Structure and membrane compartments

Team Publications

Year of publication 2018

Léa Ripoll, Xavier Heiligenstein, Ilse Hurbain, Lia Domingues, Florent Figon, Karl J Petersen, Megan K Dennis, Anne Houdusse, Michael S Marks, Graça Raposo, Cédric Delevoye (2018 Jun 8)

Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.

The Journal of cell biology : 2709-2726 : DOI : 10.1083/jcb.201709055 Learn more
Summary

Vesicular and tubular transport intermediates regulate organellar cargo dynamics. Transport carrier release involves local and profound membrane remodeling before fission. Pinching the neck of a budding tubule or vesicle requires mechanical forces, likely exerted by the action of molecular motors on the cytoskeleton. Here, we show that myosin VI, together with branched actin filaments, constricts the membrane of tubular carriers that are then released from melanosomes, the pigment containing lysosome-related organelles of melanocytes. By combining superresolution fluorescence microscopy, correlative light and electron microscopy, and biochemical analyses, we find that myosin VI motor activity mediates severing by constricting the neck of the tubule at specific melanosomal subdomains. Pinching of the tubules involves the cooperation of the myosin adaptor optineurin and the activity of actin nucleation machineries, including the WASH and Arp2/3 complexes. The fission and release of these tubules allows for the export of components from melanosomes, such as the SNARE VAMP7, and promotes melanosome maturation and transfer to keratinocytes. Our data reveal a new myosin VI- and actin-dependent membrane fission mechanism required for organelle function.

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Philip D Stahl, Graça Raposo (2018 May 17)

Exosomes and extracellular vesicles: the path forward.

Essays in biochemistry : 119-124 : DOI : 10.1042/EBC20170088 Learn more
Summary

Over the course of the past several decades, the concept that extracellular vesicles, exosomes and microvesicles, operate as cellular “housekeepers” and as agents for communication between and among cells and tissues, has emerged into one of the most promising yet vexing problems facing the biomedical community. Already, extracellular vesicles from biological fluids are being used for diagnostic purposes and hopes abound for their use as therapeutic agents. However, the most basic mechanistic questions surrounding their biogenesis and function in cellular and tissue homeostasis remain largely unexplored. In this issue of , the rise of a new intercellular communications pathway is considered from many perspectives-cell biology, physiology, and pathophysiology.

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Cécile Campagne, Léa Ripoll, Floriane Gilles-Marsens, Graça Raposo, Cédric Delevoye (2018 Feb 15)

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

International journal of molecular sciences : DOI : E568 Learn more
Summary

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

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Guillaume van Niel, Gisela D'Angelo, Graça Raposo (2018 Jan 18)

Shedding light on the cell biology of extracellular vesicles.

Nature reviews. Molecular cell biology : 213-228 : DOI : 10.1038/nrm.2017.125 Learn more
Summary

Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively. They are present in biological fluids and are involved in multiple physiological and pathological processes. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles.

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Frederik Johannes Verweij, Maarten P Bebelman, Connie R Jimenez, Juan J Garcia-Vallejo, Hans Janssen, Jacques Neefjes, Jaco C Knol, Richard de Goeij-de Haas, Sander R Piersma, S Rubina Baglio, Matthijs Verhage, Jaap M Middeldorp, Anoek Zomer, Jacco van Rheenen, Marc G Coppolino, Ilse Hurbain, Graça Raposo, Martine J Smit, Ruud F G Toonen, Guillaume van Niel, D Michiel Pegtel (2018 Jan 18)

Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling.

The Journal of cell biology : 1129-1142 : DOI : 10.1083/jcb.201703206 Learn more
Summary

Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

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Year of publication 2017

Ilse Hurbain, Maryse Romao, Peggy Sextius, Emilie Bourreau, Céline Marchal, Françoise Bernerd, Christine Duval, Graça Raposo (2017 Oct 22)

Melanosome Distribution in Keratinocytes in Different Skin Types: Melanosome Clusters Are Not Degradative Organelles.

