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The Journal of Neuroscience, December 15, 1998, 18(24):10241-10249
Synaptic Physiology and Ultrastructure in comatose
Mutants Define an In Vivo Role for NSF in Neurotransmitter
Release
Fumiko
Kawasaki,
Annette M.
Mattiuz, and
Richard W.
Ordway
Department of Biology, The Pennsylvania State University,
University Park, Pennsylvania 16802
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ABSTRACT |
N-Ethylmaleimide-sensitive fusion protein
(NSF) is a cytosolic protein thought to play a key role in vesicular
transport in all eukaryotic cells. Although NSF was proposed to
function in the trafficking of synaptic vesicles responsible for
neurotransmitter release, only recently have in vivo
experiments begun to reveal a specific function for NSF in this
process. Our previous work showed that mutations in a
Drosophila NSF gene, dNSF1, are responsible for the
temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, we perform
electrophysiological and ultrastructural analyses in three different
comt alleles to investigate the function of dNSF1 at
native synapses in vivo. Electrophysiological analysis
of postsynaptic potentials and currents at adult neuromuscular synapses
revealed that in the absence of repetitive stimulation,
comt synapses exhibit wild-type neurotransmitter release
at restrictive (paralytic) temperatures. In contrast, repetitive
stimulation at restrictive temperatures revealed a progressive,
activity-dependent reduction in neurotransmitter release in
comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the
target membrane but rather functions in maintaining the pool of readily
releasable vesicles competent for fast calcium-triggered fusion. To
define dNSF1 function further, we used transmission electron microscopy
to examine the distribution of vesicles within synaptic terminals, and
observed a marked accumulation of docked vesicles at restrictive
temperatures in comt. Together, the results reported
here define a role for dNSF1 in the priming of docked synaptic vesicles
for calcium-triggered fusion.
Key words:
NSF; neurotransmitter release; Drosophila; comatose; synaptic vesicle; neuromuscular transmission
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INTRODUCTION |
The molecular machinery underlying
chemical synaptic transmission represents a fundamental element of
neural function. One critical aspect of this process is the highly
regulated and rapid form of exocytosis responsible for the release of
neurotransmitter. A number of proteins has now been identified as
components of the neurotransmitter release apparatus, and recent
progress has begun to address their specific roles.
The N-ethylmaleimide-sensitive fusion protein (NSF) is a
soluble, oligomeric ATPase that served as one of the primary starting points from which this progress has developed. NSF was identified as an
N-ethylmaleimide (NEM)-sensitive factor required for Golgi transport in vitro (Wilson et al., 1989 ; Rothman, 1994) and
was termed a fusion protein on the basis that NEM caused an
accumulation of transport vesicles at target membranes (Orci et al.,
1989). The biochemical connection to membranes was made by the elegant characterization of an NSF-containing protein complex including the
soluble NSF attachment proteins (SNAPs) (Clary et al., 1990; Whiteheart
et al., 1993) and their membrane receptors, SNAP receptors (SNAREs)
(Söllner et al., 1993; Südhof, 1995). In parallel with these studies, genetic analysis of constitutive secretion in yeast (Schekman, 1992; Bennett and Scheller, 1993; Ferro-Novick and Jahn,
1994), together with biochemical and functional analysis of synaptic
proteins (Huttner, 1993; Südhof, 1995), independently identified
and characterized the NSF, SNAP, and SNARE proteins. Thus a convergence
of results revealed a set of core proteins functioning in intracellular
vesicle trafficking in perhaps all eukaryotes.
The SNARE hypothesis (Rothman, 1994; Rothman and Wieland, 1996) was
developed to explain the docking and fusion of transport vesicles with
their target membranes. According to this hypothesis, vesicle docking
involves assembly of a protein complex including vesicle SNAREs (e.g.,
synaptobrevin) and target SNAREs (e.g., SNAP-25 and syntaxin). Because
different SNAREs seemed to function at distinct stages of the secretory
pathway in yeast (Ferro-Novick and Jahn, 1994), the specificity of
vesicle targeting was attributed to these proteins. It was further
proposed that after docking, the SNARE complex is joined by the
cytosolic SNAP and NSF proteins to form a fusion particle. Finally,
hydrolysis of ATP by NSF was proposed to disassemble or rearrange this
structure in a subsequent step necessary for fusion. The SNARE
hypothesis has provided an excellent working model and has been both
supported and challenged by subsequent functional analysis of
constitutive membrane trafficking (Mayer et al., 1996; Nichols et al.,
1997; Ungermann et al., 1998).
