Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNAmediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion. Phosphatidic acid (PA)2 is a pleiotropic bioactive lipid that has been proposed to activate selected enzymes (1), recruit proteins to membrane surfaces (2), and serve as a substrate for the formation of other signaling lipids (3). Most intriguingly, PA has also been shown to promote negative curvature in bi-layer membranes due to its small polar head-group in combination with two fatty-acyl side chains (4). The bulk of cellular PA is synthesized via two different acylation pathways, the glycerol 3-phosphate pathway and the dihydroxy acetone phosphate pathway, which are named according to their respective precursors. However, PA is also produced via hydrolysis of phosphatidylcholine by phospholipase D (PLD) (5) on a much faster time scale, and this latter source is thought to underlie the dynamic regulation of PA that allows it to function as a signaling lipid in agonist-stimulated cell biological responses such as secretion and changes in cellular morphology.In mammals, the classic PLD family is composed of a pair of membrane-associated proteins, PLD1 and PLD2. Both PLD isoforms require phosphatidylinositol 4,5-bisphosphate for their enzymatic activity. However, whereas PLD2 exhibits relatively high basal activity in isolation, full activation of PLD1 requires its stimulation by small GTPases of the ADP-ribosylation factor (ARF), Rho and Ral families, and protein kinase C (3, 6). PLD enzymes have been proposed to be involved in a number of cellular processes, including cell growth and survival, cell differentiation, and vesicular trafficking (3)....

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-andrun, cavicapture and full-collapse fusion. During kissand-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain.Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.