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Cells constantly balance two important activities, breaking down unwanted material and releasing molecules into their surroundings. These processes may appear separate, but they share many of the same membranes and trafficking pathways inside the cell.
A research team the National Institute of Environmental Health Sciences, National Institutes of Health, in Durham, North Carolina, investigated how two autophagy proteins, ATG9A and ATG2A/B, influence the production and release of small extracellular vesicles.
Their findings show that these proteins help determine whether cellular material is directed toward degradation through autophagy or released from the cell through extracellular vesicles.
Connecting autophagy with extracellular vesicle secretion
Autophagy is a cellular recycling process. Cells surround damaged proteins, organelles, and other material with a membrane structure called an autophagosome, which eventually delivers its contents to lysosomes for degradation.
Extracellular vesicles, or EVs, follow a different route. These small membrane-bound particles are released from cells and can carry proteins, RNA, lipids, and other molecules to neighboring or distant cells.
The two pathways intersect through structures called amphisomes. These form when autophagosomes fuse with endosomal compartments involved in extracellular vesicle production.
The researchers focused on ATG9A and ATG2A/B, proteins that supply and transfer lipids needed to build autophagosome membranes.
Autophagy-deficient cells show alterations in gene expression for key lipid processing pathways
a Heat map showing the differential expression of fatty acid metabolic genes in ATG KO cells compared to WT HeLa cells (n = 3 independent biological replicates). Log2-scaled abundances were median-centered within each sample by subtracting the sample-specific median log2 abundance, thereby aligning sample medians to zero while preserving within-sample rank structure. Data shown as log2 fold changes represented by its normalized centered expression intensity on a −8 to +8 scale from blue (decreased level) to red (increased level). b Volcano plot depicting differentially expressed genes described in (a), showing the significant upregulated and downregulated genes in ATG KO cells compared to WT. y-axis depicts -log10(adjusted p-value), while x-axis depicts log2(fold change). Differential expression was assessed using two-sided empirical Bayes moderated t-tests implemented in limma (version 3.56.2). Multiple-testing correction was performed using the Benjamini-Hochberg method, with adjusted P-values reported as false discovery rates. c Schematic representation of the main biochemical reactions with key genes involved in fatty acid processing, ceramide and sphingomyelin regulation, and phospholipid balance in the cell. Lys: lysosome; PM: plasma membrane; ER: endoplasmic reticulum. d, e Expression level of selected fatty acid metabolic genes by qRT-PCR in WT and ATG KO cells. Data are shown as mean ± SEM (n = 3 independent biological replicates). Statistical significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test, and only significant results are shown: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. f, g A model summarizing a putative mechanism of sEV biogenesis and the role of amphisome-mediated secretion in WT cells (f). The scheme also highlights the increase in amphisome formation and secretion in the absence of functional lysosomes. g, The increase in sEV biogenesis in the absence of lipid -handling proteins ATG9A and ATG2 due to impaired flux of lipids from the ER (LD accumulation), depletion of amphisomes, and an increase in CD63-positive ILVs in MVEs.
Removing ATG2 or ATG9 increases small EV secretion
When the researchers deleted ATG9A or ATG2A/B from cells, normal autophagosome formation was disrupted. At the same time, the cells released substantially more small extracellular vesicles.
These vesicles were enriched in CD63, a protein commonly associated with small EVs and exosomes, and were generally smaller than those released by normal cells.
The researchers also observed an accumulation of intraluminal vesicles inside multivesicular endosomes, the cellular compartments that can eventually fuse with the plasma membrane and release their internal vesicles as exosomes.
The increased secretion depended on RAB27A, a protein involved in moving multivesicular endosomes toward the cell surface for extracellular vesicle release.
Together, these findings indicate that disrupting autophagy can redirect cellular membranes toward EV production and secretion.
Autophagy also influences what EVs carry
The researchers found that ATG2 and ATG9 affected more than the number of extracellular vesicles released. They also influenced the molecular cargo packaged inside them.
When lysosomal degradation was inhibited in normal cells, small EVs contained LC3 and several proteins normally involved in recognizing and transporting autophagy cargo.
In cells lacking ATG9A or ATG2A/B, however, these proteins were largely absent from the extracellular vesicles even though overall EV secretion increased.
This suggests that the missing autophagy proteins disrupted amphisome formation and prevented certain autophagy-related materials from entering the EV secretion pathway.
Proteomic analysis supported this conclusion. Extracellular vesicles from cells lacking ATG2 or ATG9 contained fewer autophagy receptors and proteins associated with ferritinophagy, a process that helps cells recycle iron-storage proteins. At the same time, the vesicles contained more RNA-binding proteins and proteins involved in endosomal trafficking.
Lipids help determine whether cells degrade or secrete material
One of the most important findings involved cellular lipids.
ATG9A and ATG2 proteins help manage the movement of lipids needed to build cellular membranes. Removing these proteins caused major changes in the cellular lipidome, the complete collection of lipids within a cell.
Cells lacking ATG9A or ATG2A/B accumulated ceramides and neutral lipids and showed changes in the balance of phospholipids. The expression of enzymes involved in lipid metabolism was also altered.
These changes appear to contribute directly to increased extracellular vesicle secretion.
When the researchers inhibited neutral sphingomyelinase, an enzyme involved in producing ceramide and promoting EV formation, small EV secretion returned closer to normal levels. This finding connects changes in lipid metabolism with the increased vesicle release caused by loss of ATG2 or ATG9.
Choosing between degradation and secretion
Cells have limited membrane resources, and many internal trafficking pathways rely on the same pools of lipids and membrane structures.
The results suggest that ATG9A and ATG2A/B act as molecular gatekeepers that help decide how these resources are distributed.
When these proteins function normally, they support autophagosome and amphisome formation, allowing material to move through degradative autophagy or, under certain conditions, into extracellular vesicles.
When ATG2 or ATG9 is missing, autophagosome formation is blocked and lipid metabolism changes. More membrane material is then directed toward multivesicular endosomes and small EV secretion.
This connection provides a clearer picture of how autophagy, lipid metabolism, and extracellular vesicle biology are linked. Because both autophagy and EV signaling are involved in cancer, neurodegenerative diseases, immune responses, and other conditions, understanding how cells balance these pathways could help researchers better interpret changes in extracellular vesicle production during disease.
