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The Journal of Neuroscience, February 15, 2000, 20(4):1281-1289
Comparison of Cysteine String Protein (Csp) and Mutant
 -SNAP Overexpression Reveals a Role for Csp in Late Steps of
Membrane Fusion in Dense-Core Granule Exocytosis in Adrenal
Chromaffin Cells
Margaret E.
Graham and
Robert D.
Burgoyne
The Physiological Laboratory, The University of Liverpool,
Liverpool L69 3BX, United Kingdom
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ABSTRACT |
Assembly of the SNARE complex and its disassembly caused by
the action of soluble N-ethylmaleimide-sensitive factor
(NSF) attachment protein (SNAP) and NSF is crucial for the
maintenance of vesicular traffic, including fusion of regulated
exocytotic vesicles. Various other proteins may also have important
roles in the processes leading to membrane fusion via interaction with the SNARE proteins, including the secretory vesicle cysteine string protein (Csp). Here we have examined the effect of overexpression of a
dominant negative  -SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to
detect released catecholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granules was substantially inhibited by
expression of -SNAP(L294A). The amplitude and characteristics of the
individual release events were unaffected by expression of
-SNAP(L294A), consistent with an essential role for -SNAP in
early steps of priming but not in the fusion process. In contrast, Csp
overexpression, which also inhibited the extent of exocytosis, also
modified the kinetics of the individual release events seen as
an increase in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike -SNAP, Csp plays a key role in the protein interactions close to the fusion process or fusion pore opening during
Ca2+-regulated exocytosis.
Key words:
exocytosis; secretion; SNAP; NSF; Csp; amperometry
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INTRODUCTION |
An essential role for proteins of
the SNARE family in vesicular traffic is well established, and
syntaxin 1, synaptosomal associated protein of 25 kDa (SNAP-25), and
vesicle-associated membrane protein (VAMP) are essential for
vesicle fusion in regulated exocytosis in synapses and in
neuroendocrine and endocrine cells (Schiavo et al., 1992 ; Hay and
Scheller, 1997; Burgoyne and Morgan, 1998; Lang, 1999). Syntaxin 1, SNAP-25, and VAMP are able to form a stable complex (the SNARE complex)
(Hayashi et al., 1994; Sutton et al., 1998). The assembly of the SNARE
complex is regulated by many factors, and considerable attention has
been given to the ability of the ATPase NSF to disassemble the SNARE
complex after its recruitment by -soluble
N-ethylmaleimide-sensitive factor attachment protein
(-SNAP) (Sollner et al., 1993a,b). The original suggestion that
disassembly of the complex by SNAP/NSF provided a driving force for
membrane fusion (Sollner et al., 1993b) is inconsistent with various
data (Morgan and Burgoyne, 1995a), and a current view is that assembly
of the SNARE complex drives fusion (Sutton et al., 1998; Weber et al.,
1998).
In the yeast homotypic vacuole fusion system, SNAP/NSF have a role in
priming the SNAREs on each vacuole membrane before membrane docking and
fusion (Mayer et al., 1996; Nichols et al., 1997; Ungermann et al.,
1998). It has been assumed that the role of SNAP/NSF in the synapse is
to disassemble the SNARE complex only after synaptic vesicle
fusion. This would allow recycling of the SNARE proteins for subsequent
rounds of fusion (Lin and Scheller, 1997; Rizo and Sudhof, 1998; Sutton
et al., 1998; Weber et al., 1998). Synaptic vesicles are reused up to
1000 times, and factors that affect the recycling of the vesicles and
the fusion machinery will have a major functional impact on exocytosis.
In contrast, dense-core granule exocytosis in adrenal chromaffin cells
involves a one-shot fusion event of a granule derived from the
trans-Golgi network (TGN) (Winkler, 1977), which in the absence of its
released content cannot effectively resequester catecholamine
(Phillips, 1982). Chromaffin cells possess a large pool of mature
granules, which in the absence of stimulation are stable, with their
catecholamine content having a half-life greater than 15 d
(Corcoran et al., 1984). Addition of -SNAP stimulated dense-core
granule exocytosis in permeabilized chromaffin cells (Chamberlain et
al., 1995; Morgan and Burgoyne, 1995b), demonstrating that
SNAP/NSF-mediated SNARE priming could occur before fusion. Data from
patch-clamp capacitance analyses suggest that -SNAP has an early
function in recruitment of vesicles into the releaseable pool (Kibble
et al., 1996; Xu et al., 1999); however, an essential requirement for
SNAP/NSF function before dense-core granule fusion has not been
demonstrated. The -SNAP mutant, -SNAP(L294A) (Barnard et al.,
1997), which is a dominant negative inhibitor of endosome fusion
(Christoforidis et al., 1999), did not inhibit exocytosis in
permeabilized (Barnard et al., 1997) or patch-clamped chromaffin cells
(Xu et al., 1999), which we have argued is attributable to insufficient
time for exchange with endogenous -SNAP. If SNARE priming by
SNAP/NSF is essential in the early steps leading to fusion, then
-SNAP(L294A) should inhibit the extent of exocytosis of the
preformed granules if exchange can occur but not affect the
characteristics of the fusion process.
