.2014.01.002 En savoir plus
Résumé

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.

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Joel R Ho, Elodie Chapeaublanc, Lisa Kirkwood, Remy Nicolle, Simone Benhamou, Thierry Lebret, Yves Allory, Jennifer Southgate, François Radvanyi, Bruno Goud (2012 Jun 23)

Deregulation of Rab and Rab effector genes in bladder cancer.

PloS one : e39469 : DOI : 10.1371/journal.pone.0039469 En savoir plus
Résumé

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer.

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Tarn Duong, Bruno Goud, Kristine Schauer (2012 May 16)

Closed-form density-based framework for automatic detection of cellular morphology changes.

Proceedings of the National Academy of Sciences of the United States of America : 8382-7 : DOI : 10.1073/pnas.1117796109 En savoir plus
Résumé

A primary method for studying cellular function is to examine cell morphology after a given manipulation. Fluorescent markers attached to proteins/intracellular structures of interest in conjunction with 3D fluorescent microscopy are frequently exploited for functional analysis. Despite the central role of morphology comparisons in cell biological approaches, few statistical tools are available that allow biological scientists without a high level of statistical training to quantify the similarity or difference of fluorescent images containing multifactorial information. We transform intracellular structures into kernels and develop a multivariate two-sample test that is nonparametric and asymptotically normal to directly and quantitatively compare cellular morphologies. The asymptotic normality bypasses the computationally intensive calculations used by the usual resampling techniques to compute the P-value. Because all parameters required for the statistical test are estimated directly from the data, it does not require any subjective decisions. Thus, we provide a black-box method for unbiased, automated comparison of cell morphology. We validate the performance of our test statistic for finite synthetic samples and experimental data. Employing our test for the comparison of the morphology of intracellular multivesicular bodies, we detect changes in their distribution after disruption of the cellular microtubule cytoskeleton with high statistical significance in fixed samples and live cell analysis. These results demonstrate that density-based comparison of multivariate image information is a powerful tool for automated detection of cell morphology changes. Moreover, the underlying mathematics of our test statistic is a general technique, which can be applied in situations where two data samples are compared.

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Olena Pylypenko, Bruno Goud (2012 Mar 28)

Posttranslational modifications of Rab GTPases help their insertion into membranes.

Proceedings of the National Academy of Sciences of the United States of America : 5555-6 : DOI : 10.1073/pnas.1202494109 En savoir plus
Résumé

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Laurent Chesneau, Daphné Dambournet, Mickaël Machicoane, Ilektra Kouranti, Mitsunori Fukuda, Bruno Goud, Arnaud Echard (2012 Jan 10)

An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis.

Current biology : CB : 147-53 : DOI : 10.1016/j.cub.2011.11.058 En savoir plus
Résumé

Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo. Membrane trafficking is crucial for animal cell cytokinesis, and several endocytic pathways regulated by distinct GTPases (Rab11, Rab21, Rab35, ARF6, RalA/B) contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis. Here, we report that the ARF6 GTPase negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis.

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Année de publication : 2011

Benoît Sorre, Andrew Callan-Jones, John Manzi, Bruno Goud, Jacques Prost, Patricia Bassereau, Aurélien Roux (2011 Dec 21)

Nature of curvature coupling of amphiphysin with membranes depends on its bound density.

Proceedings of the National Academy of Sciences of the United States of America : 173-8 : DOI : 10.1073/pnas.1103594108 En savoir plus
Résumé

Cells are populated by a vast array of membrane-binding proteins That execute critical functions. Functions, like signaling and intracellular transport require the abilities to bind to highly curved membranes and membrane deformation to trigger. Among proteins thesis is Amphiphysin 1 Implicated in clathrin mediated endocytosis,. It contains a Bin-Amphiphysin Rvs-membrane-binding domain with an N-terminal amphipathic helix and senses That Generates membrane curvature. HOWEVER, an understanding of the parameters distinguishing thesis two functions is missing. By pulling a highly curved nanotube radius of controlled from a giant vesicle in a solution Containing Amphiphysin, we Observed que la actions of the protein depends on icts Directly density on the membrane. At low densified of protein on the vesicle Nearly flat, the distribution of proteins and the mechanical effects are induced Described by a model based on spontaneous curvature induction. The tube radius and strength are modified by protein binding but still depends on membrane voltage. In the dilute limit, When Were Practically no proteins present on the vesicle, no mechanical effects Were detected, strong goal protein enrichment proportional to curvature Was seen on the tube. At high densified, the radius is independent of voltage and vesicle protein density, resulting and from the formation of a scaffold around the tube. As a result, the scaling of the power with voltage is modified. For the entire density range, protein enriched Was on the tube as Compared to the vesicle. Our approach shows que le strength of curvature sensing and mechanical effects on the tube depends on the protein density.

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Alexandre Gidon, Sabine Bardin, Bertrand Cinquin, Jerome Boulanger, François Waharte, Laurent Heliot, Henri de la Salle, Daniel Hanau, Charles Kervrann, Bruno Goud, Jean Salamero (2011 Sep 23)

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

Traffic (Copenhagen, Denmark) : 815-33 : DOI : 10.1111/j.1600-0854.2012.01354.x En savoir plus
Résumé

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.

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