Stable SNARE Complex Prior to Evoked Synaptic Vesicle Fusion
Revealed by Fluorescence Resonance Energy Transfer*
Zongping
Xia
,
Qiong
Zhou
,
Jialing
Lin§, and
Yuechueng
Liu
¶
From the
Department of Pathology and the
§ Department of Biochemistry and Molecular Biology, the
University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73190
Received for publication, September 25, 2000
 |
ABSTRACT |
Although it is clear that soluble
N-ethylmaleimide-sensitive factor (NSF) attachment
protein receptor (SNARE) complex plays an essential role in synaptic
vesicle fusion, the dynamics of SNARE assembly during vesicle fusion
remain to be determined. In this report, we employ fluorescence
resonance energy transfer technique to study the formation of SNARE
complexes. Donor/acceptor pair variants of green fluorescent protein
(GFP), cyan fluorescent protein (CFP), and yellow fluorescent
protein (YFP) are fused with the N termini of SNAP-25 and
synaptobrevin, respectively. In vitro assembly of SNARE
core complex in the presence of syntaxin shows strong fluorescence
resonance energy transfer (FRET) between the CFP-SNAP-25 and
YFP-synaptobrevin. Under the same conditions, CFP fused to the C
terminus of SNAP-25, and YFP- synaptobrevin have no FRET.
Adenovirus-mediated gene transfer is used to express the fusion
proteins in PC12 cells and cultured rat cerebellar granule cells.
Strong FRET is associated with neurite membranes and vesicular
structures in PC12 cells co-expressing CFP-SNAP-25 and
YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET
between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with
sites presumed to be synaptic junctions. Neurosecretion in PC12 cells
initiated by KCl depolarization leads to an increase in the extent of
FRET. These results demonstrate that significant amounts of stable
SNARE complex exist prior to evoked synaptic vesicle fusion and that
the assembly of SNARE complex occurs during vesicle docking/priming
stage. Moreover, it demonstrates that FRET can be used as an effective
tool for investigating dynamic SNARE interactions during synaptic
vesicle fusion.
 |
INTRODUCTION |
The SNARE1 core complex
involved in synaptic vesicle fusion consists of target SNARE syntaxin
and SNAP-25 and vesicle SNARE synaptobrevin/VAMP (1-3). It has been
shown by in vitro binding studies that the SNAREs interact
directly and form a tight ternary complex (4-9). The recently solved
crystal structure of the SNARE core complex and additional biophysical
studies of the core complex demonstrate that the N- and C-terminal
helix domains of SNAP-25 participate in the formation of a parallel
coiled-coil structure with syntaxin and synaptobrevin (10-13). Limited
proteolysis and in vitro binding assay suggest that the two
SNAP-25
-helix domains may act independently and contribute equally
to form the SNARE complex with syntaxin and synaptobrevin (10). Upon
association with additional proteins, e.g. synaptotagmin,
NSF, and
-SNAP, the complex undergoes further conformational changes
and disassembly (14).
Although the importance of SNARE complex in neurosecretion and the
basic structure of the SNARE core complex have been well studied and
established, the molecular mechanisms underlying its involvement in the
membrane fusion process are less clear. Contrary to the earlier belief
that SNARE assembly attaches vesicles to plasma membrane and the
subsequent disassembly of the complex in the presence of NSF and
-SNAP drives membrane fusion, recent findings suggest that the
formation of SNARE core complex and membrane fusion is tightly linked
and the assembly of SNARE complex may provide the energy needed for
membrane fusion (15-17). By using a reconstituted liposome system, the
formation of SNARE complex is able to drive membrane fusion (18, 19).
Nevertheless, the timing and dynamics of SNARE complex assembly prior
to vesicle fusion remain to be determined. By using antibodies specific
for SNAP-25 peptides, Xu et al. (20) have presented evidence
in chromaffin cells, suggesting that SNARE complex exists in two dynamically distinctive "loose" and "tight" states that both
contribute to calcium-dependent exocytosis. In addition,
studies using neurotoxins also suggest the presence of stable SNARE
complex prior to fusion. Therefore, the assembly of SNARE complex may
be a rate-limiting step during neurotransmission.
