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ABSTRACT: Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
The Journal of Cell Biology 02/2013; 200(4):373-83. · 10.26 Impact Factor
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ABSTRACT: PMEL is a pigment cell-specific protein responsible for the formation of fibrillar sheets within the pigment organelle, the melanosome. The fibrillar sheets serve as a template upon which melanins polymerize as they are synthesized. The PMEL fibrils are required for optimal pigment cell function, as animals that either lack PMEL expression or express mutant PMEL variants show varying degrees of hypopigmentation and pigment cell inviability. The PMEL fibrils have biophysical properties of amyloid, a protein fold that is frequently associated with neurodegenerative and other diseases. However, PMEL is one of a growing number of non-pathogenic amyloid proteins that contribute to the function of the cell and/or organism that produces them. Understanding how PMEL generates amyloid in a non-pathogenic manner might provide insights into how to avoid toxicity due to pathological amyloid formation. In this review we summarize and reconcile data concerning the fate of PMEL from its site of synthesis in the endoplasmic reticulum to newly formed melanosomes and the role of distinct PMEL subdomains in trafficking and amyloid fibril formation. We then discuss how its progression through the secretory pathway into the endosomal system might allow for the regulated and non-toxic conversion of PMEL to an ordered amyloid polymer. © 2013 John Wiley & Sons A/S.
Pigment Cell & Melanoma Research 01/2013; · 5.06 Impact Factor
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11/2012;
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Anand Sitaram,
Megan K Dennis,
Rittik Chaudhuri,
Wilfredo De Jesus-Rojas,
Danièle Tenza,
Subba Rao Gangi Setty,
Christopher S Wood,
Elena V Sviderskaya,
Dorothy C Bennett, Graça Raposo,
Juan S Bonifacino,
Michael S Marks
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ABSTRACT: Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.
Molecular biology of the cell 06/2012; 23(16):3178-92. · 5.98 Impact Factor
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ABSTRACT: Myosin VI has been implicated in various steps of organelle dynamics. However, the molecular mechanism by which this myosin contributes to membrane traffic is poorly understood. Here, we report that myosin VI is associated with a lysosome-related organelle, the melanosome. Using an actin-based motility assay and video microscopy, we observed that myosin VI does not contribute to melanosome movements. Myosin VI expression regulates instead the organization of actin networks in the cytoplasm. Using a cell-free assay, we showed that myosin VI recruited actin at the surface of isolated melanosomes. Myosin VI is involved in the endocytic-recycling pathway, and this pathway contributes to the transport of a melanogenic enzyme to maturing melanosomes. We showed that depletion of myosin VI accumulated a melanogenic enzyme in enlarged melanosomes and increased their melanin content. We confirmed the requirement of myosin VI to regulate melanosome biogenesis by analysing the morphology of melanosomes in choroid cells from of the Snell's waltzer mice that do not express myosin VI. Together, our results provide new evidence that myosin VI regulates the organization of actin dynamics at the surface of a specialized organelle and unravel a novel function of this myosin in regulating the biogenesis of this organelle.
Traffic 02/2012; 13(5):665-80. · 4.92 Impact Factor
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ABSTRACT: Rab GTPases are key coordinators of eukaryotic intracellular membrane trafficking. In their active states, Rabs localise to the cytoplasmic face of intracellular compartments where they regulate membrane trafficking processes. Many Rabs have been extensively characterised whereas others, such as Rab30, have to date received relatively little attention.
Here, we demonstrate that Rab30 is primarily associated with the secretory pathway, displaying predominant localisation to the Golgi apparatus. We find by time-lapse microscopy and fluorescence recovery after photobleaching studies that Rab30 is rapidly and continuously recruited to the Golgi. We also show that Rab30 function is required for the morphological integrity of the Golgi. Finally, we demonstrate that inactivation of Rab30 does not impair anterograde or retrograde transport through the Golgi.
Taken together, these data illustrate that Rab30 primarily localises to the Golgi apparatus and is required for the structural integrity of this organelle.
Biology of the Cell 12/2011; 104(2):84-101. · 3.60 Impact Factor
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Sandrine Alais,
Ricardo Soto-Rifo,
Vincent Balter,
Henri Gruffat,
Evelyne Manet,
Laurent Schaeffer,
Jean Luc Darlix,
Andrea Cimarelli, Graça Raposo,
Théophile Ohlmann,
Pascal Leblanc
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ABSTRACT: The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.
Cellular and Molecular Life Sciences CMLS 11/2011; 69(8):1331-52. · 6.57 Impact Factor
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ABSTRACT: Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for lysosome-related organelle (LRO) biogenesis. PMEL-a component of melanocyte LROs (melanosomes)-is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.
