IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Molecular mechanisms and signaling pathways during sperm's dense core granule exocytosis
Autor/es:
BUSTOS MA; LUCCHESI O; RUETE MC; MAYORGA LS; TOMES CN
Lugar:
San Carlos de Bariloche
Reunión:
Simposio; The Second South American Spring Symposium in Signal Transduction and Molecular Medicine; 2012
Institución organizadora:
SISTAM
Resumen:
Exocytosis of secretory vesicles consists of multiple stages that include their tethering and docking to the plasma membrane, priming of the fusion machinery and calcium-triggered opening of fusion pores through which the vesicular contents diffuse to the extracellular milieu. Our lab is interested in unveiling the molecular mechanisms that drive exocytosis in sperm, a model where signaling cascades are mostly unidirectional and irreversible. Like all exocytosis, that of sperm?s single dense core granule (the acrosome) is a multistage process consisting of the opening of ion ? mainly calcium ? channels, the activation of multiple signaling pathways, the orchestration of many protein-protein interactions to put the fusion machinery in place, etc. Morphologically, it relies on the swelling of the dense-core granule and a dramatic remodeling of the acrosomal membrane that lead to the latter contacting the plasma membrane at the points where they will eventually fuse. We have established a 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. Thanks to this protocol, we now know that human sperm exocytosis relies on the assembly of a highly conserved proteinaceous fusion machinery that includes α-SNAP, NSF, SNAREs, complexin, Munc18 and synaptotagmin as well as kinases and phosphatases, cAMP and calcium (from the extracellular medium and from IP3-sensitive intracellular stores); the relevant cAMP target is Epac, a GEF for Rap. This exocytosis also requires Rabs3 and 27, two ?secretory Rabs? that localize to vesicles and secretory granules in a variety of secretory cell types. We found that these secretory Rabs do not cycle constantly as described for families that drive intracellular membrane traffic but their activation state and/or localization are coupled to exocytotic stimuli. By means of a fluorescence microscopy- and protein-protein interaction-based protocol developed in our laboratory, we were able to determine the subcellular localization of GTP-bound Rabs and found that Rab3A-GTP and Rab27-GTP localize to the acrosomal region of the sperm head. More importantly, we learnt that active Rab27 increases the exchange of GDP for GTP on Rab3A. The molecular mechanism for this activation appears to be through a Rab GEF cascade, as active Rab27 recruits a Rab3 GEF from human sperm extracts. Therefore, we propose that Rab27 and Rab3A act sequentially during sperm dense core granule exocytosis, with Rab27 playing the earliest role.Our findings have important potential implications with regards to the field of sperm biology and of that of regulated exocytosis at large.