RESUMEN
Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~ 40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~ 90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon depolarization with high K+, label for clathrin became scattered among de-clustered synaptic vesicles and moved closer to the presynaptic active zone. In contrast to axons, clathrin-labeled CCVs and CCPs were prominent in soma/dendrites under resting conditions, and became inconspicuous upon depolarization with high K+. Thus, EM examination suggests that the regulation and mechanism of clathrin-mediated endocytosis differ between axon and dendrite, and that clathrin redistributes differently in these two neuronal compartments upon depolarization.
Asunto(s)
Axones/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/metabolismo , Dendritas/metabolismo , Animales , Axones/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Vesículas Cubiertas por Clatrina/ultraestructura , Dendritas/ultraestructura , Ratones , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Densidad Postsináptica/metabolismo , Terminales Presinápticos/metabolismo , RatasRESUMEN
Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental, or stress signals. Sorting of transporters from their site of synthesis, the endoplasmic reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the multivesicular bodies (MVB)/lysosomes/vacuole system. In specific cases, internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review, we present evidence that shows that transporter traffic to the PM takes place through Golgi bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale of why transporters and possibly other housekeeping membrane proteins 'avoid' routes of polar trafficking.
Asunto(s)
Aspergillus nidulans/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Aspergillus nidulans/genética , Membrana Celular/ultraestructura , Endocitosis/genética , Retículo Endoplásmico/ultraestructura , Endosomas/metabolismo , Endosomas/ultraestructura , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Aparato de Golgi/ultraestructura , Lisosomas/metabolismo , Lisosomas/ultraestructura , Proteínas de Transporte de Membrana/genética , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vías Secretoras/genética , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Pancreatic cancer (PC) is one of the deadliest cancers worldwide. Cancer cells secrete excessive numbers of exosomes that play essential roles in tumorigenesis. Long non-coding RNAs (lncRNAs) are essential non-coding RNAs for cancer progression. However, the role of lncRNA plasmacytoma variant translocation 1 (PVT1) in exosome secretion of PC remains to be comprehensively investigated. Thus, nanoparticle tracking analysis and transmission electron microscopy were performed to determine exosome secretion. Confocal microscopy, western blots, real-time PCR, immunofluorescence, pull-down and RNA immunoprecipitation assays, and rescue experiments were applied to investigate the mechanism underlying the role of PVT1 in exosome secretion. The results showed that PVT1 was upregulated in PC cells, along with increased levels of YKT6 v-SNARE homolog (YKT6), ras-related protein Rab-7 (RAB7), and vesicle-associated membrane protein 3 (VAMP3). Also, PVT1 promoted the transportation of multivesicular bodies (MVBs) towards the plasma membrane. In addition, PVT1 promoted the docking of MVBs by altering RAB7 expression and localization. Moreover, PVT1 promoted the fusion of MVBs with the plasma membrane through regulating YKT6 and VAMP3 colocalization and the palmitoylation of YKT6. Taken together, the results suggest that PVT1 promoted exosome secretion of PC cells and thus, can expand the understanding of PVT1 in tumor biology.
Asunto(s)
Exosomas/metabolismo , Neoplasias Pancreáticas/patología , ARN Largo no Codificante/metabolismo , Microambiente Tumoral/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Exosomas/ultraestructura , Regulación Neoplásica de la Expresión Génica , Humanos , Lipoilación/genética , Microscopía Electrónica de Transmisión , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Neoplasias Pancreáticas/genética , Proteínas R-SNARE/genética , Regulación hacia Arriba , Proteína 3 de Membrana Asociada a Vesículas/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7Asunto(s)
Infecciones por Coronavirus/diagnóstico , Riñón/ultraestructura , Cuerpos Multivesiculares/ultraestructura , Neumonía Viral/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Betacoronavirus , COVID-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Estudios Retrospectivos , SARS-CoV-2RESUMEN
Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.