In the present study, we have used fluorescence resonance energy
transfer (FRET) to dissect the dynamics of SNARE assembly in PC12 cells
and cultured cerebellar neurons. FRET is a process in which energy from
a fluorescent donor molecule is transferred to a fluorescent acceptor
without the involvement of a proton. One result is that the
fluorescence emission of the acceptor is enhanced by the excitation of
the donor molecule. The efficiency of energy transfer is dependent on
the molecular distance at an inverse 6th power. Therefore, FRET is a
highly specific "molecular ruler." FRET has been increasingly used
for in vivo studies of molecular interactions including
calmodulin structure and function, (21), Bcl-2-Bax interaction
(22), and synaptic activity in the synaptic spine (23). By using a pair
of green fluorescent protein (GFP) variants CFP and YFP, we measured
FRET between SNAP-25 fused with CFP and synaptobrevin fused with YFP
under both in vitro and in vivo conditions. Our
results suggest that an intermediate and a stable form of SNARE complex
are present prior to fusion or during vesicle docking stage.
 |
EXPERIMENTAL PROCEDURES |
Mutagenesis and Preparation of GFP Fusion Proteins--
A mouse
cDNA encoding synaptobrevin-2 was purchased from the I.M.A.G.E.
Consortium. cDNA encoding mouse SNAP-25b was a generous gift from Dr. Michael C. Wilson (Scripps Research Institute, San Diego,
CA) (24). cDNA encoding rat syntaxin 1A was a generous gift from
Dr. Richard Scheller (Howard Hughes Medical Institute, Stanford, CA).
pECFP-N1, pECFP-C1, and pEYFP-C1 were from
CLONTECH. A BglII site was introduced at
the ATG start codon of synaptobrevin cDNA by site-directed
mutagenesis, and the resulting BglII-EcoRI fragment containing the coding sequence of synaptobrevin was ligated into the same sites of pEYFP-C1. Similarly, a KpnI site and
an EcoRI site were introduced into the SNAP-25 cDNA at
its start or stop site, respectively. A KpnI-ApaI
and an EcoRI fragment encoding SNAP-25 were ligated into the
corresponding sites of pECFP-N1 and pECFP-C1, respectively. Recombinant
adenoviruses were prepared using the pAdEasy system as described by He
et al. (25). ECFP-SNAP-25 (C-SN), SNAP-25-ECFP (SN-C), and
EYFP-synaptobrevin (Y-SB) were cloned into pAdShuttle-CMV.
Recombination with pAdEasy-1 backbone plasmid was performed in
Escherichia coli strain BJ5183. Recombinant viruses were
obtained by transfection of HEK293 cells, and the viruses were
amplified and purified to ~1012 particles/ml by
ultracentrifugation on a CsCl gradient.
Cell Culture and Viral Infection--
PC12 cells were plated in
35-mm tissue culture dishes coated with 50 µg/ml
poly-D-lysine, and the cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and
5% bovine calf serum. NGF (Life Technologies, Inc.) was added to 50 ng/ml final concentration to induce differentiation. Cerebellar granule
cells from 5- to 8-day-old Harlan Sprague-Dawley rats were cultured as
described previously (26). Briefly, cells were dissociated from the
cerebellum by mechanical chopping and treatment with 0.5% trypsin and
1 mM EDTA for 5 min at 37 °C. The cells were dissociated
by trituration and plated onto poly-D-lysine (50 µg/ml,
Sigma)-coated dishes. The cells were cultured in minimum essential
medium containing 10% fetal bovine serum and 25 mM KCl. To
inhibit the growth of non-neuronal cells, cytosine arabinoside was
added 24 h later to 10 µM. The granule cells were
cultured for 4-5 days, and recombinant adenoviruses were added
directly to the neuron cultures at an m.o.i. of 200-250. The infected
cells were maintained for 48 h before use. All of the cells were
maintained in a humidified 35 °C incubator with 5%
CO2.
Immunoprecipitation and Immunoblot Analysis--
For
immunoprecipitation, the cells were washed once with 50 mM
Na2HPO4, pH 7.4, 100 mM NaCl (PBS)
and lysed with the same buffer containing 1% Nonidet P-40.
