Chemistry Reference
In-Depth Information
at these ionic strengths. However, at these high salt concentrations the binding of
synaptotagmin-1 to SNAREs and anionic membranes is strongly reduced or even
absent [
5
,
7
,
21
,
22
,
25
]. Furthermore, these high concentrations potentially screen
important electrostatic interactions which might very well be expected due to the
charged lipids that are ubiquitously present in membranes.
We sought to address this important issue—what happens when the electrostatic
interactions are not screened? We do this by reducing the ionic strength of the buffer
solution. When the charges are not screened, the electrostatic interactions due to
the charged lipids in the membranes play a very important role, as we will see.
In the following sections, we show that under such conditions, in the presence of
anionic lipids, SNARE-dependent membrane fusion is completely blocked. Further,
we show that the fusion strictly depends on the presence of
Ca
2
+
and synaptotagmin-
1 when the ionic strength of the medium is reduced. It is then demonstrated that
synaptotagmin-1, by regulating the distance between the membranes in a
Ca
2
+
-
dependent manner, acts even before SNARE nucleation occurs, which is a significant
departure from the current understanding of its role.
3.2 Experimental Techniques
In vitro experiments help to understand the minimal model for membrane fusion
by controlled trials. A typical assay for studying membrane fusion in vitro is to
reconstitute functional proteins in liposomes that are formed by fluorescently tagged
lipids. Vesicles containing the complementary fusion proteins are respectively tagged
with fluorescent tags that form a FRET pair. When the vesicles are separate, there
is no resulting FRET signal due to the large distance between them. However, as
the vesicles fuse, their constituent lipids mix in the newly formed membrane, thus
bringing themclose enough to get a FRET signal. The efficiency of the fusion between
the complementary vesicles is then followed by recording the FRET fluorescence
signal over time. As the fusion proceeds, the donor fluorescence decreases and the
acceptor fluorescence correspondingly increases.
Liposomes with a size in the range of 30-40nm are prepared using size exclusion
chromatography as described extensively [
25
]. Briefly, Lipids (Avanti, Alabaster,
AL) were mixed in chloroform to yield (molar ratios): phosphatidylcholine (5),
l phosphatidylethanolamine (2), phosphatidylserine (1), phosphatidylinositol (1),
cholesterol (1). After drying, they were resuspended in hybridization buffer (HB100)
solution containing 5% (wt/vol) cholate at a total lipid concentration of 13.5mM.
SNARE-proteins in 1.5% cholate were added (lipid to protein ratio of 100:1 n/n),
followed by chromatography on Sephadex G-50 superfine equilibrated in HB100
buffer by using a sample-to-column volume ratio of 1:30. For the preparation of
liposomes containing Synataxin and SNAP-25, the proteins were preincubated for
1h before addition to the phospholipid mixture.
Q-SNARE population of liposomes contained 1:2,000 (protein:lipid molar ratio)
of syntaxin-1A and SNAP-25 and a synaptobrevin-2
49
−
96
fragment. R-SNARE pop-
ulation of liposomes contained a 1:1,000 (protein:lipid molar ratio) synaptobrevin-2.
