RESUMEN
Approximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides. These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522-1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522-1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522-1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut. IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522-1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.
Asunto(s)
Bacteroides thetaiotaomicron/metabolismo , Ceramidas/biosíntesis , Inositol/metabolismo , Vesículas Transportadoras/metabolismo , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides thetaiotaomicron/genética , Vías Biosintéticas , Ceramidas/química , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , Lipidómica , Espectrometría de Masas , Operón , Esfingolípidos/química , Esfingolípidos/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/genéticaRESUMEN
Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Membrana Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Microfilamentos/genética , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Transportadoras/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Autophagy is a versatile degradation system for maintaining cellular homeostasis whereby cytosolic materials are sequestered in a double-membrane autophagosome and subsequently delivered to lysosomes, where they are broken down. In multicellular organisms, newly formed autophagosomes undergo a process called 'maturation', in which they fuse with vesicles originating from endolysosomal compartments, including early/late endosomes and lysosomes, to form amphisomes, which eventually become degradative autolysosomes. This fusion process requires the concerted actions of multiple regulators of membrane dynamics, including SNAREs, tethering proteins and RAB GTPases, and also transport of autophagosomes and late endosomes/lysosomes towards each other. Multiple mechanisms modulate autophagosome maturation, including post-translational modification of key components, spatial distribution of phosphoinositide lipid species on membranes, RAB protein dynamics, and biogenesis and function of lysosomes. Nutrient status and various stresses integrate into the autophagosome maturation machinery to coordinate the progression of autophagic flux. Impaired autophagosome maturation is linked to the pathogenesis of various human diseases, including neurodegenerative disorders, cancer and myopathies. Furthermore, invading pathogens exploit various strategies to block autophagosome maturation, thus evading destruction and even subverting autophagic vacuoles (autophagosomes, amphisomes and autolysosomes) for survival, growth and/or release. Here, we discuss the recent progress in our understanding of the machinery and regulation of autophagosome maturation, the relevance of these mechanisms to human pathophysiology and how they are harnessed by pathogens for their benefit. We also provide perspectives on targeting autophagosome maturation therapeutically.
Asunto(s)
Autofagosomas/genética , Autofagia/genética , Enfermedades Neurodegenerativas/genética , Vesículas Transportadoras/genética , Endosomas/genética , Humanos , Lisosomas/genética , Enfermedades Neurodegenerativas/patología , Fagosomas/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas SNARE/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues-very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD.
Asunto(s)
Transporte Axonal/genética , Epigénesis Genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Vesículas Transportadoras/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Muerte Celular , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Metilación , Ratones , Ratones Transgénicos , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Vesículas Transportadoras/genética , Vesículas Transportadoras/patologíaRESUMEN
During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As initiators of endocytosis, Eps15 and Fcho1/2 concentrate downstream components, while permitting dynamic rearrangement within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1/2 rely on weak, liquid-like interactions to catalyse endocytosis. In vitro, these weak interactions promote the assembly of protein droplets with liquid-like properties. To probe the physiological role of these liquid-like networks, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15-knockout cells. By contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels, which stalled vesicle budding, probably owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of initiator proteins provides an optimal catalytic platform for endocytosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Membrana Celular/genética , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de la Membrana/genética , Vesículas Transportadoras/genética , Animales , Proteínas de Unión al Calcio/genética , Catálisis , Clatrina/genética , Endocitosis/genética , Humanos , Ratones , Fosfoproteínas/genéticaRESUMEN
Shengmai Formula (SMF) is one of the traditional Chinese medicine representative formulas and is widely used for the treatment of cardio- and cerebrovascular disease. Previous studies demonstrated that the major effective ingredients in SMF can interact with each other based on some uptake transporters. However, the role of the efflux transporter breast cancer resistance protein (BCRP) in these interactions involving SMF remains unclear. The purpose of this study was to investigate the interactions of the major active components of SMF with BCRP and the compatibility mechanism of these complex components in SMF based on BCRP. We selected 4 main fractions, including ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), and fructus schisandrae total lignans (STL), and 12 bioactive components, including ginsenosides Re, Rd, Rb1, and Rg1, ophiopogonins D and D', methylophiopogonanones A and B, schizandrins A and B, and schizandrols A and