Immunoprecipitation was performed using a mouse monoclonal antibody
against syntaxin (clone HPC-1, Sigma) and protein G-plus agarose
(Oncogene Science). The immunocomplex was pelleted by centrifugation at
1,000 × g for 2 min and washed 3 times with 50 mM Na2HPO4, pH 7.4, 100 mM NaCl, and 0.5% Nonidet P-40. The immunoprecipitated
proteins were heated to 80 °C for 3 min in 1% SDS, 10 mM EDTA, 10 mM dithiothreitol, 15% glycerol, 20 mM Tris-HCl, pH 6.8, and 0.01% bromphenol blue. The
precipitated proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and transferred onto nitrocellulose membrane for
immunoblot analysis. The following primary antibodies were used: the
HPC-1 monoclonal anti-syntaxin (1:1,000), a goat anti-SNAP-25 antibody
(1:10,000) (27), and a goat anti-synaptobrevin (1:5,000) raised against a GST-synaptobrevin fusion protein. Alkaline phosphatase-conjugated secondary anti-mouse and anti-goat antibodies from Sigma were used.
In Vitro SNARE Core Complex Formation and Spectrofluorometer
Assay--
COS-7 cells were infected with recombinant adenoviruses to
express C-SN, SN-C, and Y-SB. The cells were lysed with binding buffer
containing 20 mM Hepes, pH 7.4, 100 mM KCl, 2 mM EDTA, 1% Triton X-100, and 1 mM
phenylmethylsulfonyl fluoride. GST-Syntaxin bound to
glutathione-agarose beads were incubated with C-SN/Y-SB or SN-C/Y-SB
for 3 h at room temperature. Beads were then washed three times
with the binding buffer, and the bound SNARE complexes were eluted with
5 mM reduced glutathione in binding buffer. The eluted
SNARE complexes were immediately measured for FRET using a
spectrofluorometer (SLM 8100, SLM-Aminco, Rochester, NY). The samples
were excited at 425 nm, and emission spectra were collected from 450 to
550 nm. The final emission spectra were corrected for background,
smoothed, and normalized.
Microscopy, Image Analysis, and FRET Calculation--
PC12 cells
or primary cerebellar neurons were infected with adenoviruses
expressing ECFP and EYFP fusion proteins. For detection of ECFP, cells
were viewed with an inverted fluorescence microscope (Leica DMIL) under
a filter set with an excitation filter of 440/21 nm, a dichroic beam
splitter of 455, and an emission filter of 480/30 nm. EYFP expressing
cells were viewed under a filter set with an excitation filter of
500/25 nm, a dichroic beam splitter of 525 nm, and an emission filter
of 545/35 nm. The filters for FRET were an excitation filter of 440/21
nm, a dichroic beam splitter of 455, and an emission filter of 535/26
nm. Images were captured with a cooled CCD camera (Quantix 57, Photometrics, Tucson, AZ) controlled by IPlab 3.5 (Scanalytics,,
Fairfax, VA) and analyzed with the IPlab software.
FRET was quantified with the three-filter set system to normalize the
FRET intensity for ECFP and EYFP concentration in each region of
interest (ROI) (28). ROIs were selected manually. For an ROI, intensity
(I) from the three filter sets was obtained after background
subtraction. Then FRETN was calculated as follows: FRETN = (IFRET
IECFP·55%
IEYFP·15%)/(IECFP·IEYFP),
where 55% is the percentage of ECFP contribution to FRET intensity,
and 15% is the percentage of EYFP contribution to FRET intensity. In
some cases, the images were adjusted pixel-by-pixel using a reference
channel of ROI.
 |
RESULTS |
FRET between CFP-SNAP-25 and YFP-Synaptobrevin in Vitro--
To
express effectively the recombinant proteins, the CFP-SNAP-25 (C-SN)
and YFP-synaptobrevin (Y-SB) were cloned into an adenovirus vector as
described by He et al. (25). A control fusion protein SNAP-25-CFP (SN-C) in which the C terminus of SNAP-25 was fused with
CFP was also prepared. The fusion with SNAP-25 or synaptobrevin did not
affect the spectrum properties of CFP and YFP (Fig.
1A). To determine whether the
fusion proteins still retained the function in SNARE complex assembly,
Y-SB was co-expressed with C-SN or SN-C in cultured rat cerebellar
granule cells. The cells were lysed with nonionic detergent, and the
SNARE complexes were immunoprecipitated using an anti-syntaxin
antibody. As shown in Fig. 2, the fusion proteins were co-immunoprecipitated with the SNARE complex containing endogenous SNAP-25 and synaptobrevin. This result suggests that the
fusion proteins are able to participate in the formation of SNARE
complex.

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Fig. 1.
