CONICET | Buscador de Institutos y Recursos Humanos

The two secretory Rabs, Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single, large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization and upon exposing sperm to inducers. Sperm exocytosis utilizes the same fusion machinery as neurons and neuroendocrine cells. Here, we show for the first time that Rab27 is required for the acrosome reaction as is Rab3. Inducers were unable to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with anti-Rab27 antibodies or with Slac2-b, an effector that specifically binds Rab27-GTP. Pull down assays showed a substantial proportion of sperm Rab27 in its GTP-bound form; the levels increased even further upon initiation of exocytosis. Likewise, challenging with acrosome reaction triggers augmented the magnitude of GTP-bound Rab3A. We describe here a new fluorescence microscopy-based method suitable to determine the subcellular localization of GTP-bound Rabs. By means of this technology we detected endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Treatment with calcium caused a dramatic increase in the population of cells exhibiting GTP-bound Rabs. Interestingly, the introduction of recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells depicting GTP-bound Rab3A. In the converse condition, recombinant, persistently activated Rab3A did not modify the percentage of Rab27-GTP-containing cells. Results from functional assays suggest that Rab27 is required earlier than Rab3A during calcium-triggered sperm exocytosis. The novelty of our findings is two fold: we are the first to show simultaneously the GTP status and membrane targeting of endogenous Rab27 and Rab3 and to establish that these Rabs act in a sequence during dense-core granule exocytosis.