B to explore the interactions of SMF with BCRP in LLC-PK1 and LLC-PK1/BCRP cells and BCRP membrane vesicles. The results showed that ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A can be transported by BCRP into LLC-PK1/BCRP cells. Schisandrol B exhibited a markedly inhibitory effect on the transport function of BCRP and can significantly inhibit the uptake of methylophiopogonanone B and schizandrin A into LLC-PK1/BCRP cells. In "Inside-Out" BCRP membrane vesicles, BCRP mediated the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A, with Km values of 111.9⯱â¯31.26⯵M, 82.01⯱â¯16.72⯵M, 57.06⯱â¯8.789⯵M, and 37.19⯱â¯6.512⯵M, respectively. GTS, STL, ginsenosides Rd and Rb1, and schisandrol B were potent inhibitors of BCRP and showed different degrees of inhibition on the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A via BCRP. In conclusion, GTS, STL, ginsenosides Rd and Rb1, and schizandrol B are potential inhibitors of BCRP. Ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A are potential substrates of BCRP, and their transport, which is mediated by BCRP, may be inhibited by potential inhibitors in SMF. There are potential interactions of these main effective components of SMF at the cellular and vesicular levels that are mediated by BCRP. The interplay of these bioactive components based on BCRP may be an important compatibility mechanism in SMF.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Antineoplásicos Fitogénicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Vesículas Transportadoras/efectos de los fármacos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Antineoplásicos Fitogénicos/análisis , Antineoplásicos Fitogénicos/metabolismo , Transporte Biológico , Combinación de Medicamentos , Medicamentos Herbarios Chinos/análisis , Medicamentos Herbarios Chinos/metabolismo , Células LLC-PK1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Porcinos , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismoRESUMEN
Rubber tree is an economically important tropical crop. Its endophytic bacterial strain Serratia marcescens ITBB B5-1 contains an intracellular macrovesicle and red pigment. In this research, the red pigment was identified as prodigiosin by quadrupole time-of-flight mass spectrometry. Prodigiosin has a wide range of potential medical values such as anticancer and antiorgan transplant rejection. The strain ITBB B5-1 accumulated prodigiosin up to 2000 mg/L, which is higher production compared to most known Serratia strains. The formation of the macrovesicle and prodigiosin biosynthesis were highly associated and were both temporal- and temperature-dependent. A mutant strain B5-1mu that failed to produce prodigiosin was obtained by ultraviolet mutagenesis. Whole genome sequencing of wild-type and mutant strains indicated that the PigC gene encoding the last-step enzyme in the prodigiosin biosynthesis pathway was mutated in B5-1mu by a 17-bp deletion. Transmission electron microscopy analysis showed that the macrovesicle was absent in the mutant strain, indicating that formation of the macrovesicle relied on prodigiosin biosynthesis. Immunoelectron microscopy using prodigiosin-specific antiserum showed the presence of prodigiosin in the macrovesicle, the cell wall, and the extracellular vesicles, while immuno-reaction was not observed in the mutant cell. These results indicate that the macrovesicle serves as a storage organelle of prodigiosin, and secretes prodigiosin into cell envelop and culture medium as extracellular vesicles.
Asunto(s)
Endófitos/genética , Endófitos/metabolismo , Hevea/microbiología , Prodigiosina/biosíntesis , Serratia marcescens/genética , Serratia marcescens/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Endófitos/ultraestructura , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Microscopía Inmunoelectrónica , Mutación , Serratia marcescens/ultraestructura , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructuraRESUMEN
Endocytosis of the amyloid precursor protein (APP) is critical for generation of ß-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.
Asunto(s)
Enfermedad de Alzheimer/genética , Secuencias de Aminoácidos/genética , Precursor de Proteína beta-Amiloide/genética , Proteínas de Unión al GTP rab5/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Animales , Proteínas Portadoras/genética , Endocitosis/genética , Endosomas/genética , Humanos , Ratones , Transporte de Proteínas/genética , Vesículas Transportadoras/genéticaRESUMEN
Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.
Asunto(s)
Núcleo Celular/metabolismo , Meiosis , Profase Meiótica I , Modelos Biológicos , Oocitos/metabolismo , Vesículas Transportadoras/metabolismo , Actinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/genética , Células Cultivadas , Simulación por Computador , Difusión , Femenino , Gotas Lipídicas/metabolismo , Ratones , Análisis Numérico Asistido por Computador , Tamaño de los Orgánulos , Tamaño de la Partícula , Factores de Tiempo , Vesículas Transportadoras/genéticaRESUMEN
Actin dynamics generate forces to deform the membrane and overcome the cell's high turgor pressure during clathrin-mediated endocytosis (CME) in yeast, but precise molecular details are still unresolved. Our previous models predicted that actin filaments of the endocytic meshwork continually polymerize and disassemble, turning over multiple times during an endocytic event, similar to other actin systems. We applied single-molecule speckle tracking in live fission yeast to directly measure molecular turnover within CME sites for the first time. In contrast with the overall ~20 s lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 s, and similarly high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins' motions. These results indicate that endocytic proteins turn over up to five times during the formation of an endocytic vesicle, and suggest revising quantitative models of force production.
