title: Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis.
abstract: Two so-called "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 on exposure to inducers in vitro. Sperm exocytosis uses 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, as demonstrated by the inability of inducers 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 on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge 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 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.
authors: Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología, Facultad de Ciencias Médicas,  Universidad Nacional de Cuyo, 5500 Mendoza,  Argentina.
authors: Matías A
authors: MA
authors: Bustos
authors: Ornella
authors: O
authors: Lucchesi
authors: María C
authors: MC
authors: Ruete
authors: Luis S
authors: LS
authors: Mayorga
authors: Claudia N
authors: CN
authors: Tomes
mesh_terms: D000177:Acrosome / Q000502:physiology*; D020101:Acrosome Reaction / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001426:Bacterial Proteins; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D054876:Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism / Q000502:physiology*; D012685:Sepharose; D013301:Streptolysins; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; C514892:EXPH5 protein, human; D011994:Recombinant Proteins; D013301:Streptolysins; D000075745:rab27 GTP-Binding Proteins; C025964:streptolysin O; D006160:Guanosine Triphosphate; D012685:Sepharose; D005982:Glutathione Transferase; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins