Distinct Roles of Phospholipase D Isoforms in Exosome Release

Abstract

Multivesicular endosome (MVE) fusion with the plasma membrane is a key step in the release of small extracellular vesicles (sEVs), including exosomes, which mediate intercellular communication and contribute to disease processes. Lipid signaling has been implicated in regulating membrane fusion, with phospholipase D (PLD) enzymes and their product phosphatidic acid (PA) proposed to promote exocytosis. However, the spatial and mechanistic roles of distinct PLD isoforms in MVE–plasma membrane fusion remain unclear. In this study, we investigated the roles of PLD1 and PLD2 in MVE–plasma membrane fusion in A549 lung epithelial cells, a well-established model with robust small extracellular vesicle secretion, using live-cell total internal reflection fluorescence (TIRF) microscopy combined with quantitative analysis of vesicle dynamics. Functional perturbations, including genetic knockdown and pharmacological inhibition, were used to define isoform-specific contributions. PLD1 and PLD2 regulate exosome release through distinct mechanisms. PLD1 associates with CD63-positive vesicles, with CD63 serving as a marker of multivesicular endosomes and exosomes and promotes fusion through local PA generation at docking and fusion sites, consistent with a role in establishing fusion competence. In contrast, PLD2 is required for efficient fusion and sEV secretion but does not accumulate at vesicle–plasma membrane docking or fusion sites. Consistent with this, prefusion PA levels at fusion sites are not reduced under PLD2 perturbation, and fusion probability following vesicle–membrane contact remains unchanged. Instead, PLD2 perturbation reduces vesicle availability at the plasma membrane, identifying vesicle availability as a key determinant of fusion output. In addition, PLD2 perturbation alters post-fusion CD63 decay dynamics, shifting cargo release toward faster-dispersing modes. Together, these findings support a model in which PLD1 acts locally at fusion sites through PA production, whereas PLD2 functions upstream to regulate vesicle availability and post-fusion cargo behavior. These findings establish a mechanistic distinction between PLD isoforms, in which membrane fusion and vesicle supply are governed by distinct regulatory layers.