RESUMEN
Mosaic variegated aneuploidy (MVA) is a rare condition in which abnormal chromosome counts (that is, aneuploidies), affecting different chromosomes in each cell (making it variegated) are found only in a certain number of cells (making it mosaic). MVA is characterized by various developmental defects and, despite its rarity, presents a unique clinical scenario to understand the consequences of chromosomal instability and copy number variation in humans. Research from patients with MVA, genetically engineered mouse models and functional cellular studies have found the genetic causes to be mutations in components of the spindle-assembly checkpoint as well as in related proteins involved in centrosome dynamics during mitosis. MVA is accompanied by tumour susceptibility (depending on the genetic basis) as well as cellular and systemic stress, including chronic immune response and the associated clinical implications.
RESUMEN
Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Animales , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclo Celular , Fosforilación/fisiología , Mitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , MamíferosRESUMEN
Interferon stimulated gene 15 (ISG15) is an ubiquitin-like protein whose expression and conjugation to targets (ISGylation) is induced by infection, interferon (IFN)-α and -ß, ischemia, DNA damage and aging. Attention has historically focused on the antiviral effects of ISGylation, which blocks the entry, replication or release of different intracellular pathogens. However, recently, new functions of ISGylation have emerged that implicate it in multiple cellular processes, such as DNA repair, autophagy, protein translation and exosome secretion. In this Review, we discuss the induction and conjugation of ISG15, as well as the functions of ISGylation in the prevention of infections and in cancer progression. We also offer a novel perspective with regard to the latest findings on this pathway, with special attention to the role of ISGylation in the inhibition of exosome secretion, which is mediated by fusion of multivesicular bodies with lysosomes. Finally, we propose that under conditions of stress or infection, ISGylation acts as a defense mechanism to inhibit normal protein translation by modifying protein kinase R (PKR, also known as EIF2AK2), while any newly synthesized proteins are being tagged and thus marked as potentially dangerous. Then, the endosomal system is re-directed towards protein degradation at the lysosome, to effectively 'lock' the cell gates and thus prevent the spread of pathogens, prions and deleterious aggregates through exosomes.
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Células/metabolismo , Interferones/metabolismo , Animales , Infecciones Bacterianas/metabolismo , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Virosis/metabolismoRESUMEN
The transfer of molecules between cells during cognate immune cell interactions has been reported, and recently a novel mechanism of transfer of proteins and genetic material such as small RNA between T cells and antigen-presenting cells (APCs) has been described, involving exchange of extracellular vesicles (EVs) during the formation of the immunological synapse (IS). EVs, a term that encompasses exosomes and microvesicles, has been implicated in cell-cell communication during immune responses associated with tumors, pathogens, allergies, and autoimmune diseases. This review focuses on EV transfer as a mechanism for the exchange of molecules during immune cell-cell interactions.
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Células Presentadoras de Antígenos/inmunología , Inmunidad Celular , Sinapsis Inmunológicas/inmunología , Vesículas Secretoras/inmunología , Linfocitos T/inmunología , Animales , Comunicación Celular/inmunología , Humanos , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Extracellular vesicles (EVs), a term that includes both exosomes of endocytic origin and vesicles derived from plasma membranes, are continuously secreted by cells to the extracellular environment, and represent a novel vehicle for cell-cell communication. Exosomes contain specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Although the molecular mechanisms that regulate the loading of proteins into exosomes have been studied for years, the sorting of RNA has been elusive until recently. Here we review the molecular mechanisms that control the sorting of molecules into exosomes, with special attention to the sorting of RNA. We also discuss how the cellular context affects the composition of exosomes, and thus the outcome of the communication between the exosome-producer and recipient cells, with particular focus on the communication between tumor cells and with cells of the tumor microenvironment.
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Exosomas/metabolismo , Transporte Biológico/fisiología , Comunicación Celular/fisiología , Humanos , Proteínas/metabolismo , ARN/metabolismo , Microambiente Tumoral/fisiologíaRESUMEN
Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.
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Antígeno Ki-67 , Mitosis , Proteína Fosfatasa 2 , Animales , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Ratones , Antígeno Ki-67/metabolismo , Fosforilación , Cromosomas de los Mamíferos/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas/metabolismoRESUMEN
Numerical chromosomal aberrations are highly frequent in cancer cells. However, tumor-associated mutations in regulators of the mitotic machinery that controls chromosome segregation are rather rare. By sequencing families with hereditary cancer, Chen and colleagues report two novel heterozygous mutations in CDC20, a coactivator of the anaphase-promoting complex (APC/C) and a target of the spindle assembly checkpoint (SAC) that prevents chromosome missegregation during mitosis. CDC20 mutations result in partial SAC functionality and segregate with tumor susceptibility in families with aneuploid ovarian cancers and other malignancies. The expression of these mutations in a knock-in mouse model accelerates the development of Myc-induced lymphomas and mortality, strongly supporting the notion that partial dysfunction of mitotic regulators may have profound implications in spontaneous and hereditary cancer. See related article by Chen et al., p. 3499.
