Sequence of events leading to SNARE complex assembly during acrosomal exocytosis

TOMES, CLAUDIA N.; DE BLAS, GERARDO A.; ROGGERO, CARLOS M.; MAYORGA, LUIS S.

Tallinn, Estonia

Congreso; Meeting of International Research Scholars Howard Hughes Medical Institute.; 2004

Acrosomal exocytosis is a calcium-dependent secretory event resulting in the release of the acrosoma1 contents and the loss of the membranes surrounding the acrosome. By using a permeabilized sperm model, we have shown that SNARES (SNAP receptors) and SNARE-interacting proteins, such as NSF (N-ethlymaleimide-sensitive factor), alphaSNAP (soluble NSF attachment protein), and synaptotagmin VI, are necessary for this complex process. We have developed two strategies to study the sequence of steps governed by these proteins that leads to membrane fusion. First, we used the acetoxymethyl cster of 0-nitrophenyl EGTA (EGTA-NP), a photosensitive reagent that accumulates in the acrosome in permeabilized spermatozoa and inhibits exocytosis by chelating calcium in the dark. Upon illumination, EGTA-NP is inactivated, the acrosomal calcium pool is replenished, and exocytosis can proceed. By using this inhibitor, we have evidence that NSF and alphaSNAP are required before and that SNAREs and synaptotagmin VI are required after the release of intra-acrosomal calcium. The second approach is based on the fact that neurotoxins are specific zinc-dependent proteases and SNARES are resistant to neurotoxins when assembled in stable complexes. By adding botuli´nu´m ´ neurotoxin E and suppressing its activity with the zinc chelator N,N,N´,Nr-tetrakis(2-pyridymethyl)ethylenediamine, we can assess whether SNAREs are engaged in stable complexes at different steps during the exocytotic process. The results indicate that SNARES are inaccessible to the toxin in resting sperm but become toxin-sensitive upon stimulation with calcium or Rab3A. Combinations of both methods show that SNARES remain toxin-sensitive until the release of intra-acrosomal calcium. These results suggest that SNAREs forming stable cis ccmplexes are disassembled by NSF and alphaSNAP upon Rab3A activation. Afterwards, the SNARE proteins assemble in loose trans complexes until the release of intra-acrosomal calcium activates synaptotagmin VI, which in turn promotes full assembly of the complexes and triggers membrane fusion.