Asunto(s)
Actinas/genética , Clatrina/genética , Endocitosis/genética , Vesículas Transportadoras/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de la Cápside/genética , Membrana Celular/genética , Clatrina/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Schizosaccharomyces/genética , Imagen Individual de Molécula , Vesículas Transportadoras/metabolismoRESUMEN
Desulfovibrio alaskensis is a Gram-negative bacterial species that belongs to the group of Sulphate Reducing Bacteria (SRB) and presents prophages in genomes, a common characteristic of the genus Desulfovibrio. Genetic material can be transported by outer membrane vesicles, however, no data regarding the production of these vesicles has been reported for D. alaskensis. To verify the expression of D. alaskensis prophages and their involvement with outer membrane vesicles, the DSM16109 strain was used. The DSM16109 strain had three prophages and presented reduced growth after mitomycin C addition when compared to the control culture. This reduction was accompanied by the presence of virus-like particles (VLPs), indicating mitomycin C dependent prophage induction. The increase in the number of cap gene copies and transcriptions of the three prophages was verified in the control sample, however, without the formation of VLPs. Prophage genes were identified in outer membrane vesicles from cultures treated and not treated with mitomycin C. DSM16109 prophages are expressed spontaneously but only in the presence of mitomycin C was it possible to observe VLP formation. Due to the genetic material detection from the prophages within outer membrane vesicles, this property may be related to the horizontal transfer of viral genes.
Asunto(s)
Desulfovibrio/virología , Transferencia de Gen Horizontal , Profagos/genética , Vesículas Transportadoras/genética , Desulfovibrio/crecimiento & desarrollo , Mitomicina/farmacología , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
The role of vesicle-mediated gene transfer in Acinetobacter baumannii populations has been investigated in the last decade. Importantly, outer membrane vesicles (OMVs) secreted from A. baumannii cells have proven to be efficient agents of transfer of antimicrobial resistance genes to other bacterial species. However, the measurement of vesicle-mediated transfer depends on many experimental parameters. Here, we describe an experimental method useful to study transfer of DNA via membrane vesicles of A. baumannii in various bacterial populations.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/genética , Transferencia de Gen Horizontal , Vesículas Transportadoras , Transporte Biológico , Humanos , Reacción en Cadena de la Polimerasa , Vesículas Transportadoras/genéticaRESUMEN
Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-ß (TRIF).
Asunto(s)
Herpesvirus Humano 1/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Vesículas Transportadoras/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 1/genética , Humanos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Vesículas Transportadoras/genética , Vesículas Transportadoras/virología , Células Vero , Proteínas Virales/genéticaRESUMEN
Emerging evidence from various studies indicates that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when it is used for electrotransfection. To provide morphological evidence of the process, we investigated ultrastructures in cells that were associated with the electrotransfected pDNA, using immunoelectron microscopy. The results demonstrate that four endocytic pathways are involved in the uptake of the pDNA, including caveolae- and clathrin-mediated endocytosis, macropinocytosis, and the clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endosomal compartment (CLIC/GEEC) pathway. Among them, macropinocytosis is the most common pathway utilized by cells having various pDNA uptake capacities, and the CLIC/GEEC pathway is observed primarily in human umbilical vein endothelial cells. Quantitatively, the endocytic pathways are more active in easy-to-transfect cells than in hard-to-transfect ones. Taken together, our data provide ultrastructural evidence showing that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA.
Asunto(s)
Endocitosis/fisiología , Transfección/métodos , Vesículas Transportadoras/genética , Vesículas Transportadoras/ultraestructura , Transporte Biológico/genética , Transporte Biológico/fisiología , Proteínas de Ciclo Celular , Línea Celular , Clatrina , ADN/metabolismo , Digoxina , Electricidad , Células Endoteliales , Proteínas Ligadas a GPI , Técnicas de Transferencia de Gen , Humanos , Microscopía Inmunoelectrónica/métodos , Pinocitosis , Plásmidos/genética , Plásmidos/metabolismo , Adhesión del Tejido , Vesículas Transportadoras/fisiología , VenasRESUMEN
The plasma membrane of eukaryotic cells is not a simple sheet of lipids and proteins but is differentiated into subdomains with crucial functions. Caveolae, small pits in the plasma membrane, are the most abundant surface subdomains of many mammalian cells. The cellular functions of caveolae have long remained obscure, but a new molecular understanding of caveola formation has led to insights into their workings. Caveolae are formed by the coordinated action of a number of lipid-interacting proteins to produce a microdomain with a specific structure and lipid composition. Caveolae can bud from the plasma membrane to form an endocytic vesicle or can flatten into the membrane to help cells withstand mechanical stress. The role of caveolae as mechanoprotective and signal transduction elements is reviewed in the context of disease conditions associated with caveola dysfunction.