Spectrum properties of C-SN, SN-C, and Y-SB
in solution and in PC12 cells. A, excitation and
emission spectra of the fusion proteins. The fusion proteins were
expressed in COS-7 cells by adenoviral vectors for 24 h, and the
cells were lysed with PBS containing 1% Triton X-100. The cell lysate
was measured using a spectrofluorometer. Solid lines
indicate the excitation spectra, and dashed lines indicate
the emission spectra. B, fluorescence images of PC12 cells
expressing the fusion proteins. Differentiated PC12 cells were infected
with recombinant adenoviruses at 10 m.o.i. for 48 h. Images
were acquired under FRET and CFP/YFP filters. Notice that the C-SN and
SN-C were localized to the plasma membranes of the cell bodies and
neurites, whereas Y-SB had a punctate localization.
Bar, 30 µm.
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Fig. 2.
Co-immunoprecipitation of C-SN, SN-C, and
Y-SB with SNARE complexes from rat cerebellar neuron cultures.
A, immunoblot analysis of total cell lysates from cerebellar
neurons infected with recombinant adenoviruses expressing 1) C-SN and
Y-SB or 2) SN-C and Y-SB. B, immunoprecipitation using
anti-syntaxin antibodies. The immunoprecipitated proteins were blotted
against goat anti-SNAP-25, goat anti-synaptobrevin, and monoclonal
anti-syntaxin antibodies. Lane 1, cells expressing C-SN and
Y-SB; lane 2, cells expressing SN-C and Y-SB; and lane
3, cells expressing C-SN and Y-SB, control sample without
anti-syntaxin added during immunoprecipitation. Molecular mass markers
are 73, 47, 33, 28, and 20 kDa (arrowheads).
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The assembly of SNARE core complex was first examined by FRET in
vitro. Y-SB, C-SN, and SN-C were overexpressed in COS-7 cells by
recombinant adenovirus infection. Purified GST-syntaxin was incubated
with Triton X-100-solubilized membrane preparations containing similar
amounts of Y-SB and C-SN/SN-C. The Y-SB-GST-syntaxin-C-SN or
Y-SB-GST-syntaxin-SN-C ternary complexes were eluted with excess glutathione and measured for fluorescence absorption at excitation of
425 nm. There are two advantages of using the crude COS-7 preparations instead of purified proteins expressed in bacteria. It allows the
correct folding and post-translational modifications of the proteins,
and the presence of other cellular proteins further reduced nonspecific
absorption by GST-agarose. As shown in Fig. 3A, no FRET between SN-C and
Y-SB was observed. However, a significant amount of FRET was detected
between C-SN and Y-SB, which exhibited an increased absorption peak at
530 nm (Fig. 3A). For control experiments, an equal aliquot
of COS-7 lysate containing C-SN was incubated with the same amount of
GST-syntaxin and unlabeled synaptobrevin, and the resulting ternary
complex was eluted for fluorescence measurement, which showed a
significantly higher absorption at 480 nm in the absence of
fluorescence acceptor (data not shown). Assuming that the affinity of
Y-SB for syntaxin and SNAP-25 was the same as the unlabeled
synaptobrevin, the FRET efficiency between C-SN and Y-SB was estimated
to be ~44%. These results are consistent with the crystal structure
of SNARE core complex model, in which the two
-helices of SNAP-25
form a parallel 4-
-helical bundle with syntaxin and synaptobrevin
(10-13).

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Fig. 3.
FRET between C-SN and Y-SB in
vitro and in vivo. A,
preformed SNARE core complexes measured for FRET using a
spectrofluorometer. The SNARE core complexes composed of GST-syntaxin,
Y-SB, and C-SN/SN-C were eluted with 5 mM glutathione and
immediately measured for FRET. Arrow indicates FRET between
C-SN and Y-SB at the emission peak of Y-SB (533 nm). No FRET was
observed between SN-C and Y-SB. B and C, PC12
cells expressing SN-C and Y-SB (B) or C-SN and Y-SB
(C) viewed under CFP, YFP, or FRET filter sets.
Arrows indicate FRET-negative sites; arrowheads
indicate FRET-positive sites, as determined by the method described
under "Experimental Procedures." Notice that in cells expressing
C-SN and Y-SB, most of plasma membrane and neuritic varicosities were
positive for FRET, whereas intracellular structures were
FRET-negative. Bar, 40 µm.
