RESUMEN
To combat microbial infections, mammalian cells use a variety of innate immune response pathways to induce synthesis of anti-microbial proteins. The cGAS/STING pathway recognizes cytoplasmic viral or cellular DNA to elicit signals that lead to type I interferon and other cytokine synthesis. cGAMP, synthesized by DNA-activated cGAS, activates the ER-associated protein, STING, which oligomerizes and translocates to other intracellular membrane compartments to trigger different branches of signaling. We have reported that, in the ER, EGFR-mediated phosphorylation of Tyr245 of STING is required for its transit to the late endosomes, where it recruits and activates the transcription factor IRF3 required for IFN induction. In the current study, we inquired whether STING Tyr245 phosphorylation per se or STING's location in the late endosomes was critical for its ability to recruit IRF3 and induce IFN. Using pharmacological inhibitors or genetic ablation of proteins that are essential for specific steps of STING trafficking, we demonstrated that the presence of STING in the late endosomal membranes, even without Tyr245 phosphorylation, was sufficient for IRF3-mediated IFN induction.
Asunto(s)
Interferones , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Inmunidad Innata/genética , ADN , Endosomas/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
The cellular endosomal sorting complex required for transport (ESCRT) system comprises five distinct components and is involved in many different physiological processes. Recent studies have shown that different viruses rely upon the host ESCRT system for viral infection. However, whether this system is involved in white spot syndrome virus (WSSV) infection remains unclear. Here, we identified 24 homologs of ESCRT subunits in kuruma shrimp, Marsupenaeus japonicus, and found that some key components were strongly upregulated in shrimp after WSSV infection. Knockdown of key components of the ESCRT system using RNA interference inhibited virus replication, suggesting that the ESCRT system is beneficial for WSSV infection. We further focused on TSG101, a crucial member of the ESCRT-I family that plays a central role in recognizing cargo and activating the ESCRT-II and ESCRT-III complexes. TSG101 colocalized with WSSV in hemocytes. The addition of N16 (a TSG101 inhibitor) markedly decreased WSSV replication. TSG101 and ALIX of the ESCRT system interact with WSSV envelope proteins. The host proteins TSG101, RAB5, and RAB7, the viral protein VP28, and DNA were detected in endosomes isolated from hemocytes of WSSV-infected shrimp. Knockdown of Rab5 and Rab7 expression reduced viral replication. Taken together, these results suggest that the ESCRT system is hijacked by WSSV for transport through the early to late endosome pathway. Our work identified a novel requirement for the intracellular trafficking and infection of WSSV, and provided novel therapeutic targets for the prevention and control of WSSV in shrimp aquaculture. IMPORTANCE: Viruses utilize the ESCRT machinery in a variety of strategies for their replication and infection. This study revealed that the interaction of ESCRT complexes with WSSV envelope proteins plays a crucial role in WSSV infection in shrimp. The ESCRT system is conserved in the shrimp Marsupenaeus japonicus, and 24 homologs of the ESCRT system were identified in the shrimp. WSSV exploits the ESCRT system for transport and propagation via the interaction of envelope proteins with host TSG101 and ALIX in an endosome pathway-dependent manner. Understanding the underlying mechanisms of WSSV infection is important for disease control and breeding in shrimp aquaculture.
Asunto(s)
Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Penaeidae , Replicación Viral , Virus del Síndrome de la Mancha Blanca 1 , Animales , Virus del Síndrome de la Mancha Blanca 1/fisiología , Virus del Síndrome de la Mancha Blanca 1/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Penaeidae/virología , Penaeidae/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Endosomas/metabolismo , Endosomas/virología , Hemocitos/virología , Hemocitos/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Interferencia de ARNRESUMEN
Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and late endosomes/lysosomes (LE/lys) are emerging as critical hubs for diverse cellular events, and changes in their extents are linked to severe neurological diseases. While recent studies show that the synaptotagmin-like mitochondrial-lipid-binding (SMP) domain-containing protein PDZD8 may mediate the formation of ER-LE/lys MCSs, the cellular functions of PDZD8 remain largely elusive. Here, we attempt to investigate the lipid transfer activities of PDZD8 and the extent to which its cellular functions depend on its lipid transfer activities. In accordance with recent studies, we demonstrate that PDZD8 is a protrudin (ZFYVE27)-interacting protein and that PDZD8 acts as a tether at ER-LE/lys MCSs. Furthermore, we discover that the SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and that the SMP domain can transport lipids between membranes in vitro. Functionally, PDZD8 is required for LE/lys positioning and neurite outgrowth, which is dependent on the lipid transfer activity of the SMP domain.