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Puntos de Control de la Fase M del Ciclo Celular , Neoplasias , Animales , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Predisposición Genética a la Enfermedad , Células Germinativas/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/genética , Ratones , Mitosis/genética , Neoplasias/genéticaRESUMEN
Germline mutations leading to aneuploidy are rare, and their tumor-promoting properties are mostly unknown at the molecular level. We report here novel germline biallelic mutations in MAD1L1, encoding the spindle assembly checkpoint (SAC) protein MAD1, in a 36-year-old female with a dozen of neoplasias. Functional studies demonstrated lack of full-length protein and deficient SAC response, resulting in ~30 to 40% of aneuploid blood cells. Single-cell RNA analysis identified mitochondrial stress accompanied by systemic inflammation with enhanced interferon and NFκB signaling both in aneuploid and euploid cells, suggesting a non-cell autonomous response. MAD1L1 mutations resulted in specific clonal expansions of γδ T cells with chromosome 18 gains and enhanced cytotoxic profile as well as intermediate B cells with chromosome 12 gains and transcriptomic signatures characteristic of leukemia cells. These data point to MAD1L1 mutations as the cause of a new variant of mosaic variegated aneuploidy with systemic inflammation and unprecedented tumor susceptibility.
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Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate anti-pathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.
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Antígenos/inmunología , Células Dendríticas/inmunología , Vesículas Extracelulares/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Antígenos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/virología , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Expresión Génica/inmunología , Células HEK293 , Humanos , Interferones/inmunología , Interferones/metabolismo , Células Jurkat , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/metabolismo , Linfocitos T/virología , Virus/inmunologíaRESUMEN
Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. Posttranslational modifications are involved in the sorting of specific proteins into exosomes. Here we identify ISGylation as a ubiquitin-like modification that controls exosome release. ISGylation induction decreases MVB numbers and impairs exosome secretion. Using ISG15-knockout mice and mice expressing the enzymatically inactive form of the de-ISGylase USP18, we demonstrate in vitro and in vivo that ISG15 conjugation regulates exosome secretion. ISG15 conjugation triggers MVB co-localization with lysosomes and promotes the aggregation and degradation of MVB proteins. Accordingly, inhibition of lysosomal function or autophagy restores exosome secretion. Specifically, ISGylation of the MVB protein TSG101 induces its aggregation and degradation, being sufficient to impair exosome secretion. These results identify ISGylation as a novel ubiquitin-like modifier in the control of exosome production.
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Citocinas/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Exosomas/metabolismo , Lisosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Factores de Transcripción/metabolismo , Animales , Autofagia , Células HEK293 , Humanos , Células Jurkat , Macrófagos , Ratones , Ratones Noqueados , Linfocitos T , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinas/genéticaRESUMEN
The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4(+) T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation, and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward proinflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD(+) levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify strategies for intervention in mitochondrial-related diseases.
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Proteínas de Unión al ADN/inmunología , Enfermedades por Almacenamiento Lisosomal/inmunología , Lisosomas/inmunología , Mitocondrias/inmunología , Proteínas Mitocondriales/inmunología , Esfingolipidosis/inmunología , Linfocitos T/inmunología , Factores de Transcripción/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Respiración de la Célula , Proteínas de Unión al ADN/genética , Eliminación de Gen , Inmunidad Celular , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/genética , Lisosomas/patología , Ratones , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Esfingolipidosis/genética , Esfingolipidosis/patología , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Transcripción/genéticaRESUMEN
Exosomes mediate intercellular communication and participate in many cell processes such as cancer progression, immune activation or evasion, and the spread of infection. Exosomes are small vesicles secreted to the extracellular environment through the release of intraluminal vesicles contained in multivesicular bodies (MVBs) upon the fusion of these MVBs with the plasma membrane. The composition of exosomes is not random, suggesting that the incorporation of cargo into them is a regulated process. However, the mechanisms that control the sorting of protein cargo into exosomes are currently elusive. Here, we review the post-translational modifications detected in exosomal proteins, and discuss their possible role in their specific sorting into exosomes.
RESUMEN
Immune cells release microRNA-containing exosomes that can be taken up by recipient cells. Exosomes can thus act as mediators of cell-cell communication through direct exchange of genetic material between cells. Exosome-mediated transfer of miRNAs between T cells and antigen-presenting cells (APCs) can take place over long distances. Our work has shown that this transfer is enhanced by the formation of a functional immune synapse. Here we give a detailed description of the isolation of exosomes produced by immune cells by ultracentrifugation, their quantification by flow cytometry, and the analysis of miRNA and protein exchange between T cells and APCs, both at a distance and after the formation of an immune synapse.
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Linfocitos B/química , Células Dendríticas/química , Exosomas/química , Sinapsis Inmunológicas/química , MicroARNs/aislamiento & purificación , Linfocitos T/química , Tetraspanina 30/aislamiento & purificación , Linfocitos B/citología , Linfocitos B/inmunología , Transporte Biológico , Comunicación Celular , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/inmunología , Exosomas/genética , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Sinapsis Inmunológicas/metabolismo , Células Jurkat , Microscopía Confocal , Linfocitos T/citología , Linfocitos T/inmunología , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , UltracentrifugaciónRESUMEN
Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication. Exosomes contain specific repertoires of mRNAs, microRNAs (miRNAs) and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.
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Exosomas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , MicroARNs/metabolismo , Secuencias de Aminoácidos , Comunicación Celular , Silenciador del Gen , Humanos , Células Jurkat , Leucocitos Mononucleares/metabolismo , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , SumoilaciónRESUMEN
The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.