Neurotoxins as Tools in Dissecting the Exocytic Machinery - Fusion of Biological Membranes and Related Problems - page 53

al., 1994), in the Retzius synapse of the leech (Bruns et al., 1997) and in the
case of other large synapses suitable for injection (Stanley and Mirotznik,
1997). Such experiments allow us to combine electrophysiology and
morphology studies. For example, microinjected of BoNT/C1 into the squid
giant presynaptic terminal resulted in a complete block in transmission
while the number or distribution of synaptic vesicles at the presynaptic
active zone were not changed (O'Connor et al., 1997). In such experiments
the kinetics and the extent of inhibition by the toxins were determined.

4.2. In vitro -Tissues and Cells

The structure of clostridial toxins ensures that the toxins will exert their
activity on cellular internal targets only following the activation and expo-
sure to the cytoplasm. In cells, various methodologies were used. They are
summarized in the following scheme (Figure 5).
Stable transfection. Introducing toxins to cells by stable transfection
of the active toxin (the LC) is an alternative way of introducing the toxin
to the cell milieu. In contrast to the microinjection procedure (see above),
transfection is performed on the entire culture and the genetic material
used for transfection may be genetically altered. For example, BoNT/A LC
was transfected into AtT-20 cells. This manipulation resulted in a complete
cleavage of SNAP-25. The transfected cells could not be induced to secrete
ACTH upon stimulation (Aguado et al., 1997). A more complex setting was
performed in insulin-secreting HIT-T15 cells. There, BoNT/A was intro-
duced into HIT-T15 cells following overexpressing SNAP-25. Transient
expression of BoNT/A cleaved the endogenous as well as overexpressed
SNAP-25 proteins and caused reduction in insulin secretion. This trans-
fected system was also used to introduce the cell with SNAP-25 from which
the C-terminal 9 amino acids had been deleted to mimic the effects of the
toxin (see Figure 3). This modified SNAP-25 was as effective as the toxin
at inhibiting secretion. The results of such manipulations are consistent with
the view that the inhibitory action of the BoNT/A toxin is caused by the
production of the cleavage product, which interferes with insulin granule
fusion (Huang et al., 1998). Morphological studies showed that upon treat-
ment of the toxin or the transfected truncated SNAP-25 fragment, higher
numbers of docked insulin granules are seen. This result suggests that block
in secretion occurs following docking and that the toxin effect provided by
a fragment of SNAP-25 that interact itself with the core complex in an
unproductive manner.
Liposome transfection. An alternative mode of introducing active
toxins to the cell interior is by liposome transfection of the active LC of the
toxin with or without a peptide that overlaps the putative cleavage sites.