Arf6 promotes membrane fusion by generating PIP and PA during acrosomal exocytosis.

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

Tucumán

Congreso; SAIB XLV; 2009

SAIB

During sperm acrosome reaction (AR) the plasma and the outeracrosomal membrane fuse at multiple points releasing hybridvesicles and exposing the inner acrosomal membrane. AR is aspecial type of Ca -regulated exocytosis. Our previous resultssuggested that AR needs phosphatidylinositol 4,5-bisphosphate(PIP ) and phosphatidic acid (PA) after the opening of SOC 2channels. ADP-ribosylation factors (Arfs) are a family ofmonomeric GTP-binding proteins. In chromaffin cells, Arf6activates both PI4P5Kinase and PLD1; both enzymes are thoughtto be coupled during signaling to support simultaneous increases inPIP and PA. We analyzed if this small GTPase was involved in AR 2regulation. Western blot and indirect immunofluorescencedemonstrated that Arf6 was present in human spermatozoa andlocalizes to the acrosomal region. Functional assays using thepermeabilized sperm model demonstrated that myristoylated andGTPgS loaded-Arf6 triggered AR and that the GTPase is absolutelyrequired for calcium-regulated membrane fusion. We defined thatArf6-induced membrane fusion requires PIP , PLD1 activity and 2consequently PA synthesis. By thin layer chromatography wedemonstrated that Arf6-GTPgS increased PIP concentration. We 2propose a model where Arf6 regulates PLD1 and activates a kinaseto maintain a pool of PIP in spermatozoa necessary for IP - 2 3dependent acrosomal calcium release.