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Due to their ability to shuttle proteins, lipids and genetic material between distant cells, exosomes promote extensive phenotypic changes in recipient cells, modulating immune responses, cellular migration, cancer metastasis or the spreading of neurotoxic protein aggregates in neurodegenerative diseases. Besides intercellular communication, exosome biogenesis and secretion permit the rapid release of a selective repertoire of compounds, conferring cells with an additional mechanism to fight alterations in protein, lipid or RNA homeostasis during stress or pathological conditions. Here, we review the dual role of the different quality control mechanisms arising from the endolysosomal system and the diverse situations that control the decision between degradation or secretion. The crosstalk between exosome secretion and the different cellular degradation mechanisms confers an additional layer of protection to maintain cellular integrity and homeostasis in a number of physiological and pathological conditions.

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.

Background: Eosinophils secrete several granules that are involved in the propagation of inflammatory responses in patients with pathologies such as asthma., Objective: We hypothesized that some of these granules are exosomes, which, when transferred to the recipient cells, could modulate asthma progression., Methods: Eosinophils were purified from peripheral blood and cultured with or without IFN-γ or eotaxin. Multivesicular bodies (MVBs) in eosinophils were studied by using fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry. Exosome secretion was measured and exosome characterization was performed with TEM, Western blotting, and NanoSight analysis., Results: Generation of MVBs in eosinophils was confirmed by using fluorescence microscopy and flow cytometry and corroborated by means of TEM. Having established that eosinophils contain MVBs, our aim was to demonstrate that eosinophils secrete exosomes. To do this, we purified exosomes from culture medium of eosinophils and characterized them. Using Western blot analysis, we demonstrated that eosinophils secreted exosomes and that the discharge of exosomes to extracellular media increases after IFN-γ stimulation. We measured exosome size and quantified exosome production from healthy and asthmatic subjects using nanotracking analysis. We found that exosome production was augmented in asthmatic patients., Conclusion: Our findings are the first to demonstrate that eosinophils contain functional MVBs and secrete exosomes and that their secretion is increased in asthmatic patients. Thus exosomes might play an important role in the progression of asthma and eventually be considered a biomarker., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Exosomes are extracellular vesicles that transport different molecules between cells. They are formed and stored inside multivesicular bodies (MVB) until they are released to the extracellular environment. MVB fuse along the plasma membrane, driving non-polarized secretion of exosomes. However, polarized signaling potentially directs MVBs to a specific point in the plasma membrane to mediate a focal delivery of exosomes. MVB polarization occurs across a broad set of cellular situations, e.g. in immune and neuronal synapses, cell migration and in epithelial sheets. In this review, we summarize the current state of the art of polarized MVB docking and the specification of secretory sites at the plasma membrane. The current view is that MVB positioning and subsequent exosome delivery requires a polarizing, cytoskeletal dependent-trafficking mechanism. In this context, we propose scenarios in which biochemical and mechanical signals could drive the polarized delivery of exosomes in highly polarized cells, such as lymphocytes, neurons and epithelia., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

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.

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.