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Retículo Endoplásmico , Endosomas , Lípidos , Lisosomas , Proyección NeuronalRESUMEN
The endoplasmic reticulum (ER) is the largest organelle contacting virtually every other organelle for information exchange and control of processes such as transport, fusion, and fission. Here, we studied the role of the other organelles on ER network architecture in the cell periphery. We show that the co-migration of the ER with other organelles, called ER hitchhiking facilitated by late endosomes and lysosomes is a major mechanism controlling ER network architecture. When hitchhiking occurs, emerging ER structures may fuse with the existing ER tubules to alter the local ER architecture. This couples late endosomal/lysosomal positioning and mobility to ER network architecture. Conditions restricting late endosomal movement-including cell starvation-or the depletion of tether proteins that link the ER to late endosomes reduce ER dynamics and limit the complexity of the peripheral ER network architecture. This indicates that among many factors, the ER is controlled by late endosomal movement resulting in an alteration of the ER network architecture.
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Retículo Endoplásmico , Endosomas , Transporte Biológico , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismoRESUMEN
Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.
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Exosomas , Vesículas Extracelulares , Holothuria , Pepinos de Mar , Animales , Evolución Biológica , Vesícula , MamíferosRESUMEN
The human Niemann-Pick C1 (NPC1) gene encoding a 1278 amino acid protein is very heterogeneous. While some variants represent benign polymorphisms, NPC disease carriers and patients may possess rare variants, whose functional importance remains unknown. An NPC1 cDNA construct known as NPC1 wild-type variant (WT-V), distributed between laboratories and used as a WT control in several studies, also contains changes regarding specific amino acids compared to the NPC1 Genbank reference sequence. To improve the dissection of subtle functional differences, we generated human cells stably expressing NPC1 variants from the AAVS1 safe-harbor locus on an NPC1-null background engineered by CRISPR/Cas9 editing. We then employed high-content imaging with automated image analysis to quantitatively assess LDL-induced, time-dependent changes in lysosomal cholesterol content and lipid droplet formation. Our results indicate that the L472P change present in NPC1 WT-V compromises NPC1 functionality in lysosomal cholesterol export. All-atom molecular dynamics simulations suggest that the L472P change alters the relative position of the NPC1 middle and the C-terminal luminal domains, disrupting the recently characterized cholesterol efflux tunnel. These results reveal functional defects in NPC1 WT-V and highlight the strength of simulations and quantitative imaging upon stable protein expression in elucidating subtle differences in protein function.
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Colesterol , Péptidos y Proteínas de Señalización Intracelular , Proteínas , Transporte Biológico , Colesterol/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Lisosomas/metabolismo , Simulación de Dinámica Molecular , Proteína Niemann-Pick C1 , Proteínas/metabolismoRESUMEN
ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.
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Adenosina Trifosfatasas , Fosfolípidos , Fosfolípidos/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de la Membrana/metabolismo , LisosomasRESUMEN
KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.
Asunto(s)
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis , Aparato de Golgi/metabolismo , Receptores de Péptidos/metabolismo , Línea Celular Tumoral , Endosomas/metabolismo , Edición Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cancer cells undergo drastic metabolic adaptions to cover increased bioenergetic needs, contributing to resistance to therapies. This includes a higher demand for cholesterol, which often coincides with elevated cholesterol uptake from low-density lipoproteins (LDL) and overexpression of the LDL receptor in many cancers. This implies the need for cancer cells to accommodate an increased delivery of LDL along the endocytic pathway to late endosomes/lysosomes (LE/Lys), providing a rapid and effective distribution of LDL-derived cholesterol from LE/Lys to other organelles for cholesterol to foster cancer growth and spread. LDL-cholesterol exported from LE/Lys is facilitated by Niemann-Pick Type C1/2 (NPC1/2) proteins, members of the steroidogenic acute regulatory-related lipid transfer domain (StARD) and oxysterol-binding protein (OSBP) families. In addition, lysosomal membrane proteins, small Rab GTPases as well as scaffolding proteins, including annexin A6 (AnxA6), contribute to regulating cholesterol egress from LE/Lys. Here, we summarize current knowledge that links upregulated activity and expression of cholesterol transporters and related proteins in LE/Lys with cancer growth, progression and treatment outcomes. Several mechanisms on how cellular distribution of LDL-derived cholesterol from LE/Lys influences cancer cell behavior are reviewed, some of those providing opportunities for treatment strategies to reduce cancer progression and anticancer drug resistance.
