[
International Worm Meeting,
2013]
At the active zone (AZ) of chemical synapses depolarization of presynaptic terminals triggers the opening of Voltage-Dependent Calcium Channels (VDCCs). The rise of calcium concentration that can trigger the fusion of synaptic vesicles (SVs) is restricted to nanodomains within less than a 100 nm from the VDCCs. Therefore, both the number and the distribution of VDCCs are crucial parameters that control synaptic efficiency. Using electron tomography we previously demonstrated that SVs docked at the plasma membrane are retained in close contact with the dense projection of C. elegans neuromuscular junctions. However, the relative distribution of docked SVs and VDCCs at these synapses is still enigmatic. To localize VDCCs in 3D with a nanometer resolution we are implementing a novel approach combining in vivo labeling of VDCCs via genetically-encoded extracellular epitope tags and electron tomography. In a first step we genetically introduced a GFP tag at the extracellular N-terminus of UNC-36, the a-2-d subunit of VDCCs, without impairing channel function. We then use an improved generation of multishell quantum dot (QD) as both fluorescent and electron dense probe. QDs are coupled to anti-GFP antibodies and injected in the pseudoceolomic cavity of C. elegans to label the VDCCs present at NMJs. In order to have access to near-to-native ultrastructure, worms are instantly immobilized by high pressure freezing and subsequently freeze substituted. Worms are imaged with light microscopy just before freezing, which provides a means to later image region of interest by transmission electron microscopy and visualize QD distribution. Using electron tomography we should eventually get access to the tridimensional distribution of VDCCs at the active zone with a few nanometer resolution. Current progress will be presented at the meeting.