The Journal of investigative dermatology : 647-656 : DOI : S0022-202X(17)33062-2 Learn more
Summary

The melanosome pattern was characterized systematically in keratinocytes in situ in highly, moderately, and lightly pigmented human skin, classified according to the individual typological angle, a colorimetric measure of skin color phenotype. Electron microscopy of skin samples showed qualitatively and quantitatively that in highly pigmented skin, although melanosomes are mostly isolated and distributed throughout the entire epidermis, clusters are also observed in the basal layer. In moderately and lightly pigmented skin, melanosomes are concentrated in the first layer of the epidermis, isolated-but for most of them, grouped as clusters of melanocores delimited by a single membrane. Electron tomography resolving intracellular three-dimensional organization of organelles showed that clustered melanocores depict contacts with other cellular compartments, such as endoplasmic reticulum and mitochondria. Additionally, immunogold labelling showed that clusters of melanocores do not correspond to autophagosomes or melanophagosomes but that they present, similarly to melanosomes in melanocytes, features of nonacidic, nondegradative organelles. Overall, these observations suggest that melanocore clusters do not correspond to autophagic organelles but represent reservoirs or protective structures for melanosome integrity and function. These results open avenues for understanding the basis of skin pigmentation in different skin color phenotypes.

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Christin Bissig, Ilse Hurbain, Graça Raposo, Guillaume van Niel (2017 Sep 1)

PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.

Traffic (Copenhagen, Denmark) : 747-757 : DOI : 10.1111/tra.12525 Learn more
Summary

The protein complex composed of the kinase PIKfyve, the phosphatase FIG4 and the scaffolding protein VAC14 regulates the metabolism of phosphatidylinositol 3,5-bisphosphate, which serves as both a signaling lipid and the major precursor for phosphatidylinositol 5-phosphate. This complex is involved in the homeostasis of late endocytic compartments, but its precise role in maintaining the dynamic equilibrium of late endosomes, endolysosomes and lysosomes remains to be determined. Here, we report that inhibition of PIKfyve activity impairs terminal lysosome reformation from acidic and hydrolase-active, but enlarged endolysosomes. Our live-cell imaging and electron tomography data show that PIKfyve activity regulates extensive membrane remodeling that initiates reformation of lysosomes from endolysosomes. Altogether, our findings show that PIKfyve activity is required to maintain the dynamic equilibrium of late endocytic compartments by regulating the reformation of terminal storage lysosomes.

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Anand Patwardhan, Sabine Bardin, Stéphanie Miserey-Lenkei, Lionel Larue, Bruno Goud, Graça Raposo, Cédric Delevoye (2017 Jun 14)

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.

Nature communications : 15835 : DOI : 10.1038/ncomms15835 Learn more
Summary

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function.

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Ilse Hurbain, Maryse Romao, Ptissam Bergam, Xavier Heiligenstein, Graça Raposo (2017 May 1)

Analyzing Lysosome-Related Organelles by Electron Microscopy.

Methods in molecular biology (Clifton, N.J.) : 43-71 : DOI : 10.1007/978-1-4939-6934-0_4 Learn more
Summary

Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.

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Perrine Paul-Gilloteaux, Xavier Heiligenstein, Martin Belle, Marie-Charlotte Domart, Banafshe Larijani, Lucy Collinson, Graça Raposo, Jean Salamero (2017 Feb 1)

eC-CLEM: flexible multidimensional registration software for correlative microscopies.

Nature methods : 102-103 : DOI : 10.1038/nmeth.4170 Learn more
Summary

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Year of publication 2016

Pascal Leblanc, Zaira E Arellano-Anaya, Emilien Bernard, Laure Gallay, Monique Provansal, Sylvain Lehmann, Laurent Schaeffer, Graça Raposo, Didier Vilette (2016 Dec 13)

Isolation of Exosomes and Microvesicles from Cell Culture Systems to Study Prion Transmission.