Regarding the role of SNARE complexes in neurotransmitter release,
in vivo work in Drosophila (Broadie et al., 1995;
Schulze et al., 1995; Sweeney et al., 1995; Deitcher et al., 1998), in Caenorhabditis elegans (Nonet et al., 1998), and at
the squid giant synapse (Hunt et al., 1994; O'Connor et al., 1997) has
been informative. Disruption of individual vesicle or target SNAREs in
these systems eliminated or reduced evoked neurotransmitter release.
However, in apparent contrast to predictions of the SNARE hypothesis,
ultrastructural analysis (Hunt et al., 1994; Broadie et al., 1995;
O'Connor et al., 1997) indicated that these SNAREs, at least
individually, are dispensable for specific targeting and docking of
synaptic vesicles. Regarding the role of NSF and SNAPs, only recently
have in vivo experiments begun to address their function in
neurotransmitter release (DeBello et al., 1995; Schweizer et al.,
1998). Our previous work has demonstrated that a Drosophila
NSF homolog, dNSF1, represents the comatose
(comt) locus (Pallanck et al., 1995a). Temperature-sensitive
comt alleles were shown previously to exhibit rapid
paralysis and a neurophysiological defect at elevated temperatures
(Siddiqi and Benzer, 1976). Here we use electrophysiological and
ultrastructural analysis in comt to define an in
vivo role for the dNSF1 gene product in neurotransmitter release.
Parts of this work have been published in preliminary form (Kawasaki
and Ordway, 1997, 1998; Ordway et al., 1998).
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MATERIALS AND METHODS |
Dissection
Male or female flies 1-4 d of age were anesthetized using
CO2 and then mounted over a hole in an air tube and secured
with wax as described (Koenig et al., 1989). Air was delivered to the tracheal system using an aquarium pump. In all experiments the chamber
was perfused at 0.5 ml/min with recording solution (see Microelectrode recordings).
Electrophysiological recordings. The fly was mounted
laterally and dissected in recording solution to expose the lateral
surface of one set of dorsal longitudinal flight muscles (DLMs)
as well as the thoracic ganglion of the CNS. The posterior
dorsal mesothoracic nerve, through which the DLM motor axons
project from the thoracic ganglion to the muscle, was cut and pulled
into a suction electrode for stimulation. Temperature was maintained at
20°C during dissection.
Ultrastructural analysis. The fly was mounted ventral
side-up after the head, wings, and the second and third pairs of legs were removed. The fly was dissected to expose the sternal anterior rotator coxal muscles of the most anterior legs as described (Koenig et
al., 1989). The same saline (recording) solution was used for these and
the electrophysiology experiments.
Temperature control. Temperature control was achieved using
a TC-202 temperature controller and PDMI
microincubator (Medical Systems Corporation, Greenvale, NY).
Temperature shifts from 20 to 36°C typically required ~4.5 min.
Microelectrode recordings. These recordings were performed
by conventional methods using an IX2-700 amplifier (Dagan
Corporation, Minneapolis, MN) and glass microelectrodes filled with 3 M KCl (~30 M ). The recording solution consisted of (in
mM): 128 NaCl, 2 KCl, 4 MgCl2, 1.8 CaCl2, 5 HEPES, and 36 sucrose. The pH was adjusted
to 7.0 using NaOH. DLM resting potentials were typically  90 to 95
mV. Under the same recording conditions, two-electrode voltage clamp
was performed using a TEV-200 amplifier (Dagan
Corporation). In the voltage-clamp recordings, deviations from
the command potential during recording of synaptic currents typically
did not exceed 2 mV.
Electrophysiological data acquisition and analysis
Data were acquired on-line using a Power Macintosh 7500 computer, Pulse software (HEKA Elektronik, Lambrecht, Germany), and an
ITC-16 laboratory interface (Instrutech Corporation, Great Neck,
NY). Data were low-pass filtered at 3 kHz and acquired at 10-30 kHz.
Stimulation was achieved using the Pulse program to trigger an
S-900/S-910 stimulator (Dagan Corporation). Current measurements were
performed in the data analysis program IGOR (WaveMetrics, Lake Oswego,
OR). Peak values were obtained using cursor measurements, and total
charge was determined using the area function. Microsoft Excel was used
for data tabulation, graphing, and statistical analysis. By the use of
an unpaired Student's t test, statistical significance was
assigned to comparisons with p values  0.05.
Transmission electron microscopy
After a 5 min incubation at the experimental temperature in
saline solution (see Dissection), fixation was initiated by exposing the preparation to the same saline solution containing 2.5%
paraformaldehyde and 1.5% glutaraldehyde (the primary fixative).
