RESUMO
Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.
Assuntos
Diferenciação Celular , Complexo de Golgi , Proteínas da Matriz do Complexo de Golgi , Queratinócitos , Lisossomos , Lisossomos/metabolismo , Queratinócitos/metabolismo , Queratinócitos/citologia , Complexo de Golgi/metabolismo , Humanos , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas de Membrana/metabolismoRESUMO
Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol.
Assuntos
Colesterol , Endossomos , Exossomos , Tetraspanina 30 , Tetraspanina 30/metabolismo , Colesterol/metabolismo , Exossomos/metabolismo , Endossomos/metabolismo , Humanos , Animais , Proteína C1 de Niemann-Pick , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Transporte Biológico , Actinas/metabolismo , CamundongosRESUMO
Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids, and genetic material between cells and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthesize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes [(A. Lo Cicero et al., Nat. Commun. 6, 7506 (2015)]. However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here, we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites and mature melanosomes and promote their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulating pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors to cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication.
Assuntos
Vesículas Extracelulares , Melanossomas , Melaninas , Melanócitos , QueratinócitosRESUMO
Transmission electron microscopy (TEM) is currently the only method that enables the observation of extracellular vesicles (EVs) at a nanometer scale. Direct visualization of the whole content of EV preparation provides not only crucial insights on the morphology of EVs but also an objective evaluation of the content and purity of the preparation. Coupled to immunogold labeling, TEM allows the detection and association of proteins at the surface of EVs. In these techniques, EVs are deposited on grids and are chemically immobilized and contrasted to withstand a high-voltage electron beam. Under high vacuum, the electron beam hits the sample and the electrons that scatter forward are collected to form an image. Here, we describe the steps needed to observe EVs by classical TEM and the extra steps required to label proteins through immunolabeling electron microscopy (IEM).
Assuntos
Vesículas Extracelulares , Microscopia Eletrônica de Transmissão , Microscopia Eletrônica , Vesículas Extracelulares/metabolismo , Proteínas/metabolismoRESUMO
Extracellular vesicles are now considered as active contributors to melanoma progression through their capacity to modify the tumor microenvironment and to favor the formation of a pre-metastatic niche. These prometastatic roles of tumor-derived EVs would pass through their interaction with the extracellular matrix (ECM) and its remodeling, in turn providing a substrate favoring persistent tumor cell migration. Nevertheless, the capacity of EVs to directly interact with ECM components is still questionable. In this study, we use electron microscopy and a pull-down assay to test the capacity of sEVs, derived from different melanoma cell lines, to physically interact with collagen I. We were able to generate collagen fibrils coated with sEVs and to show that melanoma cells release subpopulations of sEVs that can differentially interact with collagen.
Assuntos
Vesículas Extracelulares , Melanoma , Humanos , Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Microambiente TumoralRESUMO
Animals precisely control the morphology and assembly of guanine crystals to produce diverse optical phenomena in coloration and vision. However, little is known about how organisms regulate crystallization to produce optically useful morphologies which express highly reflective crystal faces. Guanine crystals form inside iridosome vesicles within chromatophore cells called iridophores. By following iridosome formation in developing scallop eyes, we show that pre-assembled, fibrillar sheets provide an interface for nucleation and direct the orientation of the guanine crystals. The macromolecular sheets cap the (100) faces of immature guanine crystals, inhibiting growth along the π-stacking growth direction. Crystal growth then occurs preferentially along the sheets to generate highly reflective plates. Despite their different physical properties, the morphogenesis of iridosomes bears a striking resemblance to melanosome morphogenesis in vertebrates, where amyloid sheets template melanin deposition. The common control mechanisms for melanin and guanine formation inspire new approaches for manipulating the morphologies and properties of molecular materials.
Assuntos
Guanina , Melaninas , Animais , Guanina/química , Substâncias MacromolecularesRESUMO
Extracellular vesicles (EVs) play a key role both in physiological balance and homeostasis and in disease processes through their ability to participate in intercellular signaling and communication. An ever-expanding knowledge pool and a myriad of functional properties ascribed to EVs point to a new language of communication in biological systems that has opened a path for the discovery and implementation of novel diagnostic applications. EVs originate in the endosomal network and via non-random shedding from the plasma membrane by mechanisms that allow the packaging of functional cargoes, including proteins, lipids, and genetic materials. Deciphering the molecular mechanisms that govern packaging, secretion and targeted delivery of extracellular vesicle-borne cargo will be required to establish EVs as important signaling entities, especially when ascribing functional properties to a heterogeneous population of vesicles. Several molecular cascades operate within the endosomal network and at the plasma membrane that recognize and segregate cargos as a prelude to vesicle budding and release. EVs are transferred between cells and operate as vehicles in biological fluids within tissues and within the microenvironment where they are responsible for short- and long-range targeted information. In this review, we focus on the remarkable capacity of EVs to establish a dialogue between cells and within tissues, often operating in parallel to the endocrine system, we highlight selected examples of past and recent studies on the functions of EVs in health and disease.