Developmental cell 09/2011; 21(4):708-21. · 13.36 Impact Factor
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ABSTRACT: PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)--which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core--are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show that the DW and HoSi mutations alter PMEL TMD oligomerization and/or association with membranes, with consequent formation of aberrantly packed fibrils. The aberrant fibrils are associated with a loss of pigmentation in cultured melanocytes, suggesting that they inhibit melanin production and/or melanosome integrity. A secondary mutation in the Smoky chicken, which reverts the dominant DW phenotype, prevents the accumulation of PMEL in fibrillogenic compartments and thus averts DW-associated pigment loss; a secondary mutation found in the Dun chicken likely dampens a HoSi-like dominant mutation in a similar manner. We propose that the DW and HoSi mutations alter the normally benign amyloid to a pathogenic form that antagonizes melanosome function, and that the secondary mutations found in the Smoky and Dun chickens revert or dampen pathogenicity by functioning as null alleles, thus preventing the formation of aberrant fibrils. We speculate that PMEL mutations can model the conversion between physiological and pathological amyloid.
PLoS Genetics 09/2011; 7(9):e1002286. · 8.69 Impact Factor
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Anders R Hellström,
Brenda Watt,
Shahrzad Shirazi Fard,
Danièle Tenza,
Paula Mannström,
Kristina Narfström,
Björn Ekesten,
Shosuke Ito,
Kazumasa Wakamatsu,
Jimmy Larsson,
Mats Ulfendahl,
Klas Kullander, Graça Raposo,
Susanne Kerje,
Finn Hallböök,
Michael S Marks,
Leif Andersson
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ABSTRACT: PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel⁻/⁻). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel⁻/⁻ melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.
PLoS Genetics 09/2011; 7(9):e1002285. · 8.69 Impact Factor
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07/2011: pages 247 - 294; , ISBN: 9783527636150
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 07/2011; 108(29):11906-11. · 9.68 Impact Factor
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ABSTRACT: The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.
Nature Cell Biology 06/2011; 13(7):779-89. · 19.49 Impact Factor
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ABSTRACT: Exosomes are small membrane vesicles, secreted by most cell types from multivesicular endosomes, and thought to play important roles in intercellular communications. Initially described in 1983, as specifically secreted by reticulocytes, exosomes became of interest for immunologists in 1996, when they were proposed to play a role in antigen presentation. More recently, the finding that exosomes carry genetic materials, mRNA and miRNA, has been a major breakthrough in the field, unveiling their capacity to vehicle genetic messages. It is now clear that not only immune cells but probably all cell types are able to secrete exosomes: their range of possible functions expands well beyond immunology to neurobiology, stem cell and tumor biology, and their use in clinical applications as biomarkers or as therapeutic tools is an extensive area of research. Despite intensive efforts to understand their functions, two issues remain to be solved in the future: (i) what are the physiological function(s) of exosomes in vivo and (ii) what are the relative contributions of exosomes and of other secreted membrane vesicles in these proposed functions? Here, we will focus on the current ideas on exosomes and immune responses, but also on their mechanisms of secretion and the use of this knowledge to elucidate the latter issue.
Traffic 06/2011; 12(12):1659-68. · 4.92 Impact Factor
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ABSTRACT: Melanosomes are lysosome-related organelles in retinal pigment epithelial cells and epidermal melanocytes in which melanin pigments are synthesized and stored. Melanosomes are generated by multistep processes in which an immature unpigmented organelle forms and then subsequently matures. Such maturation requires inter-organellar transport of protein cargos required for pigment synthesis but also recruitment of effector proteins necessary for the correct transport of melanosomes to the cell periphery. Several studies have started to unravel the main pathways and mechanisms exploited by melanosomal proteins involved in melanosome structure and melanin synthesis. A major unexpected finding seen early in melanosome biogenesis showed the similarities between the fibrillar sheets of premelanosomes and amyloid fibrils. Late steps of melanosome formation are dependent on pathways regulated by proteins encoded by genes mutated in genetic diseases such as the Hermansky-Pudlak Syndrom (HPS) and different types of albinism. Altogether the findings from the past recent years have started to unravel how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.
Medecine sciences: M/S 02/2011; 27(2):153-62. · 0.64 Impact Factor
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Bidisha Sinha,
Darius Köster,
Richard Ruez,
Pauline Gonnord,
Michele Bastiani,
Daniel Abankwa,
Radu V Stan,
Gillian Butler-Browne,
Benoit Vedie,
Ludger Johannes,
Nobuhiro Morone,
Robert G Parton, Graça Raposo,
Pierre Sens,
Christophe Lamaze,
Pierre Nassoy
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ABSTRACT: The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.
Cell 02/2011; 144(3):402-13. · 32.40 Impact Factor
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ABSTRACT: One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.
Pigment Cell & Melanoma Research 02/2011; 24(1):233-40. · 5.06 Impact Factor
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ABSTRACT: In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation.
Molecular biology of the cell 02/2011; 22(3):375-85. · 5.98 Impact Factor
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ABSTRACT: The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.
Molecular biology of the cell 10/2010; 21(19):3386-95. · 5.98 Impact Factor
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Bahne Stechmann,
Siau-Kun Bai,
Emilie Gobbo,
Roman Lopez,
Goulven Merer,
Suzy Pinchard,
Laetitia Panigai,
Danièle Tenza, Graça Raposo,
Bruno Beaumelle,
Didier Sauvaire,
Daniel Gillet,
Ludger Johannes,
Julien Barbier
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ABSTRACT: Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.
Cell 04/2010; 141(2):231-42. · 32.40 Impact Factor