Many other proteins are likely to function in exocytosis via effects on
the SNARE machinery (Sudhof, 1995; Burgoyne and Morgan, 1998). Among
these is the cysteine string protein (Csp) (Zinsmaier et al., 1990), a
protein found on synaptic vesicles (Mastrogiacomo et al., 1994) and
secretory granules (Chamberlain et al., 1996; Pupier et al., 1997).
Studies in Drosophila have shown that Csp is required for
viability and for evoked neurotransmission (Umbach et al., 1994;
Zinsmaier et al., 1994). It has been suggested that this is
attributable to regulation by Csp of Ca2+
channel function (Gundersen and Umbach, 1992; Umbach et al., 1998). In
contrast, studies on dense-core granule exocytosis have suggested a
direct role for Csp, independent of any effects on Ca2+ channels (Chamberlain and Burgoyne,
1998; Zhang et al., 1998, 1999). Csp can interact with Hsc70 Braun et
al., 1996; Chamberlain and Burgoyne, 1997a,b) and has the properties of
a molecular chaperone (Chamberlain and Burgoyne, 1997b). Its ability to
interact with syntaxin (Wu et al., 1999; Nie et al., 1999)
and/or VAMP (Leveque et al., 1998) would be consistent with a role in
regulating SNARE function. It is not clear, however, whether this would
be exerted in vesicle priming, docking, or fusion itself. Csp
overexpression has a negative effect in insulin-secreting cells (Brown
et al., 1998; Zhang et al., 1999) and in Drosophila (Nie et
al., 1999); therefore, we have examined the effect of transient
Csp overexpression on exocytosis in chromaffin cells. The results show
that both -SNAP(L294A) and Csp overexpression inhibits exocytosis.
Despite the ability of both of these proteins to interact with SNARE
proteins, only in the case of Csp was the kinetics of the individual
fusion events altered. This is consistent with -SNAP having an
essential function early in the exocytotic pathway but Csp exerting its function close to the fusion event.
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MATERIALS AND METHODS |
Reagents. Tissue culture reagents were obtained from
Life Technologies (Paisley, UK), and high-purity digitonin was from
Novabiochem (Nottingham, UK). A plasmid that encodes a
fluorescent-enhanced mutant of green fluorescent protein (pEGFP) was
obtained from Clontech (Basingstoke, UK), and the expression vector
pcDNA3.1 ( ) was from Invitrogen (Leek, The Netherlands). All other
reagents were obtained from Sigma (Poole, UK).
Buffers. Krebs-Ringer's buffer consisted of the
following (in mM): 145 NaCl, 5 KCl, 1.3 MgCl2, 1.2 NaH2PO4, 10 glucose, and 20 HEPES, pH 7.4. Amperometry bath buffer contained (in mM):
139 potassium glutamate, 20 PIPES, 0.2 EGTA, 2 ATP, 2 MgCl2, pH 6.5. Amperometry cell
permeabilization/stimulation buffer contained 139 mM
potassium glutamate, 20 mM PIPES, 5 mM EGTA, 2 mM ATP, 2 mM MgCl2, 20 µM digitonin, and 10 µM
Ca2+, pH 6.5 (total added
CaCl2 was 4.16 mM). PBS
contained (in mM): 142 NaCl, 2 KCl, 8 Na2HPO4, 1.5 NaH2PO4, pH 7.4. PBT buffer
contained PBS and 0.3% BSA and 0.1% Triton X-100.
Plasmid constructs. The his-tagged -SNAP (L294A)
construct was originally created by site-directed mutagenesis in a
pQE-9 vector (Barnard et al., 1997). For expression in mammalian cells, the his-tagged construct was amplified by PCR using Pfu polymerase (Stratagene, Amsterdam, The Netherlands) and cloned into pcDNA3.1() at EcoRI and HindIII restriction sites to create
p-SNAP(L294A). The wild-type -SNAP construct was similarly
subcloned into pcDNA3.1(), and in each case the constructs were
confirmed by automated sequencing. Csp1 in pcDNA3 was as a myc-tagged
construct (Zhang et al., 1999).
