Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that – despite the different vesicle biogenesis – the routes of vesicular export are adaptable.

During endosome maturation, neutral sphingomyelinase 2 (nSMase2, encoded by SMPD3) is involved in budding of intraluminal vesicles (ILVs) into late endosomes or multivesicular bodies (MVBs). Fusion of these with the plasma membrane results in secretion of exosomes or small extracellular vesicles (sEVs). Here, we report that nSMase2 activity controls sEV secretion through modulation of vacuolar H+-ATPase (V-ATPase) activity. Specifically, we show that nSMase2 inhibition induces V-ATPase complex assembly that drives MVB lumen acidification and consequently reduces sEV secretion. Conversely, we further demonstrate that stimulating nSMase2 activity with the inflammatory cytokine TNFα (also known as TNF) decreases acidification and increases sEV secretion. Thus, we find that nSMase2 activity affects MVB membrane lipid composition to counteract V-ATPase-mediated endosome acidification, thereby shifting MVB fate towards sEV secretion. This article has an associated First Person interview with the first author of the paper.

Extracellular vesicles (EVs) are shed by many different cell types. Their nucleic acids content offers new opportunities for biomarker research in different solid tumors. The role of EV RNA in prostate cancer (PCa) is still largely unknown. EVs were isolated from different benign and malignant prostate cell lines and blood plasma from patients with PCa (n = 18) and controls with benign prostatic hyperplasia (BPH) (n = 7). Nanoparticle tracking analysis (NTA), Western blot, electron microscopy, and flow cytometry analysis were used for the characterization of EVs. Non-coding RNA expression profiling of PC3 metastatic PCa cells and their EVs was performed by next generation sequencing (NGS). miRNAs differentially expressed in PC3 EVs were validated with qRT-PCR in EVs derived from additional cell lines and patient plasma and from matched tissue samples. 92 miRNAs were enriched and 48 miRNAs were depleted in PC3 EVs compared to PC3 cells, which could be confirmed by qRT-PCR. miR-99b-5p was significantly higher expressed in malignant compared to benign EVs. Furthermore, expression profiling showed miR-10a-5p (p = 0.018) and miR-29b-3p (p = 0.002), but not miR-99b-5p, to be overexpressed in plasma-derived EVs from patients with PCa compared with controls. In the corresponding tissue samples, no significant differences in the miRNA expression could be observed. We thus propose that EV-associated miR-10a-5p and miR-29b-3p could serve as potential new PCa detection markers.

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD_1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.

Wnt signaling is an important pathway in health and disease and a key regulator of stem cell maintenance, differentiation, and proliferation. During heart development, Wnt signaling controls specification, proliferation and differentiation of cardiovascular cells. In this regard, the role of activated Wnt signaling in cardiogenesis is well defined. However, the knowledge about signaling transmission has been challenged. Recently, the packaging of hydrophobic Wnt proteins on extracellular vesicles (EVs) has emerged as a mechanism to facilitate their extracellular spreading and their functioning as morphogens. EVs spread systemically and therefore can have pleiotropic effects on very different cell types. They are heavily studied in tumor biology where they affect tumor growth and vascularization and can serve as biomarkers in liquid biopsies. In this review we will highlight recent discoveries of factors involved in the release of Wnts on EVs and its potential implications in the communication between physiological and pathological heart cells.