Epac, Rap and Rab3 act in concert to mobilize calcium from sperm's acrosome during exocytosis

RUETE MC; LUCCHESI O; BUSTOS MA; TOMES CN

CELL COMMUNICATION AND SIGNALING

BIOMED CENTRAL LTD

Lugar: Londres; Año: 2014 vol. 12 p. 1 - 16

1478-811X

Background: Exocytosis of sperm?s single secretory granule or acrosome (acrosome reaction, AR) is a highly regulatedevent essential for fertilization. The AR begins with an influx of calcium from the extracellular milieu and continues withthe synthesis of cAMP and the activation of its target Epac. The cascade bifurcates into a Rab3-GTP-driven limb thatassembles the fusion machinery and a Rap-GTP-driven limb that mobilizes internal calcium.Results: To understand the crosstalk between the two signaling cascades, we applied known AR inhibitors in threeexperimental approaches: reversible, stage-specific blockers in a functional assay, a far-immunofluorescence protocol todetect active Rab3 and Rap, and single cell-confocal microscopy to visualize fluctuations in internal calcium stores. Ourmodel system was human sperm with their plasma membrane permeabilized with streptolysin O and stimulated withexternal calcium. The inhibition caused by reagents that prevented the activation of Rap was reversed by mobilizingintracellular calcium pharmacologically, whereas that caused by AR inhibitors that impeded Rab3?s binding to GTP wasnot. Both limbs of the exocytotic cascade joined at or near the stage catalyzed by Rab3 in a unidirectional, hierarchicalconnection in which the intra-acrosomal calcium mobilization arm was subordinated to the fusion protein arm;somewhere after Rab3, the pathways became independent.Conclusions: We delineated the sequence of events that connect an external calcium signal to internal calciummobilization during exocytosis. We have taken advantage of the versatility of the sperm model to investigate howcAMP, calcium, and the proteinaceous fusion machinery coordinate to accomplish secretion. Because the requirementof calcium from two different sources is not unique to sperm and fusion proteins are highly conserved, our findingsmight contribute to elucidate mechanisms that operate in regulated exocytosis in other secretory cell types.