A novel Epac-Rab3 signalling pathway is essential for sperm exocytosis

BUSTOS M; BRANHAM M T; RODRIGUEZ JF; RUETE C; ZARELLI V; TOMES CN

Villa Carlos Paz, Córdoba

Congreso; XLIV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

The single secretory granule present in sperm undergoes regulated exocytosis (the acrosome reaction, AR) near the time it encounters the egg’s zona pellucida at fertilization or following pharmacological stimulation in vitro. Our laboratory has identified a number of proteins required for sperm exocytosis; our current goal is to identify new components of the fusion machinery and reveal the sequence in which they act. To this end, we promote or perturb specific interactions between exogenous (e.g., neurotoxins, antibodies, recombinant proteins, second messengers, etc) and endogenous elements of the fusion machinery at different stages of the AR, and analyze the results by means of functional (exocytosis) and biochemical (pull down, subcellular fractionation, partition, Western blot, etc) assays. We show here that the AR is a bifurcated pathway consisting of two limbs, one driven by Rab3A, PTP1B, NSF, and Munc18-1, which arrives at the SNARE-mediated docking of the acrosome to the plasma membrane. The other is governed by Epac, Rap, and a PLC, and elicits an efflux of calcium from the acrosome. Rab3A is activated and targeted to membranes upon treatment with AR inducers. Subsequently, PTP1B dephosphorylates NSF, allowing it to disassemble unproductive SNARE complexes. Munc18-1 binds monomeric syntaxin and helps to nucleate SNAREs in fusion-competent complexes