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The Journal of Neuroscience, December 1, 1999, 19(23):10270-10279
Overexpression of Cysteine-String Proteins in
Drosophila Reveals Interactions with Syntaxin
Zhiping
Nie,
Ravi
Ranjan,
Julia J.
Wenniger,
Susie N.
Hong,
Peter
Bronk, and
Konrad
E.
Zinsmaier
Department of Neuroscience, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104-6974
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ABSTRACT |
Cysteine-string proteins (CSPs) are associated with secretory
vesicles and critical for regulated neurotransmitter release and
peptide exocytosis. At nerve terminals, CSPs have been implicated in
the mediation of neurotransmitter exocytosis by modulating presynaptic calcium channels; however, studies of CSPs in peptidergic secretion suggest a direct role in exocytosis independent of calcium transmembrane fluxes. Here we show that the individual expression of
various CSP isoforms in Drosophila similarly rescues the
loss of evoked neurotransmitter release at csp null
mutant motor nerve terminals, suggesting widely overlapping functions
for each isoform. Thus, the structural difference of CSP variants may
not explain the opposing putative functions of CSP in neurotransmitter
and peptide exocytosis. Consistently, the individual overexpression of
each CSP isoform in wild-type Drosophila shows similar
effects such as impaired viability and interference with wing and eye development. The dominant effects caused by the overexpression of CSP
are suppressed by the simultaneous overexpression of syntaxin-1A but
not by the coexpression of SNAP-25. Although overexpression of CSP
itself has no apparent effect on the synaptic physiology of larval
motor nerve terminals, it fully suppresses the decrease of evoked
release induced by the overexpression of syntaxin-1A. A direct
protein-protein interaction of CSP with syntaxin is further supported
by coimmunoprecipitations of syntaxin with CSP and by protein binding
assays using recombinant fusion proteins. Together, the genetic and
biochemical interactions of CSP and syntaxin-1A suggest that CSP may
chaperone or modulate protein-protein interactions of syntaxin-1A
with either calcium channels or other components of the regulatory
machinery mediating depolarization-dependent neurotransmitter exocytosis.
Key words:
cysteine-string protein; syntaxin; synaptic vesicle; exocytosis; neurotransmitter release; synaptic transmission; secretion
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INTRODUCTION |
Cysteine-string proteins (CSPs) are
highly conserved proteins associated with secretory vesicles and
characterized by a unique palmitoylated central string of cysteines
(for review, see Buchner and Gundersen, 1997 ). Extensive homology
exists between the N terminus of CSP and the J domain of DnaJ proteins
(Caplan et al., 1993), which mediate protein folding and protein
complex formation in cooperation with members of the heat-shock protein
family (Zylicz, 1993; Kelley, 1998). In a manner analogous to the
prokaryotic system, the J domain of CSPs is essential and sufficient to
bind the 70 kDa heat-shock cognate protein (Hsc70) and to activate its
intrinsic ATPase activity, suggesting that CSP may act as a cofactor of
the molecular chaperone Hsc70 (Braun et al., 1996; Chamberlain and
Burgoyne, 1997a,b; Zhang et al., 1999).
CSPs were originally described as synapse-associated antigens in the
nervous system of Drosophila (Zinsmaier et al., 1990) and,
independently, in Torpedo, as positive modulators of N-type calcium channels, because the coexpression of Torpedo RNAs
modulated the activity of ectopically expressed N-type channels in frog oocytes (Gundersen and Umbach, 1992). The subsequent localization of
CSPs to synaptic vesicles suggested that CSPs may link synaptic vesicles and presynaptic calcium channels to modulate channel activity
(Mastrogiacomo et al., 1994). Deletion of the csp gene in
Drosophila causes a temperature-sensitive loss of evoked
neurotransmitter release at adult first-order interneurons of the
visual system (Zinsmaier et al., 1994) and at larval neuromuscular
junctions (Umbach et al., 1994). Similarly, intracellular application
of CSP antibodies into presynaptic motor neurons inhibits evoked neurotransmitter release at frog neuromuscular junctions (Poage et al.,
1999). Further studies of Drosophila mutants showing normal induction of quantal release by calcium ionophores (Umbach and Gundersen, 1997; Ranjan et al., 1998) and reduced relative cytosolic calcium levels after high frequency stimulation (Umbach et al., 1998)
are consistent with a defect of depolarization-dependent calcium entry.
