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.

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.

Development and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion.However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6 RNAi-mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. We show that phosphomimicking Ykt6 accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies in a dose-dependent manner. Taken together, we show that exosomal sorting of Wnts is fine-tuned by a regulatory switch in Ykt6 shifting it from a membrane-bound to cytosolic state at the level of endosomal maturation.

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.