Asunto(s)
Movimiento Celular , Exosomas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Supervivencia Celular , Exosomas/ultraestructura , Espacio Extracelular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Nanopartículas/química , Nanopartículas/ultraestructura , Tetraspanina 30/química , Tetraspanina 30/metabolismo , Factores de TiempoRESUMEN
Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso)phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wild-type parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and (2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite.
Asunto(s)
Adaptación Fisiológica , Apicoplastos/metabolismo , División Celular , Interacciones Huésped-Parásitos , Metabolismo de los Lípidos , Parásitos/metabolismo , Toxoplasma/metabolismo , Toxoplasma/fisiología , Aciltransferasas/metabolismo , Animales , Membrana Celular/metabolismo , Citocinesis , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/biosíntesis , Eliminación de Gen , Humanos , Espacio Intracelular/parasitología , Estadios del Ciclo de Vida , Lipidómica , Masculino , Modelos Biológicos , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Mutación/genética , Nutrientes , Parásitos/crecimiento & desarrollo , Parásitos/fisiología , Parásitos/ultraestructura , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasma/ultraestructuraRESUMEN
An ultrastructural study on the lymphocytes from peripheral blood samples from 20 healthy adult Arabian oryx (Oryx leucocoryx) was undertaken. Small lymphocytes ranged in size from 2-3.5 µm and exhibited the typical repertoire of organelles found in small lymphocytes of other animals but showed no evidence of azurophilic granules. Medium-sized lymphocytes were 5-6.5 µm in diameter and occasionally demonstrated azurophilic granules. Microvilli were a common finding of lymphocytes. Of particular interest was the presence of multivesicular bodies, which have previously only been described in human lymphocytes. Both small- and medium-sized lymphocytes of the Arabian oryx were smaller than those reported for other animals. Small lymphocytes exhibited short, thick microvilli, whereas medium-sized lymphocytes had long thin microvilli, a single nucleolus and occasionally azurophilic granules and multivesicular bodies.
Asunto(s)
Antílopes , Linfocitos/ultraestructura , Animales , Antílopes/sangre , Cuerpos Multivesiculares/ultraestructuraRESUMEN
The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.
Asunto(s)
Exosomas/ultraestructura , Cuerpos Multivesiculares/ultraestructura , Túbulos Seminíferos/fisiología , Túbulos Seminíferos/ultraestructura , Espermatogénesis , Tetraspanina 30/análisis , Tortugas/fisiología , Animales , Western Blotting , Exosomas/química , Inmunohistoquímica , Masculino , Microscopía Electrónica , Microscopía Fluorescente , Cuerpos Multivesiculares/químicaRESUMEN
Exosomes are secreted from various cells by multivesicular bodies (MVBs) that fuse with the plasma membrane and are involved in the intestinal immune response to maintain intestinal homeostasis. Here, we demonstrate the ultrastructural characteristics of MVBs and their exosomes in immune-related cells of the zebrafish intestine, including goblet cells (GCs), mitochondria-rich cells (MRCs), high endothelial cells (HECs) and lymphocytes. In GCs, MVBs with a low electron density were present under the nucleus. MVBs with exosomes were observed among mucin granules. "Heterogeneous" MVBs were identified within the cytoplasm around mucin granules. MRCs were observed in the intestinal mucosa epithelium, including "open-type" MRCs and "close-type" MRCs. Typical MVBs were identified in these MRCs. MVBs with a variety of exosomes were observed in the HECs of the capillary located in the lamina propria (LP). The HEC basement membrane budded outward to LP cells to form a plurality of basal blebs, later containing a large number of exosomes. MVBs also existed in the LP lymphocytes. A schematic diagram of the ultrastructural distribution of MVBs and their exosomes in the intestinal mucosal immune-related cells was created. Our findings provide cytological evidence for the source and ultrastructural distribution of exosomes within the different intestine cells of zebrafish. Component analysis and immunological functions of exosomes require future study.
