Targeting the epithelial SNARE machinery by bacterial neurotoxins

Clostridial neurotoxins are responsible for botulism and tetanus by cleaving the synaptic SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) synaptobrevin/VAMP2 (Vesicle-Associated Membrane Protein 2) and its partners SNAP-25 (synaptosome-associated protein of 25 kDa) and syntaxin 1. SNARE proteins mediate membrane fusion, a crucial step in intracellular trafficking. There are seven isotypes of botulinic neurotoxins with different target specificities and one tetanus neurotoxin (TeNT), which targets synaptobrevin. Regarding the high sequence similarities between synaptobrevin and its nonneuronal homolog cellubrevin/VAMP3, different groups developed the use of TeNT to study cellubrevin (Cb). Here, we show how we have introduced the light chain of the TeNT into nonneuronal cells and selected clones expressing this toxin by Western blotting and by immunofluorescence. We also present how we identified which cells express TeNT by searching for a soluble green fluorescent protein (GFP) pattern of expression corresponding to cleaved GFP-tagged cellubrevin in living GFP-cellubrevin and TeNT transfected cells.