Asunto(s)
Caveolas/metabolismo , Membrana Celular/genética , Vesículas Transportadoras/genética , Caveolas/química , Caveolas/patología , Membrana Celular/química , Endocitosis/genética , Humanos , Transducción de Señal/genética , Estrés Mecánico , Relación Estructura-Actividad , Vesículas Transportadoras/químicaRESUMEN
Clathrin-mediated endocytosis is an essential cellular mechanism by which all eukaryotic cells regulate their plasma membrane composition to control processes ranging from cell signaling to adhesion, migration and morphogenesis. The formation of endocytic vesicles and tubules involves extensive protein-mediated remodeling of the plasma membrane that is organized in space and time by protein-protein and protein-phospholipid interactions. Recent studies combining high-resolution imaging with genetic manipulations of the endocytic machinery and with theoretical approaches have led to novel multifaceted phenomenological data of the temporal and spatial organization of the endocytic reaction. This gave rise to various - often conflicting - models as to how endocytic proteins and their association with lipids regulate the endocytic protein choreography to reshape the plasma membrane. In this Review, we discuss these findings in light of the hypothesis that endocytic membrane remodeling may be determined by an interplay between protein-protein interactions, the ability of proteins to generate and sense membrane curvature, and the ability of lipids to stabilize and reinforce the generated membrane shape through adopting their lateral distribution to the local membrane curvature.
Asunto(s)
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis , Vesículas Transportadoras/metabolismo , Animales , Membrana Celular/genética , Clatrina/genética , Humanos , Vesículas Transportadoras/genéticaRESUMEN
Predation is an extreme form of competition between bacteria, involving the secretion of antimicrobial substances by predators, often packaged within outer membrane vesicles (OMVs). Recent studies into the Myxococcus xanthus/Escherichia coli predator/prey relationship have illuminated transcriptional changes during predation, identifying likely targets of predatory attack in the prey and nutrient assimilation strategies of the predator. Abundant non-coding RNAs can be observed in the predator and prey transcriptomes, with evidence of predation-dependent regulation of RNA levels. Given the observed secretion of regulatory RNAs within OMVs by bacteria, it will next be exciting to test whether the intercellular trafficking of regulatory RNAs is employed by predator and/or prey in their survival struggles.
Asunto(s)
Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Myxococcus xanthus/genética , ARN Bacteriano/genética , ARN no Traducido/genética , Transporte Biológico , Escherichia coli/genética , Myxococcus xanthus/metabolismo , ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Transcriptoma , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismoRESUMEN
Exosomes are small vesicles of endocytic origin, which are released into the extracellular environment and mediate a variety of physiological and pathological conditions. Here we show that Schistosoma mansoni releases exosome-like vesicles in vitro. Vesicles were purified from culture medium by sucrose gradient fractionation and fractions containing vesicles verified by western blot analyses and electron microscopy. Proteomic analyses of exosomal contents unveiled 130 schistosome proteins. Among these proteins are common exosomal markers such as heat shock proteins, energy-generating enzymes, cytoskeletal proteins, and others. In addition, the schistosome extracellular vesicles contain proteins of potential importance for host-parasite interaction, notably peptidases, signaling proteins, cell adhesion proteins (e.g., integrins) and previously described vaccine candidates, including glutathione-S-transferase (GST), tetraspanin (TSP-2) and calpain. S. mansoni exosomes also contain 143 microRNAs (miRNA), of which 25 are present at high levels, including miRNAs detected in sera of infected hosts. Quantitative PCR analysis confirmed the presence of schistosome-derived miRNAs in exosomes purified from infected mouse sera. The results provide evidence of vesicle-mediated secretion in these parasites and suggest that schistosome-derived exosomes could play important roles in host-parasite interactions and could be a useful tool in the development of vaccines and therapeutics.
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Proteómica , Schistosoma mansoni/genética , Esquistosomiasis/genética , Vesículas Transportadoras/genética , Animales , Calpaína/sangre , Calpaína/genética , Exosomas/genética , Femenino , Glutatión Transferasa/sangre , Glutatión Transferasa/genética , Humanos , Ratones , Schistosoma mansoni/patogenicidad , Esquistosomiasis/sangre , Esquistosomiasis/microbiología , Esquistosomiasis/patología , Tetraspaninas/sangre , Tetraspaninas/genética , Vacunas/sangre , Vacunas/genéticaRESUMEN
Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.
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Antígenos/genética , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Bioingeniería , Nanopartículas/química , Antígenos/uso terapéutico , Bacterias Gramnegativas/genética , Nanopartículas/uso terapéutico , Vesículas Transportadoras/genéticaRESUMEN
Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.