PIP 2 promotes membrane curvature and is a signaling hub in human sperm acrosome exocytosis

SUHAIMAN L; BELMONTE SA; LUCCHESI O; ALTAMIRANO KN; RUETE MC

Paraná

Congreso; LIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The human sperm has a secretory vesicle that undergoes exocytosis when challenged with different stimuliknown as acrosome reaction(AR),which requires the fusion of the outer acrosome membrane and plasma membrane. We reported that DAG stimulates AR by feeding into aPKC and PLD1-dependent positive loop that supplies PIP2. We hypothesize that PIP2 synthesis is required to produce DAG and IP3, and toinduce a change in the acrosomal membrane curvature. Our Molecular Dynamics simulations and TEM experiments demonstrated that PIP2increase induces the formation of deep acrosomal membrane invaginations; although it was not able to induce acrosome swelling, AR ormembrane disruption. However, for the AR to proceed, PIP2 needs to be hydrolyzed. Previous work led us to propose the following signalingpathway: cAMP?>Epac?>Rap1?>PLCε. The hydrolysis of PIP2 generates IP3, which binds IP3-sensitive channels and releases Ca2+ from theacrosome. We demonstrate the presence of a nucleotide exchange factor activated by DAG (RasGRP1), described to activate Rap1 in secretorycells, by WB and IFI. Also, we proved RasGRP1 ability to trigger AR in a dose-dependent manner. Furthermore, as shown by Far-IFI, DAGwas able to activate Rap1 indicating that it is involved in the pathway described previously. Our findings highlights the dual role of PIP2 inexocytosis and report a direct evidence of the presence and function of RasGRP1 in the signaling module cAMP?>Epac?>Rap1?>PLCε in AR