Rab27 and Rab3 sequentially regulate the exocytosis of dense-core granule in human sperm

BUSTOS MA; LUCCHESI O; RUETE MC; MAYORGA LS; TOMES CN

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

NATL ACAD SCIENCES

Lugar: Washington DC, USA; Año: 2012 vol. 109 p. 2057 - 2066

0027-8424

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 or upon exposure to inducers in vitro. Sperm exocytosis utilizes the same fusion machinery as neurons and neuroendocrine cells with an additional requirement for active Rab3. Here, we show that Rab27 is also required for the acrosome reaction, since inducers were unable to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased upon initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method by which we can detect endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenging with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GEF activity. Our findings suggest that Rab27/Rab3A constitute a novel Rab GEF cascade, the first ever described in dense-core vesicle exocytosis.