Synapses are compartmentalized organelles containing thousands of proteins. Their precise localization at both pre- and post- synaptic elements is essential for a correct processing and transfer of information between excitable cells. To decipher cellular and molecular mechanisms that regulate and maintain neurotransmitter receptors at synapses we are using C. elegans neuromuscular junction (NMJ) as a model of synapse. In particular, we are interested in a novel mechanism of clustering acetylcholine and GABAA receptors based on extracellular scaffolding proteins in the synaptic cleft. The transparency and simple morphology of C. elegans combined with the ease of genomic engineering to tag endogenous proteins, provide a means to address the localization and organization of these extracellular scaffolding proteins.
We used 2D and 3D direct Stochastic Optical Reconstruction Microscopy (dSTORM) on immuno-stained worms and we found that acetylcholine receptor (AChR) clusters are close but not in perfect register with presynaptic boutons. We then built a knock-in strain expressing AChR tagged with the photoswitchable protein Eos and used PALM to analyze AChR content at individual synapses. We found that the number of AChRs is variable depending on synaptic size but the density or receptors is remarkably constant. We are now mapping protein distribution on synaptic ultrastructure by combining the tomo-array technique with STORM/PALM imaging.
To cite this abstract:Camilla Luccardini, Manon Perret, Sebastian Markert, Christian Stigloher, Béatrice Burdin, Jean-Louis Bessereau; Nanometric localization of synaptic proteins by combining single-molecule localization imaging with scanning electron microscopy. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/nanometric-localization-of-synaptic-proteins-by-combining-single-molecule-localization-imaging-with-scanning-electron-microscopy/. Accessed: October 31, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/nanometric-localization-of-synaptic-proteins-by-combining-single-molecule-localization-imaging-with-scanning-electron-microscopy/