The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

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Ubiquitination is a regulated post-translational modification that conjugates ubiquitin (Ub) to lysine residues of target proteins and determines their intracellular fate. The canonical role of ubiquitination is to mediate degradation by the proteasome of short-lived cytoplasmic proteins that carry a single, polymeric chain of Ub on a specific lysine residue. However, protein modification by Ub has much broader and diverse functions involved in a myriad of cellular processes. Monoubiquitination, at one or multiple lysine residues of transmembrane proteins, influences their stability, protein-protein recognition, activity and intracellular localization. In these processes, Ub functions as an internalization signal that sends the modified substrate to the endocytic/sorting compartments, followed by recycling to the plasma membrane or degradation in the lysosome. E3 ligases play a pivotal role in ubiquitination, because they recognize the acceptor protein and hence dictate the high specificity of the reaction. The multitude of E3s present in nature suggests their nonredundant mode of action and the need for their controlled regulation. Here we give a short account of E3 ligases that specifically modify and regulate membrane proteins. We emphasize the intricate network of interacting proteins that contribute to the substrate-E3 recognition and determine the substrate's cellular fate. E3 LigasesThe covalent ligation of the 76-amino acid peptide Ubiquitin (Ub) to substrate proteins is a highly conserved process that involves a plethora of enzymes and accessory proteins that are usually homologous across species (1). The reaction occurs via the sequential action of three enzymes: a Ub-activating enzyme E1, a Ub-conjugating enzyme E2 and a Ub ligase E3 (1). Although there is only one isoform of E1 in eukaryotic cells, many different E2s and E3s work together to ensure the correct timing, localization and specificity of the ubiquitination reaction (1). The coexistence of many E3 ligases in the same cell type underscores the intricacy of the selection and activation of the appropriate E2 and the recruitment of the substrate. To date, E3s have been considered the only ubiquitination components subject to regulation, but the recent finding of an E2 post-translationally modified by the Ub-like protein SUMO suggests an additional layer of regulation at the E2 level (2).E3 ligases are modular, single proteins or multiprotein complexes that contribute different motifs or domains to the catalytic core, including protein-protein interaction domains for substrate recognition. Together with the E2 enzymes, E3s determine the topology of the polyubiquitin chain. Two primary classes of E3s have been described. The first is distinguished by the presence of a HECT (homologous to E6-AP carboxyl terminus) domain; the second by a RING (really interesting new gene)-finger domain. Both bind to E2s (3). The HECT E3s participate in the catalytic reaction by forming a thioester bond with Ub, via a conserved cysteine residue within the HECT dom...

The neuropeptide Substance P (SP) is an important mediator of neuroimmunomodulatory activity. The aim of this study is to elucidate the mechanism used by SP to promote increased production of pro-inflammatory cytokines in fresh isolated rat peritoneal mast cells (rPMC). We have demonstrated that SP induces production of interleukin-6 (IL-6) in rPMC through the PI-3K, p42/44 and p38 MAP kinase pathways. SP-stimulated rPMC also exhibited an enhanced nuclear translocation of the nuclear factor kappa B (NF kappa B). The tumour necrosis factor-alpha (TNF-alpha) and IL-6 production was completely inhibited by using (E)-4-hydroxynonenal (HNE) as an inhibitor of I kappa B-alpha and -beta phosphorylation. Further, TNF-alpha and IL-6 expression was significantly inhibited by the oligonucleotides (ODNs) containing the NF kappa B element (NF kappa B decoy ODNs) but not by the scrambled control ODNs. These findings indicate that the NF kappa B pathway is involved in the transcriptional regulation of the TNF-alpha and IL-6 overexpression in primary SP-stimulated mast cells.

Cellular, inter‐organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio‐technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano‐objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Several cell types secrete small membranous vesicles that contain cell-specific collections of proteins, lipids, and genetic material. The function of these vesicles is to allow cell-to-cell signaling and the horizontal transfer of their cargo molecules. Here, we demonstrate that muscle cells secrete nano-sized vesicles and that their release increases during muscle differentiation. Analysis of these nanovesicles allowed us to characterize them as exosome-like particles and to define the potential role of the multifunctional protein Alix in their biogenesis.

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. Here we describe a RING-type E3 ligase complex specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. We identified beta-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated beta-catenin by mediating its degradation via the proteasome. Expression of beta-catenin mutants that reduce the binding of Ozz to endogenous beta-catenin leads to Mb-beta-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of Mb-beta-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation.

Potential applications of extracellular vesicles (EVs) are attracting increasing interest in the fields of medicine, cosmetics, and nutrition. However, the manufacturing of EVs is currently characterized by low yields. This limitation severely hampers progress in research at the laboratory and clinical scales, as well as the realization of successful and cost-effective EV-based products. Moreover, the high level of heterogeneity of EVs further complicates reproducible manufacturing on a large scale. In this review, possible directions toward the scalable production of EVs are discussed. In particular, two strategies are considered: i) the optimization of upstream unit operations and ii) the exploitation of well-established and mature technologies already in use in other industrial bioprocesses.