Asunto(s)
Exosomas/inmunología , Exosomas/ultraestructura , Intestinos/citología , Intestinos/inmunología , Cuerpos Multivesiculares/inmunología , Pez Cebra/inmunología , Animales , Transporte Biológico , Femenino , Microscopía Electrónica de Transmisión , Cuerpos Multivesiculares/ultraestructuraRESUMEN
The present study was designed to investigate the in vivo biological processes of multivesicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. The exosome markers, cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101), were positively expressed in intestinal villi during turtle hibernation. The distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further confirmed by transmission electron microscopy. During hibernation, abundantly secreted early endosomes (ees) were localized in the luminal and basal cytoplasm of the MRCs and ACs, and late endosomes (les) were dispersed with the supranuclear parts of the MRCs and ACs. Many "heterogeneous" MVBs were identified throughout the cytoplasm of the MRCs and ACs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of the GCs during hibernation; however, they were absent during nonhibernation. Furthermore, the exocytosis pathways of exosomes and autophagic vacuoles were observed in the MRCs, GCs, and ACs during hibernation. In addition, the number of different MVBs with intraluminal vesicles (ILVs) and heterogeneous endosome-MVB-exosome complexes was significantly increased in the MRCs, GCs, and ACs during hibernation. All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity, during hibernation.
Asunto(s)
Células Epiteliales/fisiología , Exosomas/metabolismo , Hibernación , Mucosa Intestinal/fisiología , Cuerpos Multivesiculares/metabolismo , Tortugas , Animales , Biomarcadores/análisis , Células Epiteliales/ultraestructura , Exosomas/química , Exosomas/ultraestructura , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Cuerpos Multivesiculares/química , Cuerpos Multivesiculares/ultraestructuraRESUMEN
Giardia intestinalis presents an intriguing endomembrane system, which includes endoplasmic reticulum and peripheral vesicles (PVs). The PVs have previously been considered to be organelles that display early and late endosomal and lysosomal properties. Some of these vesicles accumulate macromolecules ingested by the protozoan and show acid phosphatase activity. It has been previously shown that the parasite releases microvesicles, which contribute to giardiasis pathogenesis; however, the vesicles' origin and the way in which they are released by the parasite still remain unclear. In this study, we induced the parasites to encyst in vitro and analyzed these events using advanced electron microscopy techniques, including focused ion beam and electron microscopy tomography followed by three-dimensional reconstruction, in order to better understand protozoal multivesicular body (MVB) biogenesis. In addition, we performed an ultrastructural analysis of phosphatase activity during differentiation. We demonstrated that some vegetative trophozoites' PVs exhibited morphological characteristics of MVBs with a mean diameter of 50â¯nm, containing intraluminal vesicles (ILVs).
Asunto(s)
Giardia lamblia/metabolismo , Estadios del Ciclo de Vida , Cuerpos Multivesiculares/metabolismo , Trofozoítos/metabolismo , Fosfatasa Ácida/metabolismo , Fosfatasa Ácida/ultraestructura , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Endosomas/metabolismo , Endosomas/ultraestructura , Giardia lamblia/crecimiento & desarrollo , Giardia lamblia/ultraestructura , Microscopía Electrónica/métodos , Cuerpos Multivesiculares/ultraestructura , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/ultraestructura , Trofozoítos/crecimiento & desarrollo , Trofozoítos/ultraestructuraRESUMEN
Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis (Arabidopsis thaliana). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection.