Cell culture and transfection. Newly isolated bovine adrenal
chromaffin cells (Burgoyne, 1992) were plated on non-tissue
culture-treated 10 cm Petri dishes at a density of 1 × 106/ml and left overnight. Nonattached
cells were gently pelleted by centrifugation and resuspended in growth
medium at a density of 1 × 107/ml.
pEGFP (20 µg) and 20 µg of p-SNAP(L294A), p-SNAP, or pCsp1 were added per milliliter of cells. Cells and plasmids (1 ml) were
electroporated at 250 V and 975 µF for one pulse, using a Bio-Rad
Gene Pulser II (Bio-Rad, Hercules, CA) and 4 mm cuvettes. Cells were
then rapidly diluted to 1 × 106/ml
with fresh growth media. Cells (1 × 106) were added to 13 mm Petri dishes and
made up to a volume of 3 ml with fresh growth media and maintained in
culture for an additional 3-5 d.
Immunofluorescence. Transfections were performed as
described above except that cells were plated onto round glass
coverslips (13 mm diameter). After washing twice with PBS, cells were
fixed in 3.7% formaldehyde in PBS for 2 hr at room temperature. Cells were then washed twice in PBS, incubated for 30 min in PBT, and incubated overnight with a mouse monoclonal antibody against -SNAP (clone C1 77.2, Synaptic Systems, Gottingen, Germany) at 1:500 dilution
in PBT, anti-Csp antiserum (1:600), or rabbit antiserum against
chromogranin A at 1:1000 (a gift from Prof. G. J. Dockray, The
Physiological Lab, University of Liverpool). After they were washed
three times in PBT, cells were incubated for 1 hr in biotinylated anti-mouse or anti-rabbit IgG (Amersham, Buckinghamshire, UK) at 1:100
in PBT, washed three times with PBT, and finally incubated in
Streptavidin Texas Red (Amersham) at 1:50 dilution in PBT for 30 min.
After cells were mounted, they were viewed with the appropriate filters
to visualize the GPF fluorescence and immunofluorescence.
Amperometric recording. Cells were washed three times with
Krebs-Ringer's buffer, incubated in bath buffer, and viewed using a
Nikon TE300 inverted microscope. Transfected cells were identified as
those fluorescing green (caused by expression of green fluorescent protein) under blue light illumination. A precut 5-µm-diameter carbon
fiber electrode (NPI, Tamm, Germany) was positioned close to a cell
using an Eppendorf (Hamburg, Germany) PatchMan micromanipulator. The
fiber was moved against a cell until visible distortion of the cell
membrane could be seen. The fiber was then pulled away until the point
at which distortion was no longer visible but the fiber remained in
contact with the cell surface. For stimulation, a
digitonin-permeabilization protocol (Jankowski et al., 1992) was used.
A glass micropipette filled with cell permeabilization/stimulation buffer (with 20 µM digitonin and 10 µM free
calcium) was positioned on the opposite side of the cell from the
carbon fiber, ~60 µm from the cell. An Eppendorf Transjector was
used to pressure-eject the buffer onto the cell for a 20 sec pulse. A
holding voltage of +700 mV was applied across the carbon fiber tip and
the Ag/AgCl reference electrode in the bath. Amperometric responses
were monitored with a VA-10 amplifier (NPI Electronic),collected at 4 kHz, digitized with a Digidata 1200B acquisition system, and monitored
online with the AxoScope program (Axon Instruments). Data were
subsequently analyzed using an automated peak detection and analysis
protocol with the technical graphics program Origin (Microcal). Spikes were only analyzed in detail if they had a base width greater than 6 msec and an amplitude greater than 40 pA. This amplitude was chosen so
that analyses were confined to spikes arising immediately beneath the
carbon fiber and to limit effects on the data of diffusion times from
distant sites. All data are shown as mean ± SEM, and statistical
differences were assessed using an unpaired Student's t test.
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RESULTS |
Strategy for transfection and amperometric recording
We have tested whether SNAP/NSF function is essential before
fusion of TGN-derived dense-core granules and the role of Csp by using
transfection to overexpress the mutant -SNAP(L294A) protein and Csp.
To examine the effects on exocytosis in chromaffin cells, we used a
single-cell transfection-amperometry approach (Fisher and Burgoyne,
1999). The cells were cotransfected with control pcDNA3 vector, a
plasmid encoding -SNAP(L294A) or Csp1 and pEGFP to allow
visualization of transfected cells for recording. Electroporation
resulted in transfection, seen by GFP fluorescence, in ~1-5% of the
chromaffin cells. Cotransfection did not change the percentage of cells
that were transfected and had no detectable effect on cell morphology.
To confirm cotransfection with two plasmids, transfected cells were
fixed and stained with concentrations of antisera determined to be too
low to stain control nontransfected cells (Fig.
