The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolate EVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C. elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their .

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolate EVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C.elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on a scale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system. BACKGROUNDThe cellular secretion of small membrane-bound extracellular vesicles (EVs) into the external environment is an ancient capacity conserved throughout evolution 1,2,3 . EVs range in size from 30-1000 nm in diameter and can be internalized into recipient cells via endocytosis or membrane fusion. There is growing recognition that EVs may play important roles in facilitating intercellular communication through transferring protein, lipid, and genetic cargos 4,5 . The content of EVs are influenced by the physiological state of the cells and are thought to play critical roles in diverse cellular processes as well as multiple types of pathological conditions including cancer, immunity, and neurodegenerative diseases.Many studies have characterized the composition of mammalian EVs. Such EVs are highly enriched in the lipid raft species cholesterol, and sphingomyelin, and in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored (GPI) proteins 6,7,8 . The two main types of EVs studied so far are exosomes, which release from the endosomal network and microvesicles which bud directly from the plasma membrane 9,10 . Mammalian exosomes commonly contain membrane proteins such as CD9, CD63, and CD81, as well as lysosomal and endosomal-marking proteins, and various amounts of extracellular matrix proteins while are largely free of nuclear proteins 11 . Microvesicles have less defined protein markers but may contain proteins of mitochondria and endoplasmic reticulum origin 11 . However, the methods utilized to purify EVs do not separate these types of vesicles, so it is currently unclear how to definitively distinguish the cargos from different types of EVs 12 . EVs from diverse species, including humans, are known to carry RNA ...

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

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The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition. Video LinkThe video component of this article can be found at https://www.jove.com/video/60596/ . Small EVs (<200 nm) are increasingly studied because of their implication in pathological processes, including metabolic disorders, cancer, cardiovascular disease, and neurodegenerative diseases 3,4,5 . These pathologies have also been shown to influence the protein and genetic composition of EVs cargos of small EVs. Therefore, biomarker signatures of the pathology are increasingly being uncovered through EV cargo discovery methods such as LC-MS-MS and RNAseq 6,7,8,9 .C. elegans has been a useful invertebrate model for identifying evolutionarily conserved EV signaling pathways. For instance, a C. elegans flippase was first shown to induce the EV biogenesis in C. elegans embryos, and the human homolog was shown to influence EV release in human cells 10,11 . C. elegans EVs were reported to carry Hedgehog signals necessary for cuticle development. The delivery of Hedgehog and other morphogens was shown to play a major developmental role of EVs, and it is conserved in zebrafish, mice, and humans 12,13,14,15 . C. elegans is well suited for EV biomarker discovery because it secretes EVs outside of its body that function in animalto-animal communication 16,17 (Figure 1A). However, the methodology established through a previous study cannot be used because the nematodes' E. coli food source also secretes EVs 18

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