Samples remained in primary fixative at room temperature for 1-3 hr
and then overnight at 4°C. Further processing was performed using
conventional methods. Briefly, fixed samples were processed in 2%
aqueous osmium tetroxide and then in 2% aqueous uranyl acetate,
dehydrated using an ethanol series, and embedded in Spurr resin
(EM Sciences, Fort Washington, PA). Thin sections, ~60 nm in
thickness, were cut on an ultramicrotome and stained with uranyl
acetate/lead citrate before viewing on a Jeol 1200EXII transmission
electron microscope (TEM) housed at the Penn State University
Electron Microscopy Facility.
TEM data analysis
Vesicle counts were performed directly on TEM negatives.
Statistical significance was determined using an unpaired Student's t test. Significant differences were assigned to comparisons
with p values 0.05. Data in the manuscript are
presented as the mean ± SEM, and n represents the
number of active zones analyzed. In all cases a minimum of three
different preparations were examined. Images were acquired from
photographic prints using a UMAX flatbed scanner and Adobe
Photoshop software.
Drosophila lines and transformation rescue
All Drosophila lines used were cultured at 20°C.
The three temperature-sensitive comt alleles used in this
study contain point mutations leading to single amino acid changes.
Missense mutations in comtST17 and
comtST53 have been reported previously
(Pallanck et al., 1995a); comtTP7
contains a missense mutation converting proline 398 to serine (R. Ordway and L. Pallanck, unpublished observations). Rescue experiments used a transgene construct containing a wild-type dNSF1
cDNA under the control of an hsp70 heat-shock promoter
(Pallanck et al., 1995a). For rescue experiments, comt flies
bearing a transgene were subjected to three heat shocks separated by 24 hr intervals (all heat shocks were at 38°C). A 15 min heat shock on
the first day was followed by 30 min heat shocks on the second and
third days. Recordings were obtained ~24 hr after the last heat
shock. In the case of the comtST53 rescue
experiments, the third heat shock was omitted. As is the case for
temperature-sensitive paralytic behavior (Pallanck et al., 1995a),
controls in which comt flies lacking the transgene were
heat-shocked demonstrated that phenotypic rescue was entirely dependent
on the presence of the transgene.
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RESULTS |
Intracellular recordings from the dorsal longitudinal flight
muscles (DLMs) were used to monitor postsynaptic potentials
elicited by direct stimulation of the DLM motor axon. Typically the
postsynaptic potential resulting from neurotransmitter activation of
ligand-gated receptors triggers a DLM action potential (Fig.
1). As reported previously, a progressive
reduction in neurotransmitter release produces a graded reduction and
eventual loss of this action potential as the underlying postsynaptic
potential is reduced (Koenig et al., 1983).
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Figure 1.
Reversible reduction of the postsynaptic potential
in comtST17 as a result of repetitive
stimulation at a restrictive temperature. Recordings are of DLM
postsynaptic potentials elicited by direct stimulation of the DLM motor
axon. Each panel shows superimposed
traces representing the first 100 responses generated during a
1 Hz stimulation train. In the case of
comtST17 at 36°C, every eighth
trace is shown. The stimulation trains at the
restrictive temperature were initiated after 3 min at 36°C. Recovery
of comtST17 is shown 60 min after
return to 20°C. For both wild type (WT) and
comtST17, data at 36°C and at
20°C recovery were obtained from the same cell.
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At a permissive temperature (20°C), DLM action potentials in
comtST17 were indistinguishable from
those in wild-type flies and remained constant during 1 Hz stimulation
(Fig. 1). At restrictive temperatures in comt (e.g., 36°C
as shown), the first stimulus in the train also produced a wild-type
action potential. However, subsequent stimuli revealed a striking
phenotype at comt synapses under these conditions. During a
1 Hz stimulus train, a progressive reduction and then loss of the DLM
action potential revealed the underlying postsynaptic potential, which
continued to progressively decrease in amplitude. This
activity-dependent reduction was reversible and, like
temperature-sensitive paralysis, recovered slowly over a period of
~60 min after return to 20°C (Fig. 1).
The above recordings extend the initial neurophysiological studies in
comt (Siddiqi and Benzer, 1976) which used microelectrode recordings of DLM responses to central neural stimulation or simply compound extracellular recordings from the eye (electroretinograms or
ERGs). However, to characterize the functional role of dNSF1, additional approaches are required. To address whether the reduction in
the postsynaptic potential reflects a reduction in neurotransmitter release and to examine whether the kinetics of neurotransmitter release
are altered in comt, two-electrode voltage-clamp analysis was performed to record DLM synaptic currents.
At 36°C in comtST17, the first stimulus
elicited a synaptic current indistinguishable from wild type under
the same conditions (Fig.