1a).
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Figure 1.
Cotransfection of adrenal chromaffin cells and
expression of GFP and -SNAP. a, Immunofluorescence
demonstrates expression of GFP and overexpression of -SNAP in the
same cell. Chromaffin cells were transfected with plasmids encoding GFP
and -SNAP(L294A) and after fixation stained with anti--SNAP
antiserum at a concentration below that required to stain
nontransfected cells. A GFP-expressing cell is shown clearly stained
with anti--SNAP, but an attached GFP-negative cell
(asterisk) was barely stained by anti--SNAP. Scale
bar, 10 µm. b, Immunofluorescence staining with
anti-chromogranin A (CGA) after transfection with
plasmids encoding GFP and -SNAP(L294A). Similar extents of staining
were seen in GFP-expressing and nonexpressing cells. c,
Scheme of the recording configuration that was used. Note that the
ejection pipette was actually positioned ~60 µm from the cell to be
stimulated, and the carbon-fiber electrode was positioned in close
contact with the cell surface.
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Exocytosis was detected after transfection using a carbon-fiber
electrode for amperometric recording of catecholamine release (Wightman
et al., 1991) from the transfected (GFP-expressing) cells (Fig.
1c), from control nontransfected cells in the same dish, or
from control cells transfected with GFP and pcDNA3 vector. The cells
were maintained in the absence of Ca2+
(with 0.2 mM EGTA in the bath buffer), and
exocytosis was evoked using pressure ejection from a pipette containing
20 µM digitonin and 10 µM free Ca2+ to
both permeabilize and stimulate the cells directly (Fig.
1c). This approach was taken to bypass any effects of
transfection and expression on agonist receptors or membrane channels
and to allow direct assay of effects of expression on
Ca2+-triggered exocytosis. To optimize the
time resolution of measurements of amperometric spikes, the carbon
fiber was placed in direct contact with the cell surface. Contact did
not induce any responses from the cells and in the absence of a
stimulus, current spikes were rarely seen. Application of
digitonin/Ca2+ evoked a burst of fast,
transient current spikes (with most spikes in the first minute after
perfusion and fewer at later times) characteristic of the kinetics and
amplitude of release of catecholamine from single granule fusion events
(Wightman et al., 1991; Chow et al., 1992; Albillos et al., 1997).
Preliminary experiments established that no spikes were evoked in the
absence of digitonin in the pipette solution (n = 4 cells), demonstrating that permeabilization was essential for responses
to be evoked by perfusion with Ca2+. In
addition, the spikes were essentially indistinguishable from those
evoked in intact cells by agonists and distinct from slow release
events attributable to granule lysis seen after prolonged digitonin
treatment (Jankowski et al., 1992).
Ca2+-induced catecholamine release from
populations of digitonin-permeabilized chromaffin cells has been well
characterized (Burgoyne, 1991) and shown to occur by bona fide
exocytotic machinery. Indeed, under the conditions used here,
Ca2+-evoked amperometric spikes were
almost completely abolished by cotransfection with botulinum toxins C1
or E (Graham et al., 2000), indicating that they occurred via a
SNARE-dependent mechanism.
Expression of -SNAP(L294A) inhibits exocytosis
Cotransfection with GFP and -SNAP(L294A) plasmids resulted in
detectable expression of -SNAP(L294A) in GFP-positive cells. Fig.
1a shows that GFP-expressing cells were brightly stained with a low concentration of anti--SNAP antiserum, indicating overexpression of -SNAP(L294A) in those cells. Almost all
GFP-expressing cells showed -SNAP staining above the background
staining of nontransfected cells. We established that expression of
-SNAP(L294A) did not lead to depletion of secretory granules. Cells
cotransfected with plasmids encoding GFP and -SNAP(L294A) were
stained with an antiserum against the granule content protein
chromogranin A to a similar extent as control nontransfected cells
(Fig. 1b), ruling out the possibility of granule depletion.
It is clear from the example traces shown in Figure
2 that expression of the mutant protein
-SNAP(L294A) resulted in a marked reduction in the number of spikes
evoked after application of digitonin/Ca2+. From a series of
recordings on separate cells, the effect of -SNAP(L294A) expression
on the frequency and characteristics of the spikes was determined.
Expression of the -SNAP(L294A) mutant resulted in a marked (84%,
p < 0.001, Student's t test) reduction (to
3.61 ± 1.77 per cell) in the average number of evoked spikes per cell
compared with nontransfected cells in the same dishes (Fig.