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FRET in PC12 Cells and Cultured Neurons Expressing CFP-SNAP-25 and
YFP-Synaptobrevin--
To study SNARE interaction in vivo,
PC12 cells and rat cerebellar granule neurons were infected with
recombinant adenoviruses expressing the fusion proteins. It has been
shown that infection of PC12 cells with recombinant adenovirus at a
m.o.i. of up to 100 had no significant effect on endogenous protein
expression and norepinephrine release (29). At an m.o.i. of 10, ~10-15% cells were infected and expressed various levels of fusion
proteins after 24 h. As expected, C-SN and SN-C were localized to
the plasma membranes of cell bodies and neuritic processes of
differentiated PC12 cells, whereas Y-SB was mostly associated with
intracellular membranes and often with punctate structures clustered in
the cells as well as neurites (Fig. 1B). Digital images were
acquired under three sets of filters, a CFP set, a YFP set, and a FRET set. To establish background fluorescence, PC12 cells expressing only
one fusion protein were examined. There was no crossover between CFP
and YFP when viewed under the CFP or YFP filters. When the cells were
examined under the FRET filter set, YFP alone yielded less than
15-20% fluorescence and CFP yielded ~50-56% (Fig. 1B).
Since many factors may affect the accuracy of FRET measurement, we
adopted the method used by Gordon et al. (28), which uses
several values for each pair of fusion proteins to correct for
background and level of protein expression. As shown in Fig. 3 and
Table I, FRET was observed on the plasma
membranes and neuritic varicosities of PC12 cells expressing Y-SB and
C-SN. However, FRET was not observed in locations including the
Golgi-like intracellular membranes and some aggregates in the neurites
(Fig. 3B). No significant FRET was observed with the fusion
protein pair Y-SB and SN-C (Fig. 3B), which did not show
FRET under in vitro conditions (Fig. 3A).
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Table I
Mean FRETN values for co-expressed CFP-YFP fusion protein pairs
FRETN was calculated as described by Gordon et al.
(28). Data are from four independent experiments.
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Cultured cerebellar granule cells from 5- to 8-day-old rats undergo
synaptogenesis starting at the end of the 1st week when the expression
of synaptic proteins is rapidly increased (30). At this stage, the
cells are responsible for depolarization and release glutamate as the
major neurotransmitters (31, 32). At 6 days in vitro, the
endogenous synaptobrevin began to shift its localization from cell
bodies and general neuronal processes to a more restricted localization
at synaptic junctions (data not shown). SNAP-25, however, was more
uniformly distributed in the processes, and little was found in the
cell bodies. The Y-SB and SN-C fusion proteins, when co-expressed by
adenovirus infection, showed similar localization as their endogenous
counterparts, suggesting that the fusion proteins were correctly
targeted to the subcellular sites. The virally infected neurons often
showed strong fluorescence signals in their cell bodies, probably
indicating the transport of newly synthesized recombinant proteins
through Golgi apparatus. Three types of neuronal subcellular locations were selected for FRET measurement as follows: the plasma membranes of
the cell bodies, the punctate structures presumably to be the synaptic
sites, and the large varicosities presumably to be the constitutively
secreting vesicles transporting newly synthesized proteins. As shown in
Fig. 4 and Table I, FRET occurred mostly at the synaptic junctions, whereas intracellular aggregates in the soma
and varicosities undergoing axonal transport showed no FRET. The
percentages of synaptic junctions with FRET was >80% (p < 0.01), suggesting the presence of assembled SNARE
complex.

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Fig. 4.
FRET in cultured rat cerebellar neurons
expressing C-SN and Y-SB. Rat cerebellar granule cells were
cultured for 5 days before infection with adenoviruses at 200 m.o.i. Forty eight hours post-infection, the cells were examined under
CFP, YFP, or FRET filter sets and measured for FRET as described under
"Experimental Procedures." Arrowheads indicate
FRET-positive sites presumed to be synaptic junctions, and the
arrow indicates the axonally transported varicosities and
aggregates in the cell body where no FRET was detected. * indicates
cell bodies. Bar, 50 µm.
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One of the advantages of the FRET technique is its potential ability to
dissect dynamic protein-protein interactions in vivo (21,
23). In an attempt to determine whether or not the SNARE complex
undergoes conformational changes during exocytosis, as suggested by
several recent studies (17, 20), FRET between C-SN and Y-SB was
measured in PC12 cells treated with 56 mM KCl. A PC12 cell
line stably expressing Y-SB was infected with adenovirus expressing
C-SN. The cells were treated with NGF for 4 days before stimulation
with KCl to induce Ca2+-dependent exocytosis.