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Antineoplásicos , Neoplasias , Antineoplásicos/análisis , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteína Niemann-Pick C1/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
In Alzheimer's disease (AD), tau, a microtubule-associated protein (MAP), becomes hyperphosphorylated, aggregates, and accumulates in the somato-dendritic compartment of neurons. In parallel to its intracellular accumulation in AD, tau is also released in the extracellular space, as revealed by its increased presence in cerebrospinal fluid (CSF). Consistent with this, recent studies, including ours, have reported that neurons secrete tau, and several therapeutic strategies aim to prevent the intracellular tau accumulation. Previously, we reported that late endosomes were implicated in tau secretion. Here, we explore the possibility of preventing intracellular tau accumulation by increasing tau secretion. Using neuronal models, we investigated whether overexpression of the vesicle-associated membrane protein 8 (VAMP8), an R-SNARE found on late endosomes, could increase tau secretion. The overexpression of VAMP8 significantly increased tau secretion, decreasing its intracellular levels in the neuroblastoma (N2a) cell line. Increased tau secretion by VAMP8 was also observed in murine hippocampal slices. The intracellular reduction of tau by VAMP8 overexpression correlated to a decrease of acetylated tubulin induced by tau overexpression in N2a cells. VAMP8 staining was preferentially found on late endosomes in N2a cells. Using total internal reflection fluorescence (TIRF) microscopy, the fusion of VAMP8-positive vesicles with the plasma membrane was correlated to the depletion of tau in the cytoplasm. Finally, overexpression of VAMP8 reduced the intracellular accumulation of tau mutants linked to frontotemporal dementia with parkinsonism and α-synuclein by increasing their secretion. Collectively, the present data indicate that VAMP8 could be used to increase tau and α-synuclein clearance to prevent their intracellular accumulation.
Asunto(s)
Proteínas R-SNARE/metabolismo , Proteínas tau/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Regulación hacia Abajo , Endosomas/metabolismo , Demencia Frontotemporal/patología , Hipocampo/metabolismo , Ratones , Microscopía Fluorescente , Mutagénesis , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Proteínas R-SNARE/genética , Vesículas Secretoras/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas tau/genéticaRESUMEN
Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of an SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, ß1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Endosomas/metabolismo , Integrina beta1/metabolismo , Factores de Transcripción/metabolismo , Familia-src Quinasas/metabolismo , Animales , Proteínas de Ciclo Celular/deficiencia , Línea Celular , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proto-Oncogenes Mas , Transducción de Señal , Factores de Transcripción/deficiencia , Familia-src Quinasas/deficienciaRESUMEN
BACKGROUND: Neurotrophins can activate multiple signalling pathways in neuronal cells through binding to their cognate receptors, leading to neurotrophic processes such as cell survival and differentiation. γ-Enolase has been shown to have a neurotrophic activity that depends on its translocation towards the plasma membrane by the scaffold protein γ1-syntrophin. The association of γ-enolase with its membrane receptor or other binding partners at the plasma membrane remains unknown. METHODS: In the present study, we used immunoprecipitation and immunofluorescence to show that γ-enolase associates with the intracellular domain of the tropomyosin receptor kinase (Trk) family of tyrosine kinase receptors at the plasma membrane of differentiated SH-SY5Y cells. RESULTS: In differentiated SH-SY5Y cells with reduced expression of γ1-syntrophin, the association of γ-enolase with the Trk receptor was diminished due to impaired translocation of γ-enolase towards the plasma membrane or impaired Trk activity. Treatment of differentiated SH-SY5Y cells with a γ-Eno peptide that mimics γ-enolase neurotrophic activity promoted Trk receptor internalisation and endosomal trafficking, as defined by reduced levels of Trk in clathrin-coated vesicles and increased levels in late endosomes. In this way, γ-enolase triggers Rap1 activation, which is required for neurotrophic activity of γ-enolase. Additionally, the inhibition of Trk kinase activity by K252a revealed that increased SH-SY5Y cell survival and neurite outgrowth mediated by the γ-Eno peptide through activation of signalling cascade depends on Trk kinase activity. CONCLUSIONS: These data therefore establish the Trk receptor as a binding partner of γ-enolase, whereby Trk endosomal trafficking is promoted by γ-Eno peptide to mediate its neurotrophic signalling. Video abstract.
