Phospholipase D1-generated phosphatidic acid modulates secretory granule trafficking from biogenesis to compensatory endocytosis in neuroendocrine cells

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis. Despite the fact that lipids represent the majority of membrane constituents, little is known about their contribution to these processes. Using a combination of electrochemical measurement of single chromaffin cell catecholamine secretion and electron microscopy of roof-top membrane sheets associated with genetic, silencing and pharmacological approaches, we recently reported that diverse phosphatidic acid (PA) species regulates catecholamine release efficiency by controlling granule docking and fusion kinetics. The enzyme phospholipase D1 (PLD1), producing PA from phosphatidylcholine, seems to be the major responsible of these effects in this model. Here, we extended this work using spinning disk confocal microscopy showing that inhibition of PLD activity also reduced the velocity of granules undergoing a directed motion. Furthermore, a dopamine β-hydroxylase (DβH) internalization assay revealed that PA produced by PLD is required for an optimal recovery of vesicular membrane content by compensatory endocytosis. Thus, among numerous roles that have been attributed to PA our work gives core to the key regulatory role in secretion that has been proposed in different cell models. Few leads to explain these multiple functions of PA along the secretory pathway are discussed.