title: GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis.
abstract: Exocytosis of mammalian sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and active Rab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino-terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
authors: Instituto de Histología y Embriología, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
authors: Matías A
authors: MA
authors: Bustos
authors: Instituto de Histología y Embriología, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina Present address: Department of Biophysics, UT Southwestern Medical Center, Dallas, TX, USA.
authors: Carlos M
authors: CM
authors: Roggero
authors: Instituto de Histología y Embriología, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina Present address: Servicio de Patología, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina.
authors: Paola X
authors: PX
authors: De la Iglesia
authors: Instituto de Histología y Embriología, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
authors: Luis S
authors: LS
authors: Mayorga
authors: Instituto de Histología y Embriología, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina ctomes@fcm.uncu.edu.ar.
authors: Claudia N
authors: CN
authors: Tomes
mesh_terms: D000177:Acrosome / Q000378:metabolism; D000328:Adult; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008297:Male; D008561:Membrane Fusion / Q000502:physiology; D008969:Molecular Sequence Data; D011944:Receptors, Androgen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000378:metabolism*; D055815:Young Adult; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020826:rab3A GTP-Binding Protein / Q000378:metabolism*
chemical_list: C410798:AR protein, human; C484191:RAB22A protein, human; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D020691:rab GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein; D002118:Calcium
keywords: GTP hydrolysis; Rab3 cycle; acrosome; calcium; exocytosis; sperm