2c). In contrast, transfection with the plasmid encoding GFP
alone did not result in any reduction in mean spike number (24.0 ± 2.95 per cell) compared with control nontransfected cells (22.5 ± 3.9 per cell), indicating that the reduction in spike number with
-SNAP(L294A) was not simply a consequence of transfection occurring
in a nonfunctional subpopulation of cells or to an inhibitory effect of
expression of any exogenous protein in transfected cells. Overexpression of wild-type -SNAP had no statistically significant effect on spike number compared with control cells (data not
shown).
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Figure 2.
Amperometric recordings from control and
-SNAP(L294A) transfected cells. a,b, The traces shown
are examples of control nontransfected cells and cells transfected with
GFP and -SNAP(L294A) assayed in the same dish after stimulation with
a 20 sec pulse of digitonin/Ca2+. The recording is
shown from near the middle of the stimulation pulse (shaded
bar) and continues over the initial period after stimulation.
c, The inset shows the mean spikes per
cell (as mean ± SEM) over a 2 min period after stimulation for control
nontransfected cells (n = 17 cells), cells showing
GFP expression from cultures transfected with pEGFP and pSNAP(L294A)
(n = 18 cells), or pEGFP alone
(n = 5 cells).
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Expression of -SNAP(L294A) does not affect
spike characteristics
Because the extent of exocytosis was reduced by expression of
-SNAP(L294A), the characteristics of the individual residual spikes
were examined. The amplitude and time course of spikes (Fig.
3a) were similar to those
previously reported for both intact and permeabilized chromaffin cells
(Wightman et al., 1991; Chow et al., 1992; Jankowski et al., 1992).
Despite the reduction in spike number, the peak amplitude and the total
charge carried by the spikes that remained were not decreased compared
with control cells (Fig. 3c,d), showing that the
reduction in spike number was not caused by depletion of granule
catecholamine. The overall shape of the spikes did not appear to be
affected by expression of -SNAP(L294A), and the mean values for the
half-widths of the spikes was no different from that of control cells.
The spike characteristics were also no different in cells recorded in
parallel that were transfected with GFP alone. These results indicate
that -SNAP(L294A) expression did not affect the time course of
catecholamine release during individual granule fusion events (Fig.
3b).
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Figure 3.
Analysis of amperometric spikes from control and
-SNAP (L294A) transfected cells. a, Example spikes
from control, nontransfected, and GFP-expressing transfected cells
(L294A) are shown on an expanded time base. Mean values of the
half-width (b), the total charge carried per
spike (c), and the mean value for peak spike
height (d) are shown for spikes from control,
nontransfected cells (n = 383 spikes) and
-SNAP(L294A) transfected cells (n = 65 spikes).
Data are shown as mean ± SEM.
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Overexpression of Csp inhibits exocytosis
Overexpression of Csp by transfection with a plasmid encoding Csp1
(Chamberlain and Burgoyne, 1996) was confirmed by immunofluorescence labeling with a low concentration of anti-Csp that labeled
GFP-positive but not GFP-negative cells. (Fig.
4a).
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Figure 4.
Cotransfection of adrenal chromaffin cells showing
GFP expression and overexpression of Csp. a,
Immunofluorescence demonstrates expression of GFP and overexpression of
Csp in the same cell after staining with a concentration of
anti-Csp antiserum too low to stain surrounding nontransfected
cells (asterisk). b, Immunofluorescence
staining with anti-chromogranin A (CGA) after
transfection with plasmids encoding GFP and Csp1 showing similar levels
of chromogranin A staining in GFP-expressing and nonexpressing cells.
Scale bar, 10 µm.
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Based on the presence of chromogranin A staining, contransfection did
not deplete the granule population (Fig. 4b). After stimulation, Csp overexpression resulted in a reduction in the number
of amperometric spikes induced by
digitonin/Ca2+. Figure
5a,b shows
representative traces from a control cell transfected with the GFP
plasmid plus pcDNA3 and from a cell transfected with the GFP and the
Csp1 plasmids. Recordings from Csp-transfected and control cells were
performed on the same day and with the same carbon-fiber electrodes. In
a series of cells from the two conditions recorded in parallel, Csp
overexpression reduced the average number of evoked spikes by 82%
(p < 0.001, Student's t test), from
17.0 ± 4.2 spikes per cell in cells expressing GFP alone to 3.0 ± 0.9 for cells overexpressing Csp (Fig. 5c).
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Figure 5.
Amperometric recordings from control and
Csp-transfected cells. a, b, The traces shown are
examples of control GFP-expressing cells and Csp-transfected
GFP-expressing cells after stimulation after a 20 sec pulse of
digitonin/Ca2+. The inset shows the
mean spikes per cell (shown as mean ± SEM) over a 2 min period after
stimulation for control cells (n = 18 cells) and
Csp-transfected cells (n = 24 cells).
