The structure of active zones - the site of vesicular acetylcholine release at neuromuscular junctions is a highly organized, complex meshwork of proteins. Electron microscopy has revealed many details in the nanoscopic range but has also raised numerous questions concerning the molecular composition and precise arrangement of proteins (Harlow et al., 2001; Nagwaney et al., 2009). STED-microscopy allowed resolving active zone proteins with ~ 80 nm resolution (Kittel et al., 2006; Hallermann et al., 2010). Here we provide first insights into the structure of active zones with lateral resolution of ~ 20 nm using direct stochastic optical reconstruction microscopy (dSTORM) (Heilemann et al., 2008; van de Linde et al. 2011). We used the levator auris muscle of 5 to 8 week old C57BL/6 mice and stained nicotinic acetylcholine receptors with Alexa Fluor 647 labeled bungarotoxin. We are able to resolve postsynaptic junctional folds in whole mounts and cryosections that could so far not be detected by light microscopy due to the diffraction limit. Using the postsynaptic folds as a structural reference we explore the localization of active zone proteins such as bassoon, piccolo & dynamin using commercial primary antibodies and customized secondary antibodies labeled with photoswitchable fluorophores.

References

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Supported by DFG grants to MH (HE 2621/4-2 & B27/SFB 581)