Arf6 promotes membrane fusion by generating phosphatidylinositol 4,5-bisphosphate and phosphatidic acid during acrosomal exocytosis

PELLETÁN LEONARDO; MAYORGA LUIS; BELMONTE SILVIA

Colby-Sawyer College, New London, NH, USA

Conferencia; Gordon Research Conference in Cell-Cell Fusion; 2009

GRC

Fertilization is the process in which the egg and the spermatozoon fuse and a zygote is formed. When a spermatozoon reaches the egg, it must undergo acrosomal exocytosis (AE) to get through the zona pellucida and fertilize the egg. AE is a special type of calcium-regulated membrane fusion. During AE the plasma and outer acrosomal membrane fuse at multiple points releasing hybrid vesicles and causing the exposure of the inner acrosomal membrane. Multiple signaling pathways lead to SNAREs assembly. This protein complex assembly provides the energy required to fuse membranes through the formation of high affinity, four-alpha-helix bundles. The role of lipids is less well understood, but recent findings indicate that the shape of the lipids, determined by the size of their head group, and their charge might be important for the fusion process. Phosphatidic acid (PA, the product of PLD activity) and PIP2 are known to be important for regulated exocytosis. Our laboratory have collected strong evidence suggesting that two principal pathways are activated during stimulation of AE, one leads to SNARE complex assembly while the other produces an IP3-dependent acrosomal calcium release necessary for membrane fusion. In permeabilized human spermatozoa, PIP2 and PLD1 activity are required during AE. Considering that PLD1 has been described as an Arf6 effector, we analyzed if this small GTPase was involved in AE regulation. By using Western blot and indirect immunofluorescence we demonstrated that Arf6 was present in human spermatozoa and localizes to the acrosomal region. Functional assays by using the permeabilized sperm model demonstrated that myristoylated and GTPgammaS loaded-Arf6 triggered AE and that this GTPase is absolutely required for calcium-regulated membrane fusion. Furthermore, we defined that Arf6-induced membrane fusion requires PIP2, PLD 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