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Overexpression of Csp modifies the time course of
amperometric spikes
Analysis of the individual amperometric spikes from control cells
and the residual spikes from Csp-transfected cells revealed that
although the mean amplitude (spike height) was unaffected by Csp
overexpression (Fig. 6d), the
total charge per spike was significantly increased
(p < 0.001, Student's t test) (Fig.
6c) from 0.92 ± 0.05 pC for GFP control spikes to 1.49 ± 0.17 pC for spikes from Csp-overexpressing cells. The reason for the
increase in total charge was apparent on close inspection of the
individual spikes. Those from CSP-transfected cells were often broader
than those typically seen in nontransfected (Fig. 3) or control
GFP-expressing cells (Fig. 6a), with an overall increase in
the mean half-width of the spikes of 60% (p < 0.001, Student's t test) caused by Csp overexpression (Fig.
6b). This resulted from a change from a half-width of 6.6 ± 0.24 msec for control GFP spikes to 10.5 ± 0.87 msec for those from
Csp-overexpressing cells, whereas the mean spike amplitudes of 115.7 ± 5.6 pA for GFP control spikes and 105.9 ± 8.5 pA for
Csp-overexpressing cells were not significantly different. In addition,
overlay of the spikes revealed an apparent slowing of the rate of rise
(Fig. 7a), and this was
reflected in a mean 44% increase (p < 0.001, Student's t test) in the mean rise time in Csp-transfected
cells (Fig. 7b) from 5.4 ± 0.16 msec for GFP control spikes
to 7.82 ± 0.46 msec for those from Csp-overexpressing cells. The
differences in spike kinetics in Csp-overexpressing cells are unlikely
to be related to increased instability of granules in these cells after
digitonin application because it has been shown that spontaneous lysis
of granules attributable to prolonged digitonin exposure, for example,
results in slow (half-width 50-250 msec) low-amplitude release events
(Jankowski et al., 1992) distinct from those seen in control or
overexpressing cells in the present study. In addition, we only
analyzed spikes larger than 40 pA, which would exclude such spikes from
our analyses. These data, therefore, implicate Csp in events related to
fusion pore formation or the control of fusion pore opening.
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Figure 6.
Analysis of amperometric spikes from control and
Csp-transfected cells. a, Example spikes representing
the average spikes in control GFP-expressing cells and Csp-transfected
cells on an expanded time base. Mean values of the half-width of the
spikes (b), the total carried per spike
(c), and the mean value for peak spike height
(d) are shown for control (n = 302 spikes) and Csp-transfected (n = 64 spikes).
Data are shown as mean ± SEM.
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Figure 7.
Effect of Csp overexpression on the initial
kinetics of amperometric spikes. a, The spikes showing
the average characteristics from Figure 6 are overlaid to show the
apparent slower rising phase of the spikes from Csp-transfected cells.
b, Mean values (shown as mean ± SEM) for the rise time
of amperometric spikes from control (n = 302 spikes) and Csp-transfected (n = 64 spikes)
cells.
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DISCUSSION |
Many proteins are required to function in defined steps of the
exocytotic pathway to provide the control, speed, and specificity of
Ca2+-regulated exocytosis (Sudhof, 1995).
The sites of action of most identified proteins in this process are
still to be resolved (Burgoyne and Morgan, 1998), and a key question is
the identity of proteins involved in the actual fusion process. In this
study we have used transfection-amperometry (Fisher and Burgoyne,
1999) to analyze and compare the roles of two proteins, -SNAP and
Csp, and in particular made use of the ability of carbon-fiber
amperometry to resolve the kinetics of single fusion events (Wightman
et al., 1991; Chow et al., 1992; Jankowski et al., 1992). These two
proteins were compared because -SNAP is likely to function in early
priming steps in exocytosis (Chamberlain et al., 1995; Burgoyne and
Morgan, 1998; Xu et al., 1999). Csp was earlier suggested to function as a regulator of voltage-dependent Ca2+
channels (Gundersen and Umbach, 1992; Umbach et al., 1998); it has
recently been implicated as a potential regulator of the exocytotic machinery because of its ability to interact with the same exocyotic SNARE proteins as -SNAP, but it is not clear whether it acts in
early or late stages of the pathway. Analysis of the effect of Csp
overexpression suggests that in contrast to -SNAP, Csp interacts
with the exocytotic fusion machinery to play a late role close to the
fusion process.
