EDITOR'S CHOICE IN NEUROSCIENCE
There is very little about membrane vesicle fusion that Yale University biochemist James Rothman doesn’t know—he codiscovered SNAREs, the proteins that orchestrate the process. But one unanswered question in the field of membrane fusion has been what happens during the first milliseconds of synaptic transmission between neurons—when a vesicle full of neurotransmitters inside a neuron fuses to the cell membrane, opening a pore to release its contents into the synapse.
A fusion pore, the opening that occurs when a vesicle binds to a cell membrane, is present for just hundreds of microseconds, a thousand times shorter than the blink of an eye. Immediately after it opens, the pore rapidly expands as the vesicle membrane melts into the surrounding cell membrane. That quick transition has made it extremely difficult to study the pore, says Rothman. “We thought that if we could find a way to artificially stabilize the fusion pore, without interfering with its opening, we might be able to gain some new insights into neurotransmission,” he said.
To do so, Rothman’s group, together with Frédéric Pincet’s team at CNRS in Paris, France, created fusion pores in nanodiscs—circular discs of lipid bilayers, held together by scaffold proteins wrapped around each lipid disc like a belt. Because of the nanodiscs’ small size and rigid structure, a fusion pore can form but does not expand beyond 2 nm, essentially freezing the pore in place for analysis.
Rothman’s team added SNARE proteins, which initiate vesicle-membrane fusion, to the nanodiscs and exposed them to small vesicles embedded with different SNARE proteins, creating an artificial model of synaptic vesicle fusion. By varying the number of SNAREs in the nanodiscs, the team was able to determine that only one SNARE per disc is necessary to temporarily open a fusion pore; however, three or more are required to keep the pore open long enough for the neurotransmitter to be released through it.
“This further emphasizes the importance of these proteins in the process of membrane fusion,” says Thierry Galli, who studies membrane trafficking at the Institut Jacques Monod in Paris and was not involved in the research. It also marries two previously contradictory experiments about how many SNARE proteins are required to open a fusion pore, he adds. Two in vivo studies, conducted in 2001 and 2010, found that a minimum of three SNARE complexes are necessary for neurotransmitter release, but a 2010 in vitro analysis concluded that just one SNARE complex is sufficient for membrane fusion. Since then, scientists have debated over which minimum number of SNAREs is correct. “Now the field should be able to rest in peace,” says Rothman. “Everybody’s right!”
L. Shi et al., “SNARE proteins: one to fuse and three to keep the nascent fusion pore open,” Science, 335:1355-59, 2012.