info:eu-repo/semantics/article
GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis
Fecha
2014-08Registro en:
Bustos, Matias Alberto; Roggero Savoini, Carlos Marcelo; De la Iglesia, Paola X.; Mayorga, Luis Segundo; Tomes, Claudia Nora; GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis; Oxford University Press; Journal of Molecular Cell Biology; 6; 4; 8-2014; 286-298
1674-2788
CONICET Digital
CONICET
Autor
Bustos, Matias Alberto
Roggero Savoini, Carlos Marcelo
De la Iglesia, Paola X.
Mayorga, Luis Segundo
Tomes, Claudia Nora
Resumen
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.