| Description |
Synapses are the tiny specialized contact sites for neurotransmission, translating electrical activity of one cell to another. The rate limiting step of neurotransmission is calcium influx through voltage-gated calcium channels. Membrane depolarization causes these channels to open and calcium enters the cell, where it activates exocytosis of neighboring neurotransmitter-filled vesicles onto postsynaptic cells. The speed and reliability of neurotransmission depend on nanodomain coupling of synaptic vesicles to calcium channels. Previous studies of synaptic calcium domains have been limited by the micron-scale resolution of standard fluorescent tags, and redundancies from multiple representatives of each channel type. To determine the subsynaptic distribution of essential neurotransmission proteins, we used CRISPR/cas9 to insert geneticallyencoded, super-resolution tags suitable for imaging by single-molecule localization microscopy. C. elegans encodes only a single gene for each class of channel contributing synaptic calcium: CaV1/egl-19, CaV2/unc-2, and RyR/unc-68. To determine the consequence of calcium entry, mutant animals lacking these channels were assessed by genetic analysis, by electrophysiology, and by time-resolved electron microscopy. This approach revealed two distinct sites of calcium entry within a single synapse. CaV2 active zones contain scaffolds (RIMB-1/RIM-BP), adhesion molecules (NRX- 1/neurexin), and priming proteins (UNC-13/Munc13) and are required for exocytosis of vesicles at the center of synapses. CaV1 and RyR channels together trigger exocytosis of iv a distinct set of lateral vesicles. Lateral CaV1 sites contain LIN-7/VELI scaffolds, SYG- 1/KIRREL adhesion proteins, and the short isoform of unc-13. Consequently, central CaV2 sites and lateral CaV1 sites comprise two independent active zones. |