Asunto(s)
Espacio Extracelular/metabolismo , Cuerpos Multivesiculares/metabolismo , Hojas de la Planta/metabolismo , Potyvirus/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Espacio Extracelular/virología , Interacciones Huésped-Patógeno , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Cuerpos Multivesiculares/ultraestructura , Cuerpos Multivesiculares/virología , Hojas de la Planta/virología , Potyvirus/genética , Potyvirus/fisiología , Proteómica/métodos , ARN Viral/genética , ARN Viral/metabolismo , Nicotiana/metabolismo , Nicotiana/virología , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Intercellular communication of gut epithelial cells is critical to gut mucosal homeostasis. Exosomes are important intercellular mediators in communication between cell to cell. Although many literature focus on the immunologic roles in the gut by the exosomes, the biological process of exosomes in the absorptive cells remains unknown. Uncovering the distribution, classification and formation process of multivesicular bodies (MVBs) and their exosomes in the absorptive cells of the zebrafish gut, is urgently needed to establish a platform for immunological research of fish gut exosomes. The expression levels of CD63 and TSG101 were different among the three segments of the gut, and they were enriched at the apex of the mid gut villi. The characteristics of MVBs and their exosomes in the absorptive cells were further revealed by transmission electron microscopy (TEM). Early endosomes (ee) were mainly present in the apical and basal cytoplasm of absorptive cells. Late endosomes (le) were mostly distributed with the supranuclear part of these cells. "Heterogeneous" MVBs were detected underlying the apical membranes of absorptive cells. Many exosomes with some MVB-like structures occurred in the lumen, indicating that the release process was mainly through apical secretion. Various MVBs with exosomes and the endosome-heterogeneous MVB-exosome complex existed widely in the mid gut absorptive cells, concluding that zebrafish as a potential model for in vivo MVBs and their exosomes research. All the results were summarized in a schematic diagram illustrating the morphological characteristics of gut MVBs and their exosomes in zebrafish.
Asunto(s)
Exosomas/ultraestructura , Tracto Gastrointestinal/citología , Cuerpos Multivesiculares/ultraestructura , Pez Cebra , Animales , Tracto Gastrointestinal/inmunología , Inmunidad Mucosa , Microscopía Electrónica de TransmisiónRESUMEN
Elevated mechanical stress in glomerular hypertension is thought to damage podocytes, the loss of which leads to development of glomerulosclerosis. Applying cDNA array analysis to mechanically stressed podocytes, we have recently identified TSG101 as a stretch-induced candidate gene among others. TSG101, which is part of the ESCRT-I complex, is involved in multivesicular body (MVB) formation. Here we demonstrate that TSG101 mRNA is strongly upregulated in conditionally immortalized mouse podocytes by cyclic mechanical stress. Differentiation of podocytes does not affect TSG101 mRNA levels. TSG101 immunofluorescence is distributed in a vesicular pattern in podocytes, the staining intensity being enhanced by mechanical stress. In DOCA/salt treated rats, a model of glomerular hypertension, glomerular TSG101 mRNA levels are elevated, and an increased number of MVBs is observed by electron microscopy in podocyte processes. Our data demonstrate that mechanical stress upregulates TSG101 in podocytes, suggesting that glomerular hypertension enhances sorting of cell surface proteins and their ligands into the degradative pathway in podocytes.
Asunto(s)
Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Podocitos/metabolismo , Podocitos/patología , Estrés Mecánico , Factores de Transcripción/genética , Regulación hacia Arriba/genética , Animales , Diferenciación Celular/genética , Acetato de Desoxicorticosterona , Masculino , Ratones , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Podocitos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas WistarRESUMEN
An ultrastructural study on the platelets from peripheral blood samples from 20 healthy adult Arabian oryx (Oryx leucocoryx) was performed. Characteristic was the extreme polymorphism of both the platelets, as well as their alpha granules. They vary in size from 100 to 800 nm in diameter and their numbers typically are less than those reported for humans and other animal species. Also, the alpha granules in contrast to those of humans and animals, such as the Arabian tahr, do not have nucleoids. Typically, the oryx platelets exhibit only 1-2 electron-dense bodies per cell and they lack an open canalicular system. Of particular interest is the presence of Type I multivesicular bodies, which have previously only been described in human megakaryocytes and are hypothesized as being intermediate development stages of alpha and dense granules.