Asunto(s)
Diferenciación Celular , Neuritas , Fosfopiruvato Hidratasa , Receptor trkA , Línea Celular Tumoral , Humanos , Neuritas/fisiología , Fosfopiruvato Hidratasa/metabolismo , Receptor trkA/metabolismoRESUMEN
Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.
Asunto(s)
Anexina A6/genética , Colesterol/genética , Proteínas Activadoras de GTPasa/genética , Enfermedad de Niemann-Pick Tipo C/genética , Proteínas de Unión al GTP rab/genética , Animales , Células CHO , Proteínas Portadoras/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Endosomas/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Dominios Proteicos/genética , Transporte de Proteínas/genética , ARN Interferente Pequeño/genética , Proteínas de Unión a GTP rab7RESUMEN
As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the ß2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune , Muromegalovirus/crecimiento & desarrollo , Muromegalovirus/inmunología , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Animales , Células Cultivadas , Endosomas/metabolismo , Lisosomas/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Unión Proteica , Transporte de ProteínasRESUMEN
Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome-lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co-localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA-Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ-BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein-sorting (HOPS) complex hampered the co-localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endocitosis/fisiología , Endosomas/fisiología , Lisosomas/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Endosomas/metabolismo , Humanos , Células K562 , Lisosomas/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Cytotoxic T lymphocytes patrol our body in search for infected cells which they kill through the release of cytotoxic substances contained in cytotoxic granules. The fusion of cytotoxic granules occurs at a specially formed contact site, the immunological synapse, and is tightly controlled to ensure specificity. In this review, we discuss the contribution of two intracellular compartments, endosomes and cytotoxic granules, to the formation, function and disassembly of the immunological synapse. We highlight a recently proposed sequential process of fusion events at the IS upon target cell recognition. First, recycling endosomes fuse with the plasma membrane to deliver cargo required for the docking of cytotoxic granules. Second, cytotoxic granules arrive and fuse upon docking in a SNARE-dependent manner. Following fusion, membrane components of the cytotoxic granule are retrieved through endocytosis to ensure the fast, efficient serial killing of target cells that is characteristic of cytotoxic T lymphocytes.
Asunto(s)
Citotoxicidad Inmunológica , Endocitosis , Exocitosis , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Animales , Gránulos Citoplasmáticos/inmunología , Endosomas/inmunología , Humanos , Lisosomas/inmunología , Fusión de Membrana , Proteínas SNARE/inmunologíaRESUMEN
The spatiotemporal regulation of calcium (Ca2+) storage in late endosomes (LE) and lysosomes (Lys) is increasingly recognized to influence a variety of membrane trafficking events, including endocytosis, exocytosis, and autophagy. Alterations in Ca2+ homeostasis within the LE/Lys compartment are implicated in human diseases, ranging from lysosomal storage diseases (LSDs) to neurodegeneration and cancer, and they correlate with changes in the membrane binding behaviour of Ca2+-binding proteins. This also includes Annexins (AnxA), which is a family of Ca2+-binding proteins participating in membrane traffic and tethering, microdomain organization, cytoskeleton interactions, Ca2+ signalling, and LE/Lys positioning. Although our knowledge regarding the way Annexins contribute to LE/Lys functions is still incomplete, recruitment of Annexins to LE/Lys is greatly influenced by the availability of Annexin bindings sites, including acidic phospholipids, such as phosphatidylserine (PS) and phosphatidic acid (PA), cholesterol, and phosphatidylinositol (4,5)-bisphosphate (PIP2). Moreover, the cytosolic portion of LE/Lys membrane proteins may also, directly or indirectly, determine the recruitment of Annexins to LE. Strikingly, within LE/Lys, AnxA1, A2, A6, and A8 differentially contribute to cholesterol transport along the endocytic route, in particular, cholesterol transfer between LE and other compartments, positioning Annexins at the centre of major pathways mediating cellular cholesterol homeostasis. Underlying mechanisms include the formation of membrane contact sites (MCS) and intraluminal vesicles (ILV), as well as the modulation of LE-cholesterol transporter activity. In this review, we will summarize the current understanding how Annexins contribute to influence LE/Lys membrane transport and associated functions.
