Tetanus toxin (TNT) causes spastic paralysis by blocking synaptic neurotransmission through proteolysis of the v-SNARE protein synaptobrevin (VAMP2). The proteolytic active light chain of TNT recognizes three regions in neuronal synaptobrevin (n-syb): a cleavage region (residues 70-78) that surrounds the scissile bond and two regions that contribute to high-affinity binding to n-syb that are located N- and C-terminal of the cleavage site (Sikorra et al. 2008). Electrophysiological recordings at the Calyx of Held revealed that TNT leads to a decrease of evoked vesicle release, loss of the fast release component and to slowed recovery of the fast component (Sakaba et al. 2005). These kinetic changes can be interpreted as a TNT mediated block of vesicle release sites. If TNT mediated proteolysis of n-syb leads to cloging up of active zones by fusion-incompetent vesicles, this may be detectable in the reorganization of filamentous active zone components using super-resolution light microscopy (van de Linde et al. 2011). To address this hypothesis, TNT was conditionally expressed in Drosophila melanogaster neurons in a spatial and temporally restricted manner. This allows to target a specific set of neurons and to control for developmental effects. Assessing larval locomotion, we found that induction of TNT expression for 1200 min is sufficient to alter behaviour. After TNT expression, the organization of the filamentous active zone component Bruchpilot (Brp; Wagh et al. 2006; Kittel et al. 2006), was evaluated immunocytochemically using a monoclonal antibody, BrpNC82, directed against a C-terminal epitope of Brp (Fouquet et al. 2009).
Sikorra S, Henke T, Galli T, Binz T. (2008). Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins. J Biol Chem. 283:21145-52
Sakaba T, Stein A, Jahn R, Neher E. (2005). Distinct kinetic changes in neurotransmitter release after SNARE protein cleavage. Science. 309:491-4
van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M. (2011). Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc. 6:991-1009
Wagh DA, Rasse TM, Asan E, Hofbauer A, Schwenkert I, Dürrbeck H, Buchner S, Dabauvalle MC, Schmidt M, Qin G, Wichmann C, Kittel R, Sigrist SJ, Buchner E. (2006). Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron. 49:833-44
Kittel RJ, Wichmann C, Rasse TM, Fouquet W, Schmidt M, Schmid A, Wagh DA, Pawlu C, Kellner RR, Willig KI, Hell SW, Buchner E, Heckmann M, Sigrist SJ. (2006). Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science. 312:1051-4
Fouquet W, Owald D, Wichmann C, Mertel S, Depner H, Dyba M, Hallermann S, Kittel RJ, Eimer S, Sigrist SJ. (2009). Maturation of active zone assembly by Drosophila Bruchpilot. J Cell Biol. 186:129-45