Chemistry Reference
In-Depth Information
Fig. 3.10
Left panel
Membrane fusion at low ionic strengths is strictly dependant on the presence
of both
Ca
2
+
and synaptotagmin-1. In the absence of either of these, fusion does not proceed.
When both these are present, fusion proceeds normally even at low ionic strengths. At normal ionic
stengths however, neither
Ca
2
+
nor synaptotagmin-1 dramatically changes the kinetics. The
+
and
symbols indicate the presence and absence of
Ca
2
+
respectively.
Right panel
The fusion rate
depends on
Ca
2
+
concentration. A roughly linear dependance is seen
−
3.3.3 Synaptotagmin-1 May Be a Distance Regulator
The experiments described thus far establish a clear
Ca
2
+
dependant role of
synaptotagmin-1 in membrane fusion at low ionic strengths. We now seek to uncover
the exact mechanistic details of this interaction. As described in earlier sections,
synaptotagmin-1 has one membrane anchor and two sites for
Ca
2
+
binding, the
C2A and the C2B domains. Indeed, when the two calcium binding are disrupted,
membrane fusion does not occur at low ionic strengths even in the presence of
Ca
2
+
(Fig.
3.11
).
Further, previous studies have shown that the synaptotagmin-1 binds to anionic
lipids both with and without
Ca
2
+
and this binding leads to membrane cluster-
ing [
5
,
7
,
18
,
9
-
13
,
26
]. Indeed, negative-staining electron microscopy showed
strong instantaneous clustering of the liposomes when synaptotagmin-1 liposomes
were mixed with empty liposomes without SNAREs (Fig.
3.12
). This clustering
was
Pi
P
2
and synaptotagmin-1 specific and was reduced when a mutant of
synaptotagmin-1was used that does not bind electrostatically to anionic lipids. Identi-
cal results were obtainedwhen the SNAREs were present. This was further confirmed
by dynamic light scattering (DLS) experiments where the mean size of liposomes
without fusion or synaptotagmin was found to be 40
(
4
,
5
)
±
8nm but that increased to
110
55nm due to liposome clustering in the presence of synanptotagmin-1.
Together, these indicate that without
Ca
2
+
the polybasic patch of synaptotagmin-1
leads to the clustering of liposomes, but not to their fusion. The size of the clusters
reveals that while the liposomes are not fused, they are still close together. There-
±
