Phosphatidylinositol 4,5-bisphosphate promotes membrane curvature and is a signaling hub in human sperm acrosome exocytosis

ALTAMIRANO, KARINA N; SUHAIMAN, LAILA; LUCCHESI, ORNELLA; RUETE, CELESTE; DEL POPOLO, MARIO; BELMONTE, SILVIA A.

Mendoza

Simposio; Second International Symposium on Traslational Medicine; 2019

Universidad de Freiburg (Alemania), FCM-FCEN- Universidad Nacional de Cuyo

PRIMER PREMIO A LA MEJOR COMUNICACION ORAL INVESTIGADOR JOVEN: KARINA N. ALTAMIRANO otorgado por la UNIVERSIDAD DE FREIBURG, ALEMANIA.The human sperm has a secretory vesicle that undergoes exocytosis when challenged with different stimuli known as acrosome reaction (AR). The exocytosis requires the fusion of the outer acrosome membrane (OAM) and plasma membrane in multiple points. Previously, we reported that diacylglycerol (DAG) stimulates AR by feeding into a PKC and PLD1-dependent positive loop that supplies PIP2. We hypothesize that PIP2 synthesis is required to produce DAG and IP3, and to induce a change in the OAM curvature. Coarse-grained Molecular Dynamics simulations are being performed in order to find a correlation between membrane curvature and local lipid composition. Also, electron microscopy experiments showed that elevated levels of PIP2 induces OAM invaginations, however this increase has no effect inducing acrosome swelling, AR nor membrane disruption. Nevertheless PIP2 must be synthesized and hydrolyzed for the progression of AR. A recent publication proposed the following signaling pathway: cAMP-Epac-Rap1-PLCε. Where PIP2 is hydrolized by PLC, generating IP3. The latter binds to IP3-sensitive channels releasing Ca2+ from the acrosome. Given that DAG induces AR in the absence of cAMP and ext[Ca2+], we wondered about the mechanism triggered by DAG. Far-IFI experiments showed that DAG activates Rap1 suggesting the existence of an exchange factor for this small GTPase. Then, we thought about a nucleotide exchange factor activated by DAG (RasGRP1), described to activate Rap1 in secretory cells. By WB and IFI we demonstrated the presence and localization of RasGRP1 in human sperm. Further, we tested RasGRP1 in exocytosis assays. We observed that it triggered the AR in a dose-dependent manner. Our findings highlight the dual role of PIP2 in exocytosis: controlling membrane curvature and constituting a hub in the signaling pathway leading to exocytosis. We present direct evidence showing the presence and function of RasGRP1 which adds an alternative pathway to exocytosis.