As shown in Fig. 5 and Table I, FRET
measured after KCl treatment was 40-60% (p < 0.05)
higher than that before the treatment. A degree of heterogeneity in the
extent of FRET was also observed, which was likely due to the variance
of different membrane fusion events during neurosecretion (3). These
results suggested that a tighter cis-SNARE complex may be formed
following membrane fusion and that FRET may be used as an effective
tool for studying the dynamic interactions between individual SNAREs during synaptic transmission. FRET was also measured in PC12 cells pretreated with 1 µM PMA, a phorbol ester that activates
protein kinase C and enhances neurosecretion in PC12 cells. Since
several SNAREs are substrates for protein kinase C (33), it is possible that protein kinase C enhances neurosecretion by phosphorylation of
SNAREs. No significant changes in FRET were detected in PMA-treated cells (data not shown), suggesting that PMA treatment did not cause
major structural reorganization of the SNARE core complexes.

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Fig. 5.
FRET in differentiated PC12 cells stimulated
with high KCl. PC12 cells stably transfected with Y-SB were grown
on poly-D-lysine-coated coverslips and infected with C-SN
adenoviruses. The cells were treated with 50 ng/ml NGF for 4 days
before KCl was added to 56 mM. The cells were incubated for
5 min at 37 °C and fixed with 3.7% formaldehyde in PBS. FRET was
measured as described under "Experimental Procedures."
A-D, control cells; E H, KCl-treated cells.
Fluorescence images viewed under CFP filters (A and
E), YFP filters (B and F), and FRET
filters (C and G). D and H,
FRET signals after pixel-by-pixel adjustment and subtraction of
nonspecific contributions from CFP and YFP as described under
"Experimental Procedures." Arrowheads indicate FRET
positive sites. Bar, 15 µm.
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DISCUSSION |
Recent biochemical and biophysical studies from a number of
laboratories have provided much needed insights into the assembly and
disassembly of the SNARE core complex and its proposed role in vesicle
fusion (11-13, 17, 19). What remains to be delineated is the temporal
dynamics of SNARE complex assembly and disassembly in relation to the
vesicle fusion process. Such knowledge is essential to the
understanding of the function and regulation of SNARE complex during
vesicle trafficking. FRET, using fusion proteins of GFP variants,
provides a powerful tool for investigating such mechanisms. The CFP
(donor)-YFP (acceptor) pair has been used in a number of FRET
experiments (21, 23). The critical Forster radius for CFP-YFP is about
50 Å, suggesting that any significant FRET would indicate actual
physical interaction between two proteins. Since we have demonstrated
that FRET between C-SN and Y-SB can be effectively monitored in
vivo, similar experiments with other fusion protein pairs such as
syntaxin-synaptobrevin or syntaxin-SNAP-25 can be performed.
Furthermore, the technique provides a useful tool for dissecting SNARE
complex assembly/disassembly in real time during vesicle docking,
fusion, and recycling.
For the FRET technology to work effectively, the fusion proteins must
be able to be correctly targeted and participate in SNARE
assembly/disassembly. Many studies using GFP fusion proteins have shown
high fidelity of subcellular targeting of the fusion proteins, and the
same findings with N-terminally and C-terminally fused GFP-
synaptobrevin have been reported (34, 35). One concern is that the
overexpressed recombinant proteins may be mis-targeted or "spilled
over" to other compartments. If Y-SB was mis-targeted to the plasma
membranes, it could form cis-SNARE complexes with SNAP-25 and syntaxin
as those found in the in vitro binding experiments. Although
such a possibility cannot be ruled out, several lines of evidence
strongly suggest that the FRET observed with Y-SB and C-SN in
vivo was from trans-SNARE complexes. First, only cells moderately
expressing the fusion proteins were used for FRET measurement, and
similar results were obtained in PC12 cells that had been passed for at
least two generations following adenovirus infection, when the
expression of the recombinant proteins were comparable to the
endogenous protein levels. Second, glycerol gradient
ultracentrifugation was able to separate dense core vesicles from the
plasma membranes in PC12 cells (36), which showed that >95% Y-SB was
associated with the vesicle fractions, and >90% of SNAP-25 was
associated with the plasma membranes (data not shown). Third, the Y-SB
and C-SN/SN-C were correctly targeted in cultured cerebellar granule
cells. Fourth, we have shown that a GFP-synaptobrevin fusion protein
was exclusively targeted to neuromuscular junctions when expressed in
spinal motor neurons via adenovirus-mediated gene transfer (35).
