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Structure and Membrane Compartments

Keywords : exosomes, mélanosomes, endosomes, lysosome-related organelles, electron microscopy, immunocytochemistry, prion proteins

Group leader : Graça Raposo-Benedetti

Read the scientific activity report.

The organelles of the endocytic pathway serve many ‘housekeeping' functions such as taking up nutrients, controlling signalling pathways and degrading unwanted macromolecules. But aside from these tasks, endocytic organelles can also serve specific functions in certain circumstances. The major goals of our research are to gain a better understanding of the biogenesis and functions of two such specialised endosomal organelles: exosomes, which are secreted from multivesicular bodies, and the lysosome-related organelles called melanosomes (Fig. 1).

We hope to unravel novel trafficking pathways and to understand how different molecular machineries evolve together to control trafficking events. Altogether these integrated studies provide for a conceptual working model to analyse the modifications of intracellular trafficking occurring during cell transformation and the cellular basis of lysosomal diseases and new insights will hopefully contribute to the development of novel therapeutic strategies.

Fig. 1Fig. 1The endocytic pathway and possible modulations.
In several cell types, the multivesicular bodies (MVBs) fuse with the cell surface and release intraluminal vesicles that are then called exosomes. Lysosome-related organelles (LROs) are generated in some cells of the haematopoietic and non-haematopietic lineage. Melanosomes in melanocytes are an example of LROs.

In several cell types, the multivesicular bodies (MVBs) fuse with the cell surface and release intraluminal vesicles that are then called exosomes. Lysosome-related organelles (LROs) are generated in some cells of the hematopoietic and non-hematopoietic lineage. Melanosomes in melanocytes are an example of LROs.

Our group specializes in electron microscopy; the laboratory is equipped with high performance instruments for morphological and immunocytochemical analysis of cellular compartments, by using mainly immunogold labelling of ultrathin cryosections and whole cells. We study a variety of human and mouse cell types in culture by combining electron microscopy with light microscopy, biochemistry and siRNA methods.

Endosomes, exosomes and pathogens

In certain cell types, the multivesicular bodies of the endosomal pathway fuse with the cell surface to release into the extracellular environment small vesicles (50-90 nm) called exosomes. Many cells use exosomes to transfer proteins and lipids between them. We discovered that the glycophosphatidylinositol (GPI)-linked prion protein is released from cells in association with exosomes. Prions cause neurodegenerative disorders due to accumulation in the central nervous system of an abnormally folded form of the cellular prion protein called PrPSc, which is thought to be the infectious agent responsible for transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transmission of PrPSc are poorly understood.
Our findings suggest that exosomes might be vehicles for transmission of the infectious protein from one cell to another, so bypassing the need for direct cell-cell contact (Fig 2). Our recent studies indicate that retrovirus infections increase the release of prions associated with viral particles and exosomes.

Fig. 2Fig. 2A schematic model for release of prions via exosomes.
PrPsc (green) and PrPsc (red) pass through the endocytic compartments of a ‘donor' cell (left) and are secreted in association with exosomes into the extracellular milieu. The secreted exosomes might be targeted to a recipient cell (right). Exosomes bearing PrPsc and PrPsc might fuse with the cell surface, which would allow conversion of PrPsc on the plasma membrane of the target cell into PrPsc. Internalised PrPsc may then be routed to endosomes and enter intralumenal vesicles during the formation of a multivesicular body (MVB). Alternatively, the exosomes containing PrPsc may themselves be internalised by endocytosis and then transferred to endosomes. In forming MVBs, endocytosed exosomes might fuse with the MVB membrane, thus incorporating PrPsc into the limiting membrane and permitting conversion of PrPsc to PrPsc in the endocytic pathway.
Top panel : electron microscopy of isolated exosomes.

Our studies throw light on the complexity of the endosomal system in some specialised cell types and demonstrate how endocytic organelles may be usurped not only by viruses but also by unconventional pathogens such as prions. A better knowledge of the mechanisms involved in exosome formation, secretion and targeting should help us to develop ways to interfere with transmission of these unconventional pathogens. We are currently studying prion protein trafficking in the endosomal system, with the same goal in mind.

Melanosomes, an example of lysosome-related organelles

Melanosomes are lysosome-related organelles (LROs) that synthesise and store the pigment melanin in melanocytes. In the skin, they are also secretory organelles, transferring melanin to keratinocytes and, thereby, generating the pigmentation of skin and hair. Melanosomes develop in stages defined by their structure and the enzymes they contain (Fig 3).

Fig. 3Fig. 3Stages of melanosome formation in cultured melanocytes
MNT1 cells were fixed by freezing at high pressure, and then embedded in epon. Stage I premelanosomes (top, I) are membrane-bound compartments containing variable numbers of internal vesicles similar to multivesicular bodies. The unique features of melanosomes become more evident in stage II premelanosomes (top, II) : ellipsoid structures with intralumenal fibres. In stage III melanosomes (top, III), melanins are synthesised and deposited on the intralumenal fibres. The entire luminal space is filled with melanin in stage IV melanosomes (top, IV). Bar, 500 nm

Distinct subdomains of endosomes are involved in sorting melanosomal proteins and in generating premelanosomes and mature melanosomes. Certain diseases associated with hypopigmentation are due to mutations in proteins involved in the regulation and specialisation of the endocytic pathway in melanocytes. Our recent studies provide evidence that the heterotetrameric adaptor proteins AP-3 and AP-1, the small GTPases Rab38/Rab32 and the BLOC (biogenesis of lysosome-related organelles complexes) are essential for the post-Golgi, endosomal sorting of melanosomal enzymes and other cargo such as the ATP7A copper transporter (Fig 4).