2A). The peak current
amplitudes in wild type and comtST17 were
2.37 ± 0.19 µA (n = 5) and 2.36 ± 0.16 µA (n = 5), respectively. Furthermore, time course
measurements yielded essentially identical values for the time-to-peak
current (0.76 ± 0.05 msec in wild type and 0.74 ± 0.04 msec
in comtST17) and for the time for decay
to half amplitude (0.40 ± 0.03 msec in wild type and 0.38 ± 0.03 msec in comtST17). Thus we conclude
that the first stimulus in comtST17 at a
restrictive temperature elicits neurotransmitter release at wild-type
levels and with wild-type kinetics, indicating that regulated
exocytosis and postsynaptic receptor function are normal under these
conditions.
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Figure 2.
Activity-dependent reduction of the synaptic
current in comtST17.
A, Two-electrode voltage-clamp recordings of DLM
synaptic currents at a restrictive temperature in wild type and
comtST17 are shown. Wild type and
comtST17 were exposed to 36°C for
10 and 9 min, respectively. Each panel shows
representative traces from the first 100 responses
elicited during a 1 Hz stimulation train. The holding potential was
80 mV. Arrows indicate stimulation of the motor axon;
stimulation artifacts were removed. B, Peak amplitude
measurements of DLM EPSCs are plotted as a function of time for
1 Hz stimulation trains initiated after 8-10 min at 36°C. The 0 time
point is the beginning of the stimulus train. Each point represents the
average amplitude ± SEM for four cells from four different
preparations.
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Although the first stimulus produced a wild-type response in
comt, repetitive stimulation at a restrictive temperature
caused a progressive reduction in the synaptic current in
comtST17 but not in wild type (Fig.
2A). Quantitation of this reduction was performed by
comparing peak synaptic currents in wild-type and
comtST17 in response to 1 Hz stimulation
at 36°C. These data, shown in Figure 2B, indicate
that comt exhibits a reproducible and strictly activity-dependent decrease in the synaptic current. Because basic mechanisms of synaptic transmission seem to operate normally at a
restrictive temperature in comt, the activity-dependent
reduction in the synaptic current indicates that dNSF1 functions in
maintaining neurotransmitter release during repetitive stimulation.
As seen in Figure 2A, the activity-dependent
reduction in neurotransmitter release observed in comt was
associated with a slowing of the kinetics of the synaptic current. At
36°C, the 50th response in a 1 Hz stimulus train exhibits a 62%
decrease in current amplitude (Fig. 2B). Comparing
the kinetics of the first (see above) and the 50th responses revealed a
12% increase in the time to peak (0.84 ± 0.02 msec for the 50th
response; n = 5) and a 2.18-fold increase in the time
for decay to half amplitude (0.83 ± 0.07 msec for the 50th
response; n = 5). These changes in current kinetics
raise the possibility that peak current measurements may not accurately
represent changes in neurotransmitter release. Thus area measurements
may be used to determine the total charge flowing during the synaptic
current. To supplement the peak current measurements reported in Figure
2B, we performed area measurements on the same
currents. This method yielded a 37% decrease in the synaptic current
in comtST17, from 2.00 ± 0.15 to
1.27 ± 0.18 nC of charge for the first and 50th responses,
respectively (n = 5).
To address whether the change in the kinetics of the synaptic current
in comt is specifically related to dNSF1 function or rather
is generally characteristic of a reduction in neurotransmitter release,
we examined a temperature-sensitive paralytic mutant in which
neurotransmitter release is reduced by a different mechanism. At
restrictive temperatures, shibire (shi) mutations
disrupt synaptic vesicle endocytosis, resulting in depletion of
synaptic vesicles and an activity-dependent reduction in
neurotransmitter release (see Koenig et al., 1983, 1989).
comt and shi currents were compared at 33°C
because shi currents were greatly reduced at higher
temperatures. As seen in Figure 3, a
decrease in neurotransmitter release in shi produced a
slowing of the synaptic current similar to that observed in
comt. For the comtST17 and
shiTS1 currents shown, the amplitude of
the first response was 1.97 and 1.93 µA, the time to peak was 0.79 and 0.74 msec, and the time for decay to half amplitude was 0.43 and
0.48 msec, respectively. After neurotransmitter release was reduced to
0.79 µA in each case, the comtST17 and
shiTS1 currents exhibited time-to-peak
values of 0.86 and 0.86 msec and time for decay to half amplitude
values of 0.75 and 0.72 msec, respectively. The similarity of these
changes in current kinetics suggests that they are a general
consequence of reduced neurotransmitter release and further suggests
that dNSF1 activity does not influence the kinetics of synaptic vesicle
fusion.
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Figure 3.
Reducing neurotransmitter release in
shi mutants produces a slowing of synaptic current
kinetics similar to that observed in comt. DLM synaptic
currents were recorded in comtST17
and shiTS1 during 1 Hz stimulation at
33°C. In comtST17, the first and
43rd responses are shown. In shiTS1,
the first and 77th responses are shown.
