Neurotoxins as Tools in Dissecting the Exocytic Machinery - Fusion of Biological Membranes and Related Problems - page 52

toxification while introducing intracellularly. Such experiments were carried
out by mutating the toxins in the central histidine residues that are essen-
tial for the Zn 2+ -dependent catalysis by the toxin (as in Figure 3). While in
most studies such manipulation abolished the entire effect of the toxins
(Yamasaki et al., 1994c), in other cases a residual effect of the toxin was
remained. The latter suggests an additional independent mode of action for
the toxins, which is not attributed to their catalytic function (Ashton and
Dolly, 1997; Gobbi et al., 1996).

4. INVESTIGATING SECRETION WITH CLOSTRIDIAL
TOXINS-THE METHODOLOGIES

4.1. In vivo -Genetic Approach

To study the synaptic function at the level of the organism itself a trans-
genic Drosophila was prepared by introducing a gene encoding TeNT-LC
(Sweeney et al., 1995). Toxin expression in embryonic neurons removed any
detectable VAMP. The result was a complete elimination of evoked synap-
tic vesicle release. Interestingly, the effect was not observed on spontaneous
release. In this in vivo experiments at the organism level, only the neuronal
VAMP was cleaved by the toxin while the ubiquitous expressed protein
remained resistant to TeNT.

-Latrotoxin stimulatory effects on secretion
were not altered in TeNT expressing flies, while the release induced by
hyperosmolarity was affected. This set of experiments provides a new
tool to address fundamental questions on the molecular correspondence
between spontaneous and evoked release and on the mechanisms that are
activated by different mode of stimuli.
Another use of the clostridial toxins is in using organisms that were
genetically manipulated. Experiments were performed on Drosophila
(Littleton and Bellen, 1995), C. elegans (Nonet et al., 1993; Nonet et al.,
1998) or mice following knockout of individual SNARE or other protein
participating in neurotransmitter release (Broadie et al., 1995; Brose, 1998;
Littleton et al., 1998). The action of the different clostridial toxins in the
altered genetic background allows us to distinguish the roles of each
SNARE in the morphology and physiology of the synapse throughout the
complete life cycle of the organism.
Once the structure and the catalytic activity of the toxin were resolved,
it became possible to apply the activated toxin directly to the cell of inter-
est by microinjection. Such an application was successfully used in microin-
jection TeNT and BoNTs to identified cells in the buccal Aplysia ganglion
(Cornille et al., 1995; Poulain et al., 1996) in squid giant synapse (Hunt et

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