| The process of exocytosis mediates intercellular communication. Previous biochemical and functional studies of neurons identified the proteins VAMP, syntaxin, and SNAP-25, as critical to exocytosis. Collectively known as SNAREs, these proteins are the in vitro targets of neurotoxic proteases Clostridia botulinum types A-F. Botulinum toxins were applied to the frog neuromuscular junction to reveal properties of SNARE protein function in semi-intact tissue, in situ.; Electrophysiological and immunohistochemical experiments demonstrated that botulinum toxin types C, D, and E completely block neurotransmitter release and reduce the immunoreactivity of their targets: syntaxin, VAMP, and SNAP-25, respectively. Botulinum type B had no effect on peripheral release, but it cleaved its target VAMP in the brain and paralyzed frogs. Botulinum type A blocked transmitter release partially, and reduced the C-terminus, but not the N-terminus, immunoreactivity of its target SNAP-25. Therefore, the incomplete blockade reflecting a partial disruption of protein function corresponds to a partial change in protein conformation.; Next, I used immunohistochemistry to examine the effects of botulinum toxins on protein-protein interactions among SNAREs. Cleavage of VAMP or syntaxin influenced SNAP-25 immunoreactivity, indicating that these binary interactions (VAMP-SNAP-25 and syntaxin-SNAP-25) occur inside cells. The combined treatment with botulinum C and a lipid-modifying reagent revealed that SNAP-25 liberated from interaction with the plasma membrane remains intact due to its association with syntaxin.; Electron microscopic study of nerve terminals where each of the three SNARE protein types had been cleaved, and in which the cytoskeleton had been disrupted by latrunculin A, revealed that the SNARE proteins participate in tethering synaptic vesicle to plasma membrane. Furthermore, the presence of tethered vesicles is required to maintain the electron-dense thickening characterizing neurotransmitter release sites.; In summary, immunohistochemistry can be employed as a sensitive tool to examine protein-protein interaction in situ, and predictions about the role of proteins based on in vitro studies provide an important starting point for elucidating their function in the complex milieu inside intact cells. |