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Finally, an informative aspect of the activity-dependent phenotype in
comt is shown in Figure 4.
Five Hertz stimulation at 36°C was used to markedly reduce the
synaptic current in comtST17.
Subsequently resting the synapse while maintaining the restrictive temperature resulted in complete recovery of the current; a second 5 Hz
stimulus train then produced the same progressive reduction seen during
the first train.
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Figure 4.
Recovery of the postsynaptic current at a
restrictive temperature in the absence of stimulation. DLM postsynaptic
currents were recorded from comtST17
in response to two 5 Hz stimulation trains at 36°C. The stimulation
trains were 200 pulses in length, and each panel shows
representative traces superimposed. The first train was
initiated after 21 min at 36°C. After 2 min and 20 sec at
36°C without stimulation, the second train was initiated.
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To test the generality of the above phenotype and to verify that it can
be attributed entirely to the dNSF1 mutations, we recorded DLM action
potentials from two additional comt alleles, as well as from
comt flies in which the temperature-sensitive paralytic
phenotype has been rescued by transgenic expression of wild-type dNSF1
protein. Like in comtST17, both
comtTP7 and
comtST53 exhibited wild-type action
potentials at 20°C and a strictly activity-dependent reduction in the
action potential and postsynaptic potential at the restrictive
temperature (Fig. 5A). Unlike
in comtST17, 1 Hz stimulation at 36°C
did not produce a marked reduction in either of these alleles. At a
higher stimulation frequency of 10 Hz, however, both alleles exhibited
a clear reduction, whereas wild-type responses were constant under
these conditions (data not shown, but see rescue data in Fig.
5B). These results indicate that the activity-dependent
reduction in neurotransmitter release results from a general loss of
dNSF1 function rather than from any specific amino acid substitution in
the dNSF1 protein. DLM recordings were also performed using flies in
which a transgene expressing the wild-type dNSF1 protein was introduced
into a comt mutant background. After transgenic expression
of wild-type dNSF1 protein, the activity-dependent reduction in
neurotransmitter release, like the temperature-sensitive paralytic
behavior (Pallanck et al., 1995a), was dramatically rescued (Fig.
5B). Although not shown, the synaptic phenotype in
comtST17 was also completely rescued.
These experiments demonstrate that the reduction in neurotransmitter
release results entirely from mutations in the dNSF1 gene.
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Figure 5.
Reduction of the postsynaptic potential and
transformation rescue in two additional comt alleles.
A, DLM action potentials and postsynaptic potentials
recorded from comtTP7 and
comtST53 during 10 Hz stimulation
trains initiated after 7 min at 36°C. B, Recordings
similar to those shown in A after transgenic expression
of wild-type dNSF1 protein in a
comtTP7 or
comtST53 genetic background. In
comtTP7 Rescue and
comtST53 Rescue flies,
the comt phenotype has been rescued by transgenic
expression of wild-type dNSF1 protein. To emphasize the rescue, we
performed recordings in B at 38°C; similar results
were obtained at 36°C. In all four panels,
traces representing the first 50 responses are shown. In
A, every fourth trace is
shown.
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To determine whether the physiological defect in comt is
associated with an alteration in the distribution of synaptic vesicles, ultrastructural studies were performed at neuromuscular synapses of the
coxal muscles controlling the anterior legs. This preparation was used
because it is much more favorable for ultrastructural analysis of
neuromuscular synapses than the DLM (Koenig et al., 1989). Transmission
electron microscopy was used to image synaptic terminals and the
presynaptic membrane densities (active zones) that represent sites of
synaptic vesicle docking and fusion. Synaptic terminals in
comt and wild-type flies were examined at both a permissive
temperature (20°C) and restrictive temperatures (33 or 36°C). At
all temperatures, the general appearance of the comt and
wild-type terminals, including the total number of synaptic vesicles,
was quite similar. However, at restrictive temperatures, closer
examination of active zones revealed that comt mutants exhibited a striking elevation in the number of docked vesicles (those
in contact with the plasma membrane at active zones) compared with wild
type (Figs. 6,
7). For example, at 33°C the number of docked vesicles per active zone was 2.6-fold higher in
comtTP7 [3.57 ± 0.39 (n = 14)] than in wild type [1.35 ± 0.32 (n = 17)]; similar results were obtained in
comtST53 and
comtST17. At 36°C in comt,
extreme accumulation of docked vesicles precluded accurate vesicle
counts (Fig. 6). Thus a marked accumulation of docked vesicles occurred
in comt at restrictive temperatures. The number of undocked
synaptic vesicles located within 200 nm (four to five vesicle
diameters) of the active zone was also examined, revealing a moderate
but statistically significant increase in comtTP7 [12.62 ± 1.73 (n = 13)] relative to wild type [8.24 ± 1.11 (n = 17)] at 33°C.
