Phospholipase D1 localizes to mobile, docking and fusing secretory vesicles and multivesicular endosomes.

Abstract

Phospholipase D1 (PLD1) is a protein required for stimulated exocytosis. It is hypothesized to stabilize the fusion pore by producing phosphatidic acid, an inverse conical lipid that supports negative curvature. However, temporal evidence of PLD1 during exocytosis is currently lacking. With a GFP-labeled PLD1, we use TIRF and confocal microscopy to observe localization of PLD1 under basal, stimulating and inhibitory conditions. The localization of PLD1 to sites of membrane fusion was measured for both stimulated fusion in PC12 cells and constitutive fusion of multivesicular endosomes (MVEs) in A549 cells. In PC12 cells, PLD1 colocalizes with secretory vesicles (SVs) containing VAMP2 or NPY, and Syntaxin-1 clusters on the plasma membrane. Additionally, in A549 cells, PLD1 colocalizes with CD63, a marker for late endosomes and MVEs. PLD1 has been shown to colocalize with late endosomes and lysosomes, so we hypothesize that PLD1 may be involved in the formation of MVEs, exocytosis of MVEs, or both. With pHmScarlet-labeled VAMP2 and CD63, sites of fusion were identified by an increase in fluorescence due to dequenching of the fluorophore. These sites are located using an automated approach and the corresponding location in the PLD1 channel is then used to identify whether GFP-PLD1 is present. In PC12 cells, secretion is stimulated, however in A549 cells, secretion happens slowly and randomly in time. In both cell lines, drugs were used to inhibit both PLD1 and PLD2, or PLD1 specifically to observe effects on exocytosis and localization. Our data show that PLD1 is carried on moving SVs and MVEs prior to fusion or docking, suggesting PLD1 is trafficked to sites of fusion before SVs or MVEs fuse with the plasma membrane.