Regulation of fusogenic lipid synthesis: implication in hormonal release, neurotransmission and phagocytosis – Fusogenic lipid

Vesicular trafficking and membrane fusion is the basis of a large range of cellular activities. SNARE proteins play fundamental roles in the molecular machinery that drive two membranes in close proximity. However the fusion process itself is still poorly understood and many questions concerning the underlying molecular mechanisms, their specificity and kinetics remain unanswered. We have recently demonstrated that regulation of the lipid composition at fusion sites by phospholipase D (PLD) is necessary to various fusogenic events. PLD hydrolyzes phosphatidylcholine into choline and phosphatidic acid (PA). PA has been proposed to serve as a protein attachment/activating site or to promote negative membrane curvature and membrane fusion. Our goal is to establish the dynamic of PA in the course of membrane fusion by studying its implications in two classical cellular processes involving fusion steps, namely regulated exocytosis and phagocytosis and to define the regulatory aspects that control PA synthesis. Our hypothesis is based on the observations that PLD activity facilitates exocytosis and phagocytosis whereas extinction of endogenous PLD strongly blocks these cellular functions. Moreover, PLD is under the control of several kinases and GTPases and is therefore an ideal candidate to integrate signaling pathways to the level of membrane fusion. Using neuronal and endocrine cell models to study regulated exocytosis and macrophages to study phagocytosis, we plan to follow three lines of investigations: 1) We propose to use PA-binding domains coupled to GFP to determine with high-speed acquisition microscopy the kinetics of PA-synthesis at 'active exocytotic and phagocytic spots'. Membrane fusion will be monitored by ultrastructural analysis, capacitance measurements to follow the incorporation of membrane into the plasma membrane and the distribution of PA, electrophysiological recordings and amperometry to measure the release of neurotransmitters and hormones. Ultimately, we plan to correlate the level of PLD-derived PA modified by PLD over-expression or extinction with the degree of membrane fusion. 2) We plan to define the lipidic nature of the membrane fusion site. For instance, what is the nature of the PA required to generate or to stabilize a membrane fusion site' In other words, what are the length of the fatty acyl chain and the saturation status of the PA synthesized at the membrane fusion sites' To address these questions we will affinity purify the PA-rich domain and perform mass spectrometry analysis with a particular focus on the lipids. Since we have shown that raft micro-domains form on the exocytotic sites, we will test the hypothesis that these domains may contribute to concentrate PA at the fusion site. 3) To understand how cells adapt membrane fusion to their functional specificities i.e. membrane fusion is a high-speed calcium-regulated process in some cell types while in others it is a long lasting spatially regulated event, we will investigate the specific roles of the various signaling pathways converging on PLD using dominant negative and constitutively active mutants, RNA interference strategies, genetic knockout and acute photoinactivation using CALI, with a particular focus on the V-ATPase-ARNO-ARF6 and RSK2 pathways. Hence, unraveling the regulatory pathway leading to the control of PLD-derived PA synthesis should provide novel therapeutics to correct pathologies resulting from perturbation of vesicular trafficking and membrane fusion events.