RESUMO
Exosomes are endosome-derived extracellular vesicles involved in intercellular communication. They are generated as intraluminal vesicles within endosomal compartments that fuse with the plasma membrane (PM). The molecular events that generate secretory endosomes and lead to the release of exosomes are not well understood. We identified a subclass of non-proteolytic endosomes at prelysosomal stage as the compartment of origin of CD63 positive exosomes. These compartments undergo a Rab7a/Arl8b/Rab27a GTPase cascade to fuse with the PM. Dynamic endoplasmic reticulum (ER)-late endosome (LE) membrane contact sites (MCS) through ORP1L have the distinct capacity to modulate this process by affecting LE motility, maturation state, and small GTPase association. Thus, exosome secretion is a multi-step process regulated by GTPase switching and MCS, highlighting the ER as a new player in exosome-mediated intercellular communication.
Assuntos
Retículo Endoplasmático , Endossomos , Exossomos , Proteínas rab de Ligação ao GTP , Transporte Biológico , Comunicação Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/enzimologia , Exossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection.
Assuntos
Infecções por Citomegalovirus , Vesículas Extracelulares , Citomegalovirus/genética , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Placenta , Gravidez , ProteômicaRESUMO
Melanosomes are pigment cell-specific lysosome-related organelles in which melanin pigments are synthesized and stored. Melanosome maturation requires delivery of melanogenic cargoes via tubular transport carriers that emanate from early endosomes and that require BLOC-1 for their formation. Here we show that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases (PI4KIIα and PI4KIIß) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KIIα or PI4KIIß with shRNAs in melanocytes reduced melanin content and misrouted BLOC-1-dependent cargoes to late endosomes/lysosomes. Genetic epistasis, cell fractionation, and quantitative live-cell imaging analyses show that PI4KIIα and PI4KIIß function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation toward melanosomes by generating local pools of PtdIns4P. The data show that both type II PtdIns-4-kinases are necessary for efficient BLOC-1-dependent tubule elongation and subsequent melanosome contact and content delivery during melanosome biogenesis. The independent functions of PtdIns-4-kinases in tubule extension are downstream of likely redundant functions in BLOC-1-dependent tubule initiation.
Assuntos
1-Fosfatidilinositol 4-Quinase , Endossomos , Melaninas , Melanossomas , 1-Fosfatidilinositol 4-Quinase/metabolismo , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melaninas/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte ProteicoRESUMO
Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.
Assuntos
Endossomos , Membranas Intracelulares , Peptídeos e Proteínas de Sinalização Intracelular , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte ProteicoRESUMO
Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these.
Assuntos
Vesículas Extracelulares , Transporte Biológico , Biomarcadores/metabolismo , Comunicação Celular , Sistemas de Liberação de Medicamentos , Vesículas Extracelulares/metabolismoRESUMO
Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.
Assuntos
Proteínas de Drosophila , Vesículas Extracelulares , Antígeno AC133/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Discos Imaginais , Microvilosidades/metabolismo , Morfogênese , Asas de AnimaisRESUMO
Extracellular vesicles (EVs) are small cargo-bearing vesicles released by cells into the extracellular space. The field of EVs has grown exponentially over the past two decades; this growth follows the realisation that EVs are not simply a waste disposal system as had originally been suggested by some, but also a complex cell-to-cell communication mechanism. Indeed, EVs have been shown to transfer functional cargo between cells and can influence several biological processes. These small biological particles are also deregulated in disease. As we approach the 75th anniversary of the first experiments in which EVs were unknowingly isolated, it seems right to take stock and look back on how the field started, and has since exploded into its current state. Here we review the early experiments, summarise key findings that have propelled the field, describe the growth of an organised EV community, discuss the current state of the field, and identify key challenges that need to be addressed.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , HumanosRESUMO
Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III-IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.
Assuntos
Pigmentação , Pele/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Humanos , Concentração de Íons de Hidrogênio , Melanócitos , Melanoma Experimental , Melanossomas , Camundongos , Pele/citologia , Pele/patologiaRESUMO
Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking.
RESUMO
Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.