Asunto(s)
Anexinas/metabolismo , Colesterol/metabolismo , Endocitosis , Homeostasis , Animales , Anexinas/química , Transporte Biológico , Calcio/metabolismo , Colesterol/química , Endosomas/metabolismo , Exosomas/metabolismo , Humanos , Transducción de SeñalRESUMEN
Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA-gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal-lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal-lysosomal fusion events.
Asunto(s)
Endocitosis/fisiología , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Endosomas/ultraestructura , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Lisosomas/ultraestructura , Fusión de Membrana/fisiología , Microscopía Electrónica , Microscopía Fluorescente , Transporte de Proteínas , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phospholipid known to be associated with a wide variety of physiological functions in plants. However, the localization and dynamics of PI(3,5)P2 in plant cells remain largely unknown, partially due to the lack of an effective fluorescent probe. Using Arabidopsis transgenic plant expressing the PI(3,5)P2-labeling fluorescent probe (tagRFP-ML1N*2) developed based on a tandem repeat of the cytosolic phosphoinositide-interacting domain (ML1N) of the mammalian lysosomal transient receptor potential cation channel, Mucolipin 1 (TRPML1), here we show that PI(3,5)P2 is predominantly localized on the limited membranes of the FAB1- and SNX1-positive late endosomes, but rarely localized on the membranes of plant vacuoles or trans-Golgi network/early endosomes of cortical cells of the root differentiation zone. The late endosomal localization of tagRFP-ML1N*2 is reduced or abolished by pharmacological inhibition or genetic knockdown of expression of genes encoding PI(3,5)P2-synthesizing enzymes, FAB1A/B, but markedly increased with FAB1A overexpression. Notably, reactive oxygen species (ROS) significantly increase late endosomal levels of PI(3,5)P2. Thus, tandem ML1N-based PI(3,5)P2 probes can reliably monitor intracellular dynamics of PI(3,5)P2 in Arabidopsis cells with less binding activity to other endomembrane organelles.
Asunto(s)
Arabidopsis/metabolismo , Colorantes Fluorescentes/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Endosomas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lisosomas/metabolismo , Microscopía Confocal , Fosfatidilinositoles/análisis , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión , Vesículas Transportadoras/metabolismo , Vacuolas/metabolismo , Red trans-Golgi/metabolismo , Proteína Fluorescente RojaRESUMEN
Despite the discovery of annexins 40 years ago, we are just beginning to understand some of the functions of these still enigmatic proteins. Defined and characterized by their ability to bind anionic membrane lipids in a Ca2+-dependent manner, each annexin has to be considered a multifunctional protein, with a multitude of cellular locations and diverse activities. Underlying causes for this considerable functional diversity include their capability to associate with multiple cytosolic and membrane proteins. In recent years, the increasingly recognized establishment of membrane contact sites between subcellular compartments opens a new scenario for annexins as instrumental players to link Ca2+ signalling with the integration of membrane trafficking in many facets of cell physiology. In this chapter, we review and discuss current knowledge on the contribution of annexins in the biogenesis and functioning of the late endocytic compartment, affecting endo- and exocytic pathways in a variety of physiological consequences ranging from membrane repair, lysosomal exocytosis, to cell migration.