Fig. 4Fig. 4Melanosomes originate from the endocytic pathway but they are distinct from lysosomes.
We propose that Pmel17 (red arrows) is transported to stage II premelanosomes via coated endosomes (stage I premelanosomes). Pmel17 may be targeted directly to early endosomes from the trans Golgi network or from the cell surface. Some researchers have proposed that tyrosinase (black arrows) and TRP1 (brown arrows) are targeted directly from the TGN to pre-established premelanosomes or stage III melanosomes.
Our recent studies indicate that the enzymes are sorted from subdomains of early endosomes ; transport of TRP1 and tyrosinase results in maturation of premelanosomes to stage III and stage IV melanosomes. The adaptors AP-3 and AP-1, the BLOC complexes and the small GTPases Rab38/Rab32 are involved in the post-Golgi endosomal sorting of the melanosomal enzymes.

Our recent studies indicate that the enzymes are sorted from subdomains of early endosomes ; transport of TRP1 and tyrosinase results in maturation of premelanosomes to stage III and stage IV melanosomes. The adaptors AP-3 and AP-1, the BLOC complexes and the small GTPases Rab38/Rab32 are involved in the post-Golgi endosomal sorting of the melanosomal enzymes.

Studies of melanocytes by our team and others indicate that novel membrane trafficking regulators are involved in generating LROs. We aim to define better the different steps at which these proteins act, and how they co-ordinate with other traffic regulators such as Rabs and SNAREs. We also intend to image the three-dimensional organisation of the endosomal-melanosomal membrane system at high resolution using electron tomography.

Together, these studies provide a working model which helps further analysis of both the cellular basis of lysosomal diseases and the modifications of intracellular membrane trafficking that occur during cell transformation.

Key publications

  • Year of publication : 2015

  • Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

  • Hermansky-Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2-deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2-deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.

  • "Café-au-lait" macules (CALMs) and overall skin hyperpigmentation are early hallmarks of neurofibromatosis type 1 (NF1). One of the most frequent monogenic diseases, NF1 has subsequently been characterized with numerous benign Schwann cell-derived tumors. It is well established that neurofibromin, the NF1 gene product, is an antioncogene that down-regulates the RAS oncogene. In contrast, the molecular mechanisms associated with alteration of skin pigmentation have remained elusive. We have reassessed this issue by differentiating human embryonic stem cells into melanocytes. In the present study, we demonstrate that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterize the link between loss of heterozygosity and the typical CALMs that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cAMP-mediated PKA and ERK1/2 signaling pathways, leading to overexpression of the transcription factor MITF and of the melanogenic enzymes tyrosinase and dopachrome tautomerase, all major players in melanogenesis. Finally, the hyperpigmentation phenotype can be rescued using specific inhibitors of these signaling pathways. These results open avenues for deciphering the pathological mechanisms involved in pigmentation diseases, and provide a robust assay for the development of new strategies for treating these diseases.

  • The release of extracellular vesicles (EVs) is a highly conserved process exploited by diverse organisms as a mode of intercellular communication. Vesicles of sizes ranging from 30 to 1000nm, or even larger, are generated by blebbing of the plasma membrane (microvesicles) or formed in multivesicular endosomes (MVEs) to be secreted by exocytosis as exosomes. Exosomes, microvesicles and other EVs contain membrane and cytosolic components that include proteins, lipids and RNAs, a composition that differs related to their site of biogenesis. Several mechanisms are involved in vesicle formation at the plasma membrane or in endosomes, which is reflected in their heterogeneity, size and composition. EVs have significant promise for therapeutics and diagnostics and for understanding physiological and pathological processes all of which have boosted research to find modulators of their composition, secretion and targeting.

  • Cells release multiple, distinct forms of extracellular vesicles including structures known as microvesicles, which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

  • Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.

  • Year of publication : 2014

  • 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.

  • Year of publication : 2013

  • Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.

  • Year of publication : 2011

  • The function of signaling receptors is tightly controlled by their intracellular trafficking. One major regulatory mechanism within the endo-lysosomal system required for receptor localization and down-regulation is protein modification by ubiquitination and downstream interactions with the endosomal sorting complex responsible for transport (ESCRT) machinery. Whether and how these mechanisms operate to regulate endosomal sorting of mammalian G protein-coupled receptors (GPCRs) remains unclear. Here, we explore the involvement of ubiquitin and ESCRTs in the trafficking of OA1, a pigment cell-specific GPCR, target of mutations in Ocular Albinism type 1, which localizes intracellularly to melanosomes to regulate their biogenesis. Using biochemical and morphological methods in combination with overexpression and inactivation approaches we show that OA1 is ubiquitinated and that its intracellular sorting and down-regulation requires functional ESCRT components. Depletion or overexpression of subunits of ESCRT-0, -I, and -III markedly inhibits OA1 degradation with concomitant retention within the modified endosomal system. Our data further show that OA1 ubiquitination is uniquely required for targeting to the intralumenal vesicles of multivesicular endosomes, thereby regulating the balance between down-regulation and delivery to melanosomes. This study highlights the role of ubiquitination and the ESCRT machinery in the intracellular trafficking of mammalian GPCRs and has implications for the physiopathology of ocular albinism type 1.

  • Year of publication : 2009

  • Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1- and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type-specific positioning of endosomes that facilitate endosome-LRO contacts and are required for organelle maturation.