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Figure 6.
Docked vesicles accumulate in
comt at elevated temperatures. TEM images of active
zones from wild-type, comtTP7, and
comtST17 at permissive and
restrictive temperatures. comtTP7
images are shown at 20 and 33°C;
comtST17 is shown at 36°C. Scale
bar, 50 nm.
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Figure 7.
Quantitation of docked vesicles in wild-type and
comtTP7 flies. Docked vesicles were
counted as those in contact with the plasma membrane at the active
zone. The number of active zones analyzed for each condition is as
follows: for WT at 20°C, n = 9;
for WT at 33°C, n = 17; for
comtTP7 at 20°C,
n = 14; for
comtTP7 at 33°C,
n = 14; and for comtTP7
Rescue at 33°C, n = 15. An
asterisk denotes a value significantly different from
that of WT at the same temperature.
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Expression of wild-type dNSF1 protein in transgenic flies restored the
number of docked vesicles at 33°C to wild-type levels (Fig. 7). Thus,
as was the case for the electrophysiological phenotype, the
accumulation of docked vesicles in comt was shown to result entirely from a deficit in dNSF1 function. Furthermore, the fact that
multiple comt alleles exhibit similar ultrastructural
phenotypes indicates that the accumulation of docked vesicles is a
general consequence of disrupting dNSF1 activity.
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DISCUSSION |
The work presented here uses comt mutants, in which
single gene mutations result in temperature-sensitive dNSF1 activity, to address the function of NSF at mature, native synapses in
vivo. One key observation is that, at a restrictive temperature,
comt exhibits wild-type neurotransmitter release in the
absence of repetitive stimulation. A second important observation is
that repetitive stimulation causes a progressive, activity-dependent reduction in neurotransmitter release under the same conditions.
At a restrictive temperature in the absence of repetitive stimulation,
comt exhibits wild-type postsynaptic currents and
potentials. Thus we infer that basic synaptic mechanisms including
calcium signaling, the vesicle fusion process, and excitation of the
postsynaptic membrane can operate normally under these conditions.
Furthermore, the experiment showing recovery of the synaptic current
during a rest period at a restrictive temperature (Fig. 4) indicates that this is true even when vesicles are maintained at a restrictive temperature when recruited to the readily releasable pool (those vesicles competent to fuse in response to an action potential). Thus
persistence of normal fusion at a restrictive temperature cannot be
explained by a protected pool of readily releasable vesicles in which
the dNSF1 requirement was satisfied before temperature elevation.
Finally, experiments observing the kinetics of neurotransmitter release
in shi mutants suggest that the change in kinetics observed when synaptic currents are reduced in comt is secondary to
the reduction in neurotransmitter release. Taken together, the above observations indicate that dNSF1 does not participate directly in
vesicle fusion, an observation of particular interest given the initial
characterization of dNSF1 as a fusion protein (Rothman, 1994).
A second important finding is that at restrictive temperatures in
comt, repetitive stimulation causes a progressive,
activity-dependent reduction in neurotransmitter release, indicating
that dNSF1 functions in maintaining neurotransmitter release.
Maintenance of release requires restoration of the readily releasable
pool of synaptic vesicles. This is thought to occur by retrieval of
vesicles from the plasma membrane by endocytosis, vesicle targeting and
docking at active zones, and finally priming of docked vesicles leading to competence for fast calcium-triggered fusion (Pieribone et al., 1995). We do not attribute the comt phenotype to a
defect in synaptic vesicle endocytosis, both because the rapid
activity-dependent onset (<1 sec at 36°C; see Fig. 4) is more than
an order of magnitude faster than estimates of the minimum time
required for synaptic vesicle recycling (Betz and Wu, 1995) and because
no general depletion of vesicles was seen in the ultrastructural
studies. Thus the electrophysiological evidence indicates a role for
dNSF1 either in the targeting and/or docking of synaptic vesicles or in
the priming process after docking.
Ultrastructural analysis further defined the role of NSF by
demonstrating a marked accumulation of docked synaptic vesicles at
restrictive temperatures in comt. These results indicate
that dNSF1 activity is not required for synaptic vesicle docking but rather plays an important role in the consumption of vesicles. Taken
together, the electrophysiological and ultrastructural analyses reveal
that dNSF1 functions in the priming of docked synaptic vesicles for
fast calcium-triggered fusion.
