Chemistry Reference
In-Depth Information
syntaxin are present in a liposome. In this situation, we clearly do not expect the
repulsive interactions that are mediated by the lipids. It can be seen that the SNARE
nucleation proceeds normally regardless of the ionic strength of the solution, as
indicated by the decrease in the donor fluorescence signal. Again in the second case
(Q
sol
), when the SNAP25 and syntaxin are in solution with the synaptobrevin-2 on
a liposome, SNARE nucleation proceeds normally without any electrostatic effects.
In this case, the kinetics are somewhat reduced compared to the first case of R
sol
probably because of the reduced diffusion of the larger SNAP25-syntaxin complex.
All together, with these experiments we conclude that the high electrostatic repul-
sion between the liposomes blocks not only the fusion of the liposomes but also
SNARE nucleation, even though SNARE nucleation itself is not charge dependant.
This already indicates a clear departure from the current picture of SNARE-mediated
fusion. When charge repulsion effects are in place, SNAREs are probably not already
in a coil-coil structure before the action of
Ca
2
+
-synaptotagmin-1. Under such con-
ditions, it now allows us to clarify the role of synaptotagmin-1, the
Ca
2
+
trigger of
fusion.
3.3.2 C a
2
+
-Synaptotagmin-1 Rescues Membrane Fusion
Having established that electrostatic repulsion of lipid membranes blocks their
fusion, we introduced the
Ca
2
+
trigger synaptotagmin-1 to the process. For these
experiments, full-length synaptotagmin-1 is reconstituted at a protein-to-lipid ratio of
1:4,000 in the R-SNARE liposomes. The ratio of synaptotagmin-1 to synaptobrevin-2
in these liposomes is 1:4, which is similar to that in synaptic vesicles [
42
]. As in pre-
vious studies, at normal ionic strength, the kinetics of lipid mixing were increased
very little compared to the liposomes without synaptotagmin-1 and did not or only
weakly depended on the
Ca
2
+
concentration [
28
,
32
].
In contrast, at low ionic strength the lipid mixing was almost completely blocked
in the absence of
Ca
2
+
, in spite of the presence of synaptotagmin-1. The addition of
1mM
Ca
2
+
from a side channel in the microfluidic chip dramatically increases the
fusion kinetics (Fig.
3.9
). This liposome fusion is strictly dependent on the presence
of both synaptotagmin-1 and the SNAREs. This can be seen the left panel of Fig.
3.10
,
which shows the fusion experiments under different conditions of ionic strengths,
presence or absence of
Ca
2
+
and the presence or absence of synaptotagmin-1.
The fusion efficiency can be controlled by different amounts of added
Ca
2
+
and
a roughly linear behaviour is found as evidenced in the right panel of Fig.
3.10
.This
was further tested with synaptic vesicles, extracted from rat brains, that contain a
large fraction of about 15% anionic lipids, which yielded similar results. Membrane
fusion at low ionic strength is found to be largely
Ca
2
+
-dependent. Thus, at low
ionic strength
Ca
2
+
-synaptotagmin-1 triggers lipid mixing not only of liposomes,
but also of native synaptic vesicles.
Because the FRET assay due to the mixing of lipids does not distinguish between
hemifusion and full membrane fusion, it is conceivable that the membrane fusion