Asunto(s)
Antílopes/sangre , Plaquetas/ultraestructura , Animales , Anticoagulantes , Gránulos Citoplasmáticos/ultraestructura , Ácido Edético , Microscopía Electrónica/veterinaria , Microtúbulos/ultraestructura , Cuerpos Multivesiculares/ultraestructura , Seudópodos/ultraestructura , Valores de ReferenciaRESUMEN
Exosomes are vesicles secreted by the multivesicular bodies (MVBs), which have been shown to mediate immunity regulation and virus transmission. In this study, the dynamic distribution and function of the MVBs and their exosomes was investigated through morphological characterization and molecular analyses in duck spleens infected with duck Tembusu virus (DTMUV) at different times post infection (1hpi, 2hpi, 12hpi, 24hpi). CD63, the marker of MVBs and exosomes, was distributed in the sheathed capillaries and the periellipsoidal lymphatic sheaths (PELS) of the white pulp. The numbers of MVBs and their exosomes were dramatically increased at 2 hpi, and with the increasing infection time, the numbers of MVBs and their exosomes were gradually decreased. DTMUV proteins were associated with exosomes according to double label immunofluorescence results. Ultrastructural characterization by transmission electron microscopy revealed four developing stages of MVBs containing exosomes were detected in high endothelial cells of the sheathed capillaries, lymphocytes and the ellipsoid-associated macrophages in PELS. Free exosomes were observed in the extracellular matrix and the blood vessels. Genes and proteins related to the endocytosis pathway were obviously up-regulated at 2 hpi as confirmed by RT-qPCR and western blotting. We speculated that DTMUV mediates host invasion through the endocytosis pathway by utilizing MVBs and their exosomes. The in vivo distribution pattern of MVBs and their exosomes in DTMUV infected spleens is shown for the first time in this study. This report could lay the foundations for understanding the infection mechanism of DTMUV.
Asunto(s)
Patos , Exosomas/patología , Flavivirus , Cuerpos Multivesiculares/patología , Enfermedades de las Aves de Corral/virología , Bazo/patología , Animales , Exosomas/ultraestructura , Femenino , Cuerpos Multivesiculares/ultraestructura , Enfermedades de las Aves de Corral/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Extracellular vesicles (EVs) are a heterogeneous population involved in intercellular communication. Little attention has been paid to a peculiar EV type with the appearance of a multivesicular body: extracellular multivesicular body (EMVB), also termed matrix vesicle cluster/multivesicular cargo. The aim of this work is to assess the ultrastructural characteristics, participation, and tissue location of EMVBs in inflammation/repair and tumors (with physiopathological processes involving intense intercellular communication), for which representative specimens were used. The results showed several forms of EMVBs: a) mature EMVBs, made up of clusters of vesicles surrounded by a plasma membrane, b) pre-EMVBs, with protruding grouped vesicles under the cell membrane, and c) post-EMVBs, releasing their vesicles. In tissues with inflammation/repair, EMVBs were observed in vessel lumens, interstitial spaces of vessel walls (between endothelial cells, pericytes, and smooth muscle cells) and between inflammatory and stromal cells. In tumors, such as basal cell carcinoma, craniopharyngioma, syringocystoadenoma, fibrous histiocytoma, alveolar rhabdomyosarcoma, lymphomas, neuroblastoma, astrocytomas, meningiomas, and hydatiform mole, EMVBs were present in tumor gland lumens and between tumor cells. In conclusion, in numerous physiopathological processes, we contribute EMVB ultrastructural characteristics (including different forms of mature, pre- and post-EMVBs, suggesting a more efficient EV transport), location and relationship with different types of cells. Further studies are required to assess the role of EMVBs in these physiopathological conditions.
Asunto(s)
Exosomas/ultraestructura , Inflamación/patología , Cuerpos Multivesiculares/ultraestructura , Neoplasias/ultraestructura , Animales , Humanos , Ratas , Ratas Sprague-DawleyRESUMEN
Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.
Asunto(s)
Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/química , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/ultraestructura , Regulación de la Expresión Génica , Células HeLa , Humanos , Membranas Intracelulares/ultraestructura , Lisosomas/ultraestructura , Cuerpos Multivesiculares/ultraestructura , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl- channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.
Asunto(s)
Autofagia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Proteínas Relacionadas con la Autofagia/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Estrés del Retículo Endoplásmico , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Células HEK293 , Humanos , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.