Neurons communicate by releasing the contents of vesicles full of neurotransmitters into the space between one neuron and the next. Details of this fraction-of-a-millisecond step are now clearer, thanks to a powerful tool developed by researchers led by HHMI investigator Axel T. Brunger.
As an electrical impulse travels through a nerve cell, channels in the cell membrane open, allowing calcium ions to rush in. Researchers knew this flash flood of calcium somehow signaled vesicles to fuse with the neuron’s membrane and spill their contents into the synaptic cleft—the gap between neurons. But previous assays to illuminate the process told a misleading story. In those systems, membrane lipids were tagged with fluorescing dyes, and lipid mixing (from two distinct membranes) was used as an analog of fusion. The new assay, reported July 19, 2011, in the Proceedings of the National Academy of Sciences, demonstrates that while lipid mixing is necessary for fusion, it’s far from the whole picture.
“Those assays showed only part of the process, and they kept the calcium concentration constant,” says Brunger. “So they didn’t show the process from start to finish.”
To mimic fusion and neurotransmitter release, Brunger and colleagues at Stanford University and the University of California, Berkeley, tagged donor vesicle contents with green self-quenching dye and its membrane lipids with red self-quenching dye. Self-quenching dyes are more intense at lower concentrations; at higher concentrations, the fluorescence fades as the dye turns itself off.
The researchers mixed these tagged donor vesicles with acceptor vesicles tethered to a glass surface and then exposed them to stepwise increases in calcium. Using microscopy, they were able to distinguish between the vesicles docking closely (red lipid mixing) versus merging completely (both green and red lipid mixing) by monitoring dye intensity. As the vesicles fused, the concentration of tagged content decreased, causing brighter fluorescence.
Using the new method, Brunger’s team showed that a set of proteins called SNAREs initiates membrane fusion and that the protein synaptotagmin-1 lowers the barriers to fusion upon calcium influx while the protein complexin increases fusion efficiency. The team plans to continue to examine the roles of other proteins in the fusion process.