Methods in molecular biology (Clifton, N.J.) : 153-176 Learn more
Summary

Extracellular vesicles (EVs) are composed of microvesicles and exosomes. Exosomes are small membrane vesicles (40-120 nm sized) of endosomal origin released in the extracellular medium from cells when multivesicular bodies fuse with the plasma membrane, whereas microvesicles (i.e., shedding vesicles, 100 nm to 1 μm sized) bud from the plasma membrane. Exosomes and microvesicles carry functional proteins and nucleic acids (especially mRNAs and microRNAs) that can be transferred to surrounding cells and tissues and can impact multiple dimensions of the cellular life. Most of the cells, if not all, from neuronal to immune cells, release exosomes and microvesicles in the extracellular medium, and all biological fluids including blood (serum/plasma), urine, cerebrospinal fluid, and saliva contain EVs.Prion-infected cultured cells are known to secrete infectivity into their environment. We characterized this cell-free form of prions and showed that infectivity was associated with exosomes. Since exosomes are produced by a variety of cells, including cells that actively accumulate prions, they could be a vehicle for infectivity in body fluids and could participate to the dissemination of prions in the organism. In addition, such infectious exosomes also represent a natural, simple, biological material to get key information on the abnormal PrP forms associated with infectivity.In this chapter, we describe first a method that allows exosomes and microvesicles isolation from prion-infected cell cultures and in a second time the strategies to characterize the prions containing exosomes and their ability to disseminate the prion agent.

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Pierre Thouvenot, Lou Fourrière, Elodie Dardillac, Barbara Ben Yamin, Aurianne Lescure, Vincent Lejour, Xavier Heiligenstein, Jean-Baptiste Boulé, Maryse Romao, Graça Raposo-Benedetti, Bernard S Lopez, Alain Nicolas, Gaël A Millot (2016 Nov 2)

Yeast cells reveal the misfolding and the cellular mislocalisation of the human BRCA1 protein.

Journal of cell science : DOI : jcs.192880 Learn more
Summary

Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast/ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological interpretation of these events remains to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.

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Megan K Dennis, Cédric Delevoye, Amanda Acosta-Ruiz, Ilse Hurbain, Maryse Romao, Geoffrey G Hesketh, Philip S Goff, Elena V Sviderskaya, Dorothy C Bennett, J Paul Luzio, Thierry Galli, David J Owen, Graça Raposo, Michael S Marks (2016 Aug 3)

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.

The Journal of cell biology : 293-308 : DOI : 10.1083/jcb.201605090 Learn more
Summary

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1-dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3-dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky-Pudlak syndrome variants.

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Anand Patwardhan, Cédric Delevoye (2016 Jul 15)

Ions switch off darkness: Role of TPC2 in melanosomes.

Pigment cell & melanoma research : DOI : 10.1111/pcmr.12510 Learn more
Summary

Melanins are naturally occurring pigments that color the eye, skin and hair of mammals, thereby protecting the body from harmful solar radiation that can lead to skin cancers. Melanins are synthesized and stored within specialized organelles called melanosomes, which are generated within epidermal and uveal melanocytes as well as retinal pigment epithelium. Packaging melanins into pigment granules relies on the proper localization and activity of melanosomal components. To date, hundreds of genes and associated products have been identified as key regulators of pigmentation. Among those, a growing list of ion transporters might play an important role in melanin production (Haltaufderhyde and Oancea, 2014). This article is protected by copyright. All rights reserved.

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Ragna Sannerud, Cary Esselens, Paulina Ejsmont, Rafael Mattera, Leila Rochin, Arun Kumar Tharkeshwar, Greet De Baets, Veerle De Wever, Roger Habets, Veerle Baert, Wendy Vermeire, Christine Michiels, Arjan J Groot, Rosanne Wouters, Katleen Dillen, Katlijn Vints, Pieter Baatsen, Sebastian Munck, Rita Derua, Etienne Waelkens, Guriqbal S Basi, Mark Mercken, Marc Vooijs, Mathieu Bollen, Joost Schymkowitz, Frederic Rousseau, Juan S Bonifacino, Guillaume Van Niel, Bart De Strooper, Wim Annaert (2016 Jun 14)

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool.

Cell : 193-208 : DOI : 10.1016/j.cell.2016.05.020 Learn more
Summary

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer’s disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

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