Tetherin is an exosomal tether

Abstract

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions.DOI: http://dx.doi.org/10.7554/eLife.17180.001

A cell-based assay for CD63-containing extracellular vesicles

“…Our findings of bafilomycin-stimulated EV release are in agreement with earlier observations showing increased secretion of lysosomal enzymes upon treatment with bafilomycin [53]. Bafilomycin-mediated stimulation of EV release was also observed in a recent paper [54], wherein it was also shown that in HeLa cells, exosomes are retained on the plasma membrane by the action of tetherin. These authors also demonstrated that treatment of cells with bafilomycin interrupts cholesterol trafficking leading to cholesterol accumulation in endosomes and exosomes.…”

“…As above, we saw the most striking increase in EV release following treatment with bafilomycin, which inhibits the V-ATPase proton pump and thereby allows the lumenal pH of lysosomes to rise over seconds to minutes [52]. The observed increase in CD63-EV release is consistent with previous reports of bafilomycin enhancing various types of EV release [47,53,54], effects that have been attributed to the pH neutralizing effects of inhibiting the V-ATPase. Curiously, however, we found that treating cells with chloroquine, which rapidly neutralizes lumenal pH as a weak base, did not have any effect on HANLCD63 release.…”

Hepatic stellate cell–derived platelet‐derived growth factor receptor‐alpha‐enriched extracellular vesicles promote liver fibrosis in mice through SHP2

“…In this study, we demonstrate that HSC‐derived EVs transport PDGFRα, which is enriched in the EVs upon PDGF‐BB stimulation, suggesting a regulation mechanism for its trafficking, in addition to its degradation. Some prior studies show that degradation inhibition by bafilomycin A1 enhances EV release . In addition to this, here we demonstrate that degradation inhibition leads to further enrichment of EVs for PDGFRα and, in turn, blockage of PDGFRα enrichment in EVs enhances its degradation.…”

Exosomes: Revisiting their role as “garbage bags”

“…This is in agreement with the analysis of the cholesterol distribution in the endocytic pathway of B cells achieved by detection of cholesterol using immunoelectron microscopy, showing that cholesterol is mostly found in MVE ILVs and is released with exosomes . More recently, V‐ATPase inhibition by bafilomycin A1 was shown to induce a dramatic redistribution of cholesterol through exosome secretion in HeLa cells . Exosomes could thus be viewed as a way to regulate cellular cholesterol homeostasis, together with other cholesterol efflux mechanisms .…”

“…Various studies have recently suggested that there is cross‐regulation between the exosomal pathway and lysosomal degradation. Lysosome inhibitors such as chloroquine or bafilomycin A1 have been shown to increase exosome release. Moreover, it was demonstrated that Atg5 dissociates V 1 V 0 ‐ATPase in endosomes, in turn decreasing endosomal acidification and promoting exosome secretion .…”