Sperm exocytosis requires cAMP, Epac, intracellular calcium and multiple protein-protein interactions

TOMES CN

Potrero de los Funes, San Luis

Congreso; 47 Reunión Anual Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2011

SAIB

Sperm exocytosis requires cAMP, Epac, intracellular calcium and multiple protein-protein interactions. Claudia N Tomes, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo. Email: ctomes@fcm.uncu.edu.arExocytosis consists of multiple stages that include tethering and docking of the vesicle to the plasma membrane, priming of the fusion machinery, and calcium-triggered opening of fusion pores. Our lab is interested in unveiling the molecular mechanisms that drive exocytosis in sperm, a model where signaling cascades are mostly unidirectional and irreversible. We have established a plasma membrane permeabilization protocol that grants access to intracellular compartments and permits monitoring of the secretory activity following sequential application of different molecular tools, a procedure that is not readily feasible in more complex exocytotic cells or in intact sperm. Thanks to this protocol, we have shown that human sperm exocytosis relies on the assembly of a proteinaceous fusion machinery that includes Rab3 and 27, α-SNAP, NSF, SNAREs, complexin, Munc18 and synaptotagmin VI as well as kinases and phosphatases. This exocytosis also requires cAMP and calcium (from the extracellular medium and from IP3-sensitive intracellular stores). The relevant cAMP target is Epac, a GEF for Rap. We have identified a novel connection between Epac and another small GTPase, Rab3. Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release