Recovery of neurotransmitter release during a rest period at a
restrictive temperature in comt indicates that the priming process is not completely blocked under these conditions, raising the
interesting question of whether residual dNSF1 activity might account
for residual priming. We have sought to address this issue by testing
whether the most severe allele comtST17
behaves as a genetic null mutation (exhibiting no residual activity) under restrictive conditions. Our results (data not shown) confirm this
by showing that flies heterozygous for
comtST17 and a deletion that eliminates
dNSF1 exhibit electrophysiological and behavioral phenotypes that are
indistinguishable from those of comtST17
homozygotes. Furthermore, we have also observed recovery at the higher
restrictive temperature of 38°C. On the basis of these results, we
favor a model in which dNSF1 is critical to, but not absolutely
required for, the priming process. Regarding this possibility it is of
interest that a second Drosophila NSF, dNSF2, has been identified and exhibits 84% amino acid identity to dNSF1 (Boulianne and Trimble, 1995; Pallanck et al., 1995b).
Several recent studies have examined NSF function in regulated
secretion (Banerjee et al., 1996; Burgoyne et al., 1996; Schweizer et
al., 1998). Informative work in semi-intact pheochromocytoma PC12 cells
(Banerjee et al., 1996) indicated that the requirement for NSF function
in dense-core granule fusion can be fulfilled at a distinct step after
vesicle docking and preceding fusion. Although in this case neither the
fast kinetics nor the activity dependence of release was examined,
these results established a model in which NSF acts after docking in an
ATP-dependent priming step preceding calcium-activated exocytosis.
Additional support for models in which NSF does not participate
directly in fusion is inferred from recent results showing that
complementary vesicle and target SNAREs alone are sufficient to
catalyze membrane fusion, although slowly, when reconstituted in lipid
vesicles (Weber et al., 1998).
Another study has recently examined the in vivo role of NSF
using injection of NSF peptides into the presynaptic terminal of the
squid giant synapse (Schweizer et al., 1998). In this case two peptides
were found to inhibit neurotransmitter release, presumably by competing
for NSF binding partners. Inhibition was dependent on synaptic activity
but did not appear to recover during a rest period despite ~50%
residual NSF activity estimated at the peptide concentration used.
Ultrastructural analysis showed that the number of vesicles docked at
active zones was increased slightly by the peptides, indicating that
NSF is not required for vesicle docking. Finally, the reduction in
neurotransmitter release was associated with a slowing of the rise and
decay times of the synaptic current. In contrast to the results shown
here, in this case the slowing was reported to be a specific
consequence of inhibiting NSF activity. These observations led to a
model in which NSF functions after docking in a manner that is
dependent on vesicle turnover while also contributing to the kinetics
of vesicle fusion.
A strength of the work presented here is that it benefits from
knowledge of the specific gene product affected without concern either
for nonspecific effects on other gene products or for complications arising from the presence of wild-type protein. In addition, transgenic expression techniques allow unequivocal demonstration that the phenotypes observed result specifically from mutations in dNSF1. Another unique aspect of this work is the clear separation of priming
and fusion processes by comparison of wild type and comt at
a restrictive temperature in the absence of repetitive stimulation. These experiments, together with the observation that slowing of the
synaptic current in comt does not seem to be directly
related to NSF function, indicate that dNSF1 does not participate
directly in synaptic vesicle fusion.
Previous studies examining the role of NSF in vesicular trafficking
have led to different biochemical models of NSF action (for review, see
Hanson et al., 1997; Hay and Scheller, 1997). A role for NSF in
catalyzing rearrangement of SNAREs leading to membrane fusion, as
proposed in the SNARE hypothesis, is being reconsidered in light of an
alternative model. This second model, derived in part from in
vitro assays of homotypic vacuolar fusion in yeast (Ungermann et
al., 1998), may provide an adequate and general explanation of the
experimental observations to date. This model suggests that NSF is
required only for the generation of free, activated SNAREs and does not
directly participate in fusion. Despite the generality and appeal of
this second model, additional work is required to establish whether
either one of the above models is correct.
The molecular mechanisms of synaptic vesicle trafficking may be ideally
investigated using biochemical and functional analyses in the same
experimental system. Recent studies have pursued this objective by
carefully characterizing a neural SNARE complex in Drosophila and analyzing the status of this complex in
comt (Tolar and Pallanck, 1998). These studies show that
exposure of comt to a restrictive temperature results in
accumulation of an SDS-resistant SNARE complex and use subcellular
fractionation to demonstrate that the accumulated complex is located on
the plasma membrane (including docked vesicles). Thus the priming
function of dNSF1 appears to involve disassembly or rearrangement of
plasma membrane SNARE complex. Although these findings do not
distinguish between the two biochemical models described above, they
clearly represent significant progress in our understanding of NSF action.
The functional analysis reported here may also be interpreted in the
context of either biochemical model. Two simple schemes presented in
Figure 8 indicate that the biochemical
action of NSF occurs either before or after fusion. Regardless of where NSF acts biochemically, in both models it should be clear that NSF
action promotes the priming of docked vesicles for fast
calcium-triggered fusion.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 8.