The approach used in this paper depends on efficient
cotransfection with two plasmids. We demonstrated a high level of
co-transfection using immunofluorescence detection of protein
overexpression, and the functional consequences of -SNAP(L294A) or
Csp transfection on generation of amperometric spikes also indicated a
high (~85%) level of cotransfection. The data presented here show
that expression of -SNAP(L294A) or overexpression of Csp in
chromaffin cells after transfection significantly inhibited
Ca2+-evoked exocytosis. This could not be
caused by effects on synthesis of new granules because chromaffin cells
maintain a large (30,000 per cell) and stable pool of mature granules
(Winkler, 1977; Corcoran et al., 1984) in the absence of stimulation.
These have a half-life much longer than the transfection period that
was used, and on the basis of chromogranin A staining, transfected
cells did not appear to be depleted of secretory granules.
Synthesis of new granules in these cells can be readily detected only
after massive nonphysiological stimulation to deplete preexisting
granules and to activate pathways for granule biogenesis (Winkler and
Fischer-Colbrie, 1998). In addition, because the stimulation was based
on the use of permeabilized cells with direct activation of exocytosis
by Ca2+, the inhibitory effects cannot be
attributed to effects on membrane channels.
Study of the temperature-sensitive comatose mutant in
Drosophila has produced conflicting data on the site of
accumulation of the SNARE complex at the restrictive temperature. This
has lead to different interpretations about the site of action of NSF
either before or after fusion (Kawasaki et al., 1998; Littleton et al.,
1998; Tolar and Pallanck, 1998). It is important that in contrast to
the nerve terminal, the extent of exocytosis should not be affected by
interference with vesicle membrane or SNARE protein recycling in the
chromaffin cell in which there is one-shot usage of TGN-derived
granules. -SNAP(L294A) can recruit NSF to SNAREs but is unable to
stimulate the ATPase activity of NSF (Barnard et al., 1997) and so
cannot support SNARE priming (Barnard et al., 1997; Christoforidis et
al., 1999). The simplest interpretation of the data from
-SNAP(L294A) expression, therefore, is that SNAP-dependent SNARE
priming through NSF ATPase activity must be required before fusion of
naive secretory granules. Such a priming event could involve priming of
SNAREs on the granule or the plasma membrane or both and does not rule
out an additional role of SNAP/NSF in SNARE complex disassembly and
recycling after fusion that would also be crucial for synaptic transmission.
There is abundant evidence that the SNAREs need not be part of a full
complex to act as SNAP receptors because syntaxin alone (Hanson et al.,
1995) and also the syntaxin-SNAP-25 dimeric complex (Hayashi et al.,
1995) can bind -SNAP and support SNAP/NSF-mediated disassembly and
conformational change. It is likely, therefore, that the SNAP/NSF
chaperones (Morgan and Burgoyne, 1995a) interact with SNAREs at
multiple points of the vesicle cycle (Burgoyne and Morgan, 1998).
SNAREs (Tagaya et al., 1995; Hohne-Zell and Gratzl, 1996; Otto et al.,
1997), -SNAP, and NSF (Hong et al., 1994; Burgoyne and Williams,
1997) are present on chromaffin granules and synaptic vesicles, and so
SNAP/NSF-mediated priming could occur on undocked secretory vesicles.
Alternatively, priming could occur on vesicles already tethered to the
plasma membrane (and seen as morphologically docked) (Banerjee et al.,
1996) but in either case would be most likely to occur as a prelude to
SNARE complex formation at the site of fusion. It has recently been suggested that NSF and -SNAP can act directly as membrane fusogens (Otter-Nilsson et al., 1999), but data from chromaffin cells clearly show that late steps in Ca2+-triggered
fusion do not require ATP hydrolysis (Parsons et al., 1995; Xu et al.,
1999) and are insensitive to N-ethylmaleimide arguing
against a late role for NSF (Xu et al., 1999). Expression of
-SNAP(L294A), although depressing the number of release events, did
not affect the characteristics of the remaining amperometric spikes,
indicating that the -SNAP mutant did not affect the kinetics of
individual fusion events. The use of chromaffin cells allows experimental investigation of effects of expressed proteins on only the
outward arm of the exocytotic cycle, and therefore the data demonstrate
an essential role for SNAP-mediated SNARE priming before fusion in
Ca2+-regulated exocytosis of dense-core granules.
The data from the analysis of Csp overexpression demonstrate that not
only does this reduce the extent of exocytosis but it also modifies the
kinetics of the residual amperometric spikes, resulting in an increase
in the spike half-width and an increase in the mean rise time. An
increase in spike half-width could have resulted from an increase in
granule size or in the amount of catecholamine content per granule.
