How SNARE proteins drive fertilization

Researchers report on full picture of acrosomal exocytosis in PLoS Biology

By | September 7, 2005

Researchers describe, for the first time, the full molecular cascade that comprises acrosomal exocytosis, an essential step in fertilization, in this week's PLoS Biology.

"Exocytosis has been analyzed before, but the different steps were reconstructed from experiments done in different systems, including yeast and neurons," coauthor Luis Mayorga of the University of Cuyo, in Argentina, told The Scientist. "Now we have a complete picture of the process of membrane fusion occurring in one single system, from beginning to end. We expect our results to provide tools to better understand membrane fusion and control the process of fertilization."

Acrosomal exocytosis involves the fusion of acrosomal and sperm membranes, and the synchronized opening of hundreds of fusion pores between those membranes, explained Mayorga. As a result, enzymes contained in the acrosomal vesicle exit the cell, enabling the spermatozoon to penetrate the egg.

In the paper, a team led by Mayorga and Claudia Tomes use a functional assay and immunofluorescence techniques, as well as neurotoxins and a photosensitive calcium chelator, to unravel the sequence of events that comprise acrosomal exocytosis. They focused on the dynamics of SNARE protein complexes during membrane recognition and fusion.

The authors devised a model that starts with the resting sperm. At this stage, SNAREs on the acrosomal and sperm membranes are independently assembled in tight cis complexes, a fact revealed by their resistance to neurotoxins. When a spermatozoon reaches an egg, said Mayorga, calcium enters the sperm's cytoplasm and activates Rab3A, a GTP binding protein that triggers the disassembly of the cis SNARE complexes through an unknown process, leaving them toxin-sensitive. The chaperone NSF/alpha-SNAP is essential for this step of the process that renders monomeric proteins.

"Now, SNAREs in one membrane can interact with SNAREs in the opposite membrane, and form loose trans complexes," Mayorga told The Scientist. Finally, when calcium is released from the acrosome vesicle, it triggers the full assembly of the SNARE complex with synaptotagmin, a calcium sensor protein that is the last factor in the model. This time, SNAREs assemble in a trans configuration. According to the model, the sequential assembly and disassembly of SNAREs drives acrosomal exocytosis.

"This is really, really nice work," said Frederick Hughson of Princeton University, who was not involved in the study. "The authors were able to put together the sequence of events leading up to membrane fusion in a way that hasn't been done in other systems before."

The secret seems to lie in the system itself. "Sperm is an ideal system for studying acrosomal exocytosis," explained Mayorga. "It's an all-or-nothing process that happens once in the lifetime of the spermatozoon, and it's finely synchronized. In addition, sperm exocytosis is slower than in other systems such as neurons, which is a great advantage for our studies," he added.

Hughson was pleased with the tools developed by the authors. "They are able to circumvent the need for endogenous Rab3A by adding activated Rab3A, and they can also control the calcium influx: they deplete it using an inhibitor and then release calcium in an activated form." Hughson also remarked on the use of neurotoxins to determine the state of the SNARE complex. "To me, what's most impressive is that they gathered all these tools to have control over each step of the process, so they could start asking what's the order. It's a clever paper."

Mayorga expects this work to have an impact on the study of the fertilization process. "Many studies in the sperm physiology field are pharmacological or phenomological but don't analyze the system at the molecular level. Our study might help to change that."

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