ARF6 regulates the synthesis of fusogenic lipids for calcium-regulated exocytosis in neuroendocrine cells.
An important role for specific lipids in membrane fusion has recently emerged, but regulation of their biosynthesis remains poorly understood. Among fusogenic lipids, phosphatidic acid and phosphoinositol 4,5-bisphosphate (PIP(2)) have been proposed to act at various steps of neurotransmitter and hormone exocytosis. Using real time FRET (fluorescence resonance energy transfer) measurements, we show here that the GTPase ARF6, potentially involved in the synthesis of these lipids, is activated at the exocytotic sites in PC12 cells stimulated for secretion. Depletion of endogenous ARF6 by siRNA dramatically inhibited secretagogue-evoked exocytosis. ARF6-siRNA greatly reduced secretagogue-evoked phospholipase D (PLD) activation and phosphatidic acid formation at the plasma membrane and moderately reduced constitutive levels of PIP(2) present at the plasma membrane in resting cells. Expression of an ARF6 insensitive to short interference RNA (siRNA) fully rescued secretion in ARF6-depleted cells. However, a mutated ARF6 protein specifically impaired in its ability to stimulate PLD had no effect. Finally, we show that the ARF6-siRNA-mediated inhibition of exocytosis could be rescued by an exogenous addition of lysophosphatidylcholine, a lipid that favors negative curvature on the inner leaflet of the plasma membrane. Altogether these data indicate that ARF6 is a critical upstream signaling element in the activation of PLD necessary to produce the fusogenic lipids required for exocytosis.
Béglé
Aurélie
A
Département Neurotransmission et Sécrétion Neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives (UPR 3212), Centre National de la Recherche Scientifique et Université de Strasbourg, 5 rue Blaise Pascal, 67084 Strasbourg, France.
Tryoen-Tóth
Petra
P
de Barry
Jean
J
Bader
Marie-France
MF
Vitale
Nicolas
N
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D055099:Neuroendocrine Cells / Q000166:cytology / Q000378:metabolism*; D016716:PC12 Cells; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis*; D010739:Phospholipase D / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000089982:ADP-Ribosylation Factor 6; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D034741:RNA, Small Interfering; D010739:Phospholipase D; D020727:ADP-Ribosylation Factors; C000715349:Arf6 protein, rat; D002118:Calcium