This is unlikely, however, to explain the changes in the kinetics of
the rising phase of the spike attributable to Csp overexpression. The
effects of Csp overexpression are also unlikely to be caused by an
effect of granule biogenesis given the large number of stable preformed granules in these cells as discussed above. The rise time of the amperometric spike is likely to represent the kinetics of the initial
fusion event or more likely the kinetics of fusion pore opening. These
results, therefore, functionally demonstrate that Csp exerts its role
on the fusion machinery in chromaffin cells. Previous work has shown
that stable overexpression of Csp in PC12 cells enhanced exocytosis in
permeabilized cells (Chamberlain and Burgoyne, 1998), but as in the
present study, transient overexpression in insulin-secreting cells was
inhibitory (Zhang et al., 1999). Other examples are known in which a
protein required for exocytosis, including syntaxin, is inhibitory when
overexpressed (Schulze et al., 1994; Fujita et al., 1998; Wu et al.,
1998). The reason for the difference between transient and stable
overexpression of Csp in the effect on exocytosis is unclear, but one
possible explanation is that stable overexpression of Csp in PC12 cells resulted in compensatory changes in the exocytotic machinery. This
difference remains to be resolved. The inhibitory effect of Csp
overexpression on exocytosis does not appear to require interaction of
Csp with Hsc70. Mutations within the HPD motif of Csp prevent
activation of the ATPase activity of Hsc70 (Chamberlain and Burgoyne,
1997b) but do not prevent the inhibitory effect of Csp overexpression
on exocytosis (Zhang et al., 1999). Two other studies have examined
single fusion events in situations of modified Csp expression without
reporting changes in release kinetics. First, no changes in spontaneous
release events were reported in Drosophila Csp null mutants
(Umbach et al., 1994). Second, overexpression of Csp in INS-1
cells was found to inhibit amperometric events (Brown et al., 1998),
but no data on the kinetics of individual release events were
described. In both studies the small size of the events may have
precluded the type of analysis possible here with examination of the
release from large chromaffin granules.
Csp has been subject to debate over whether its primary action in
regulated exocytosis is to stimulate Ca2+
entry through Ca2+ channels (Gundersen and
Umbach, 1992; Umbach et al., 1998), to regulate SNARE assembly at
Ca2+ channels (Leveque et al., 1998), or
to more directly affect the exocytotic pathway (Chamberlain and
Burgoyne, 1998; Zhang et al., 1998, 1999). The data here and from
previous studies in which effects are preserved in permeabilized cells
(Chamberlain and Burgoyne, 1998; Zhang et al., 1998, 1999) argue for a
Ca2+ channel-independent function for Csp.
We are now able to extend this further from the analysis of individual
amperometric spikes in Csp-overexpressing cells. If the inhibitory
effect of Csp was caused by a blockade of vesicle priming or docking,
then we would expect to see only a reduction in spike number and no
effect on spike kinetics as seen for -SNAP(L294A). In contrast, the
data from Csp-overexpressing cells, demonstrating a change in the
kinetics of the release event, would be consistent with a postdocking
role for Csp acting directly or indirectly at the level of proteins in
the fusion machinery or involved in fusion pore expansion. An effect on
the kinetics of the release event has recently been seen resulting
from expression of a SNAP-25 mutant (Criado et al., 1999),
indicating that such changes reflect modifications to the fusion machinery.
The possession by Csp of a J domain allowing it to interact with Hsc70
and its ability to act as a general molecular chaperone (Braun et al.,
1996; Chamberlain and Burgoyne, 1997a,b) suggests interaction with
various substrate proteins. Recent work has shown that
Drosophila Csp can interact with syntaxin in
vitro (Wu et al., 1999) and in vivo (Nie et al., 1999).
The significance of this for the mammalian Csp is unclear because this
was reported not to bind to syntaxin but was immunoprecipitated with
VAMP (Leveque et al., 1998). The ability of Csp to interact with SNARE
proteins does not provide any detailed insight as to how and when its
function is exerted and whether this would be seen as changes in
vesicle priming, docking, fusion, endocytosis, or recycling. The
function of Csp in these steps has been unclear, but we now provide a
resolution to this issue and implicate Csp in late stages of the fusion
process. It seems probable that a major function of Csp is in the
correct folding of SNARE and other key proteins of the exocytotic
fusion machinery, and thus it directly influences fusion pore kinetics.
| |
FOOTNOTES |
Received Sept. 29, 1999; revised Nov. 15, 1999; accepted Nov. 19, 1999.
This work was supported by a grant from The Wellcome Trust to
R.D.B.
Correspondence should be addressed to Dr. Robert D. Burgoyne, The
Physiological Laboratory, The University of Liverpool, Crown Street,
Liverpool L69 3BX, UK. E-mail:
burgoyne{at}liv.ac.uk.
| |
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ABSTRACT
INTRODUCTION
