Arf6 promotes membrane fusión by generating PIP2 and PA during acrosomal exocytosis

PELLETÁN, LE; MAYORGA, L.S.; BELMONTE, SA

San Miguel de Tucumán, Tucumán, Argentina

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2009

SAIB

During sperm acrosome reaction (AR) the plasma and the outer acrosomal membrane fuse at multiple points releasing hybrid vesicles and exposing the inner acrosomal membrane. AR is a special type of Ca2+-regulated exocytosis. Our previous results suggested that AR needs phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) after the opening of SOC channels. ADP-ribosylation factors (Arfs) are a family of monomeric GTP-binding proteins. In chromaffin cells, Arf6 activates both PI4P5Kinase and PLD1; both enzymes are thought to be coupled during signaling to support simultaneous increases in PIP2 and PA. We analyzed if this small GTPase was involved in AR regulation. Western blot and indirect immunofluorescence demonstrated that Arf6 was present in human spermatozoa and localizes to the acrosomal region. Functional assays using the permeabilized sperm model demonstrated that myristoylated and GTPgammaS loaded-Arf6 triggered AR and that the GTPase is absolutely required for calcium-regulated membrane fusion. Furthermore, we defined that Arf6-induced membrane fusion requires PIP2, PLD1 activity and consequently PA synthesis. By thin layer chromatography we demonstrated that Arf6-GTPgammaS increased PIP2 concentration. We propose a model where Arf6 regulates PLD1 and activates a kinase to maintain a pool of PIP2 in spermatozoa necessary for IP3-dependent acrosomal calcium release