Possible models of dNSF1 action in
neurotransmitter release. In each model the action of NSF promotes
priming of docked vesicles for fast calcium-triggered fusion. Each
model also shows the SDS-resistant SNARE (SDS-Res.
SNARE) complex forming after vesicle docking. A,
This complex is resolved or rearranged preceding fusion, leading to
formation of a mature (primed) fusion apparatus. This model predicts
that the accumulated SDS-resistant SNARE complex in comt
will be located on the plasma membrane in association with docked
synaptic vesicles. B, The SDS-resistant SNARE complex
participates in fusion and is resolved after the fusion process. In
this case the role of dNSF1 is to provide free, activated SNAREs
necessary for the priming of new vesicles. This model predicts that the
SDS-resistant SNARE complex will accumulate on the plasma membrane or
in recycled synaptic vesicles in comt. Biochemical
demonstration that the complex accumulates on the plasma membrane in
comt is consistent with either model (Tolar and
Pallanck, 1998).
|
|
To clarify models of targeting and fusion mechanisms involving NSF, a
number of important questions about the SDS-resistant SNARE complex
remain to be addressed. What are the SDS-sensitive components and
interactions of the complex in vivo? Does this complex
represent or contribute to the primed fusion apparatus (postfusion
disassembly) or rather to a precursor of it (prefusion disassembly)?
Despite these questions, it is generally agreed that NSF-mediated
disassembly or rearrangement of this complex is a key step.
Determination of the precise timing and location of this event within
the synaptic vesicle trafficking cycle in vivo will be
essential to making connections between the priming function of NSF and
the well-characterized biochemical interactions of these proteins.
| |
FOOTNOTES |
Received July 2, 1998; revised Aug. 11, 1998; accepted Sept. 21, 1998.
This work was supported by National Science Foundation Grant
IBN-9514485. We thank Missy Hazen for excellent technical assistance. We thank Leo Pallanck for communication and discussions of unpublished data and the Penn State University Electron Microscopy Facility for expert training and consultation.
Correspondence should be addressed to Dr. Richard W. Ordway, Department
of Biology, The Pennsylvania State University, University Park, PA 16802.
| |
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6326 - 6332.
[Abstract]
[Full Text]
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F. Kawasaki, R. Felling, and R. W. Ordway
A Temperature-Sensitive Paralytic Mutant Defines a Primary Synaptic Calcium Channel in Drosophila
J. Neurosci.,
July 1, 2000;
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[Abstract]
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B. Dellinger, R. Felling, and R. W. Ordway
Genetic Modifiers of the Drosophila NSF Mutant, comatose, Include a Temperature-Sensitive Paralytic Allele of the Calcium Channel {alpha}1-Subunit Gene, cacophony
Genetics,
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155(1):
203 - 211.
[Abstract]
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M. E. Graham and R. D. Burgoyne
Comparison of Cysteine String Protein (Csp) and Mutant alpha -SNAP Overexpression Reveals a Role for Csp in Late Steps of Membrane Fusion in Dense-Core Granule Exocytosis in Adrenal Chromaffin Cells
J. Neurosci.,
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1281 - 1289.
[Abstract]
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B. Niemeyer and T. Schwarz
SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands
J. Cell Sci.,
January 11, 2000;
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4055 - 4064.
[Abstract]
[PDF]
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E. A. Neale, L. M. Bowers, M. Jia, K. E. Bateman, and L. C. Williamson
Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal
J. Cell Biol.,
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[Abstract]
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P. He, R. C. Southard, D. Chen, S. W. Whiteheart, and R. L. Cooper
Role of alpha -SNAP in Promoting Efficient Neurotransmission at the Crayfish Neuromuscular Junction
J Neurophysiol,
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3406 - 3416.
[Abstract]
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F. Kawasaki and R. W. Ordway
The Drosophila NSF Protein, dNSF1, Plays a Similar Role at Neuromuscular and Some Central Synapses
J Neurophysiol,
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[Abstract]
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J. T. Littleton, R. J. O. Barnard, S. A. Titus, J. Slind, E. R. Chapman, and B. Ganetzky
SNARE-complex disassembly by NSF follows synaptic-vesicle fusion
PNAS,
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[Abstract]
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S. Sanyal, A. Basole, and K. S. Krishnan
Phenotypic Interaction between Temperature-Sensitive Paralytic Mutants comatose and paralytic Suggests a Role for N-Ethylmaleimide-Sensitive Fusion Factor in Synaptic Vesicle Cycling in Drosophila
J. Neurosci.,
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Top
Abstract
Introduction