The evoked synaptic vesicle fusion is a tightly regulated process that
is triggered by calcium influx (3, 37). It is not clear if SNARE
complex assembly occurs during the vesicle docking/priming stages or
immediately proceeding the fusion. Studies by Chen et al.
(17) suggest that the assembly of SNARE complex is immediately followed
by membrane fusion; thus, the two events are practically linked as one
step. The findings in our current study suggest that stable SNARE
complexes that are fusion-incompetent are assembled. Alternatively, an
intermediate form of SNARE complexes may exist during vesicle docking
and undergo further conformational changes upon calcium influx.
Although it is difficult to estimate the actual amount of SNARE complex
based on the FRET values, the strong FRET observed between C-SN and
Y-SB indicates the existence of significant numbers of SNARE complexes.
Although spontaneous transmitter release by PC12 cells and cerebellar
granule cells occurs at the basal level, such large numbers of SNARE
complexes favor the notion that assembled yet fusion-incompetent SNARE
complexes are present during the vesicle docking/priming stage. The
existence of an intermediate form of SNARE complexes is supported by
electrophysiological studies in chromaffin cells. Based on the
secretion kinetics of chromaffin cells in response to stimulation, it
was proposed that a loose form of SNARE complexes exists prior
to vesicle fusion, and these complexes are required for the subsequent
fusion (20). According to the model, a fusion-competent vesicle can
exist in two states that are controlled by a loose or a tight SNARE
complex. Another study using neurotoxins also points to the presence of stable SNARE complexes in unstimulated cells (38). Although FRET can be
used for measuring distance between proteins, we have not attempted to
do so because the conformational property of C-SN and Y-SB has yet to
be characterized. Therefore, we could not conclude whether or not the
FRET was from a loose or tight SNARE complex. Nevertheless, because the
R0 of CFP/YFP is 50 Å, the distance between
C-SN and Y-SB would be likely less than 50 Å.
The finding that FRET increases following vesicle fusion supports the
proposal that a transition from trans- to cis-SNARE complexes occurs as
a result of vesicle fusion (17, 20). Another possibility is that the
net amount of SNARE complexes is increased due to membrane fusion under
the sustained depolarization conditions. Perhaps both the higher number
and tighter SNARE complexes have contributed to the increased FRET
observed in KCl-treated cells. This can only be resolved when we can
quantify the amount SNARE complex in cells.
In summary, the present study using FRET provides direct evidence of
the presence of stable SNARE complexes prior to evoked synaptic vesicle
fusion. More importantly, this technique can be applied to investigate
the dynamics of SNARE assembly and disassembly in real time during
neurotransmitter release in vivo, which would allow us to
dissect the molecular event involved in synaptic transmission.
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ACKNOWLEDGEMENTS |
We thank Dr. Michael C. Wilson for the
SNAP-25b clone, Dr. Richard Scheller for the syntaxin 1A cDNA
clone, and Drs. T.C. He and B. Vogelstein for the adenoviral
vectors. We also thank Drs. James Rand and Jane Jacob for critical
reading of this manuscript.
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FOOTNOTES |
*
This work was supported by the Oklahoma Center for the
Advancement of Science and Technology Grant HN6-022 and National
Institutes of Health Grant NS35167.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pathology, University of Oklahoma Health Sciences Center, P. O. Box
26901, Oklahoma City, OK 73190. Tel.: 405-271-2336; Fax: 405-271-3042; E-mail: yuechueng-liu@ouhsc.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008741200
 |
ABBREVIATIONS |
The abbreviations used are:
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor;
SNAP-25, synaptosomal associated protein of 25 kDa;
m.o.i., multiplicity of infection;
GFP, green fluorescence protein;
CFP, cyan
fluorescent protein;
YFP, yellow fluorescent protein;
C-SN, CFP fused
to the N terminus of SNAP-25;
SN-C, CFP fused to the C terminus of
SNAP-25;
Y-SB, YFP fused to the N terminus of synaptobrevin;
FRET, fluorescence resonance energy transfer;
NSF, N-ethylmaleimide-sensitive factor;
PMA, phorbol 12-myristate
13-acetate;
NGF, nerve growth factor;
ROI, region of interest;
GST, glutathione S-transferase;
PBS, phosphate-buffered
saline.
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