Micro- and nano-sized vesicles (MVs and NVs, respectively) from edible plant resources are gaining increasing interest as green, sustainable, and biocompatible materials for the development of next-generation delivery vectors. The isolation of vesicles from complex plant matrix is a significant challenge considering the trade-off between yield and purity. Here, we used differential ultracentrifugation (dUC) for the bulk production of MVs and NVs from tomato (Solanum lycopersicum L.) fruit and analyzed their physical and morphological characteristics and biocargo profiles. The protein and phospholipid cargo shared considerable similarities between MVs and NVs. Phosphatidic acid was the most abundant phospholipid identified in NVs and MVs. The bulk vesicle isolates were further purified using sucrose density gradient ultracentrifugation (gUC) or size-exclusion chromatography (SEC). We showed that SEC using gravity column efficiently removed co-purifying matrix components including proteins and small molecular species. dUC/SEC yielded a high yield of purified vesicles in terms of number of particles (2.6 × 10¹⁵ particles) and protein quantities (6.9 ± 1.5 mg) per kilogram of tomato. dUC/gUC method separated two vesicle populations on the basis of buoyant density. Proteomics and in silico studies of the SEC-purified MVs and NVs support the presence of different intra- and extracellular vesicles with highly abundant lipoxygenase (LOX), ATPases, and heat shock proteins (HSPs), as well as a set of proteins that overlaps with that previously reported in tomato chromoplast.

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.

Autophagy ensures the lysosome-mediated breakdown and recycling of self-material, as it not only degrades obsolete or damaged intracellular constituents but also provides building blocks for biosynthetic and energy producing reactions. Studies in animal models including Drosophila revealed that autophagy defects lead to the rapid decline of neuromuscular function, neurodegeneration, sensitivity to stress (such as starvation or oxidative damage), and stem cell loss. Of note, recently identified human Atg gene mutations cause similar symptoms including ataxia and mental retardation. Physiologically, autophagic degradation (flux) is known to decrease during aging, and this defect likely contributes to the development of such age-associated diseases. Many manipulations that extend lifespan (including dietary restriction, reduced TOR kinase signaling, exercise or treatment with various anti-aging substances) require autophagy for their beneficial effect on longevity, pointing to the key role of this housekeeping process. Importantly, genetic (e.g., Atg8a overexpression in either neurons or muscle) or pharmacological (e.g., feeding rapamycin or spermidine to animals) promotion of autophagy has been successfully used to extend lifespan in Drosophila, suggesting that this intracellular degradation pathway can rejuvenate cells and organisms. In this review, we highlight key discoveries and recent progress in understanding the relationship of autophagy and aging in Drosophila.

Lysosomal-dependent self-degradative (autophagic) mechanisms are essential for the maintenance of normal homeostasis in all eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified, based on how the cellular self material can incorporate into the lysosomal lumen. Ubiquitination, a well-known and frequently occurred posttranslational modification has essential role in all cell biological processes, thus in autophagy too. The second most common type of polyubiquitin chain is the K63-linked polyubiquitin, which strongly connects to some self-degradative mechanisms in the cells. In this review, we discuss the role of this type of polyubiquitin pattern in numerous autophagic processes.

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. We have previously identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells (Lőrincz et al., 2016a). Here, we show that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Lt and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.