The idea that CSP may modulate calcium channel activity is further
strengthened by the finding that CSP binds to
 1A-subunits of P/Q-type calcium channels
(Leveque et al., 1998).
Regulation of calcium channel interactions, however, is unlikely to be
the only function of CSPs, because the proteins have been localized to
secretory vesicles of neuronal and non-neuronal tissues of mammals (for
review, see Buchner and Gundersen, 1997) and Drosophila
(Eberle et al., 1998). A direct role of CSP in exocytosis independent
of calcium transmembrane fluxes has been further substantiated by the
overexpression of mammalian CSP in PC12 cells (Chamberlain and
Burgoyne, 1998) and insulin-secreting cells (Brown et al., 1998), which
both reveal defects of stimulated exocytosis independent of calcium
channel activity. Similarly, at peptidergic terminals of csp
null mutant Drosophila, calcium currents are normal,
suggesting that CSP may not be a generic modulator of calcium channels
(Morales et al., 1999). In this study, we determined whether the
differentially expressed CSP isoforms of Drosophila may show
significant functional differences to explain the multiple suggested
functions of CSP. In addition, we report genetic and biochemical
evidence that CSP may mediate depolarization-dependent neurotransmitter
release through its interaction with syntaxin-1A.
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MATERIALS AND METHODS |
Fly stocks. Flies were raised on standard
cornmeal-molasses agar medium with dry yeast at the indicated
temperatures. The homozygous viable transgene P{elav-GAL4} is
described by Lin and Goodman (1994); P{hs-syx} and
P{hs-SNAP-25} are described by Wu et al. (1998). The homozygous
semi-lethal cspR1,
cspU1, and
cspX1 deletions are all genetic
nulls (Zinsmaier et al., 1994; Eberle et al., 1998) and were kept
balanced with a TM6 Tb balancer chromosome. A strain
containing a heat-shock hsp70 promoter driving GAL4 (hs-GAL4) has been
obtained from the Bloomington Drosophila stock center.
Generation of transgenic fly strains. The three
alternatively spliced csp cDNAs, types I-III (Zinsmaier et al., 1990,
1994), were EcoRI-restricted and subcloned into the cloning
site of the pUAST P-element vector (Brand and Perrimon, 1993). For
P-element-mediated germ-line transformation, each pUAST-csp plasmid was
injected together with the helper plasmid pUChs  2-3 into 30- to
60-min-old white
(w1118) embryos by standard
methods (Spradling, 1986). At least two homozygous viable
transgenic lines, termed UAS-csp1, UAS-csp2, and UAS-csp3
(w1118;
P{UAS-csp1-3}), were established for all three individual csp cDNAs. For CSP overexpression, homozygous UAS-csp lines were crossed with homozygous elav-GAL4 or hs-GAL4 flies to obtain elav-csp (w1118,
P{elav-GAL4}/w1118;
P{UAS-csp}/+) or hs-csp progeny
(w1118; P{UAS-csp}/+;
P{hs-GAL4}/+). For csp mutant rescue experiments, UAS-csp, elav-GAL4, or hs-GAL4 transgenes were repeatedly crossed with
w1118; Cyo/Sco;
cspR1/TM6 Tb to generate
UAS-csp, elav-GAL4, or hsp70-GAL4 lines with a heterozygous
csp null background
(w1118, P{elav-GAL4};
cspR1/TM6 Tb and
w1118; P{hs-GAL4},
cspR1/TM6 Tb and
w1118; P{UAS-csp1-3};
cspR1/TM6 Tb). These were
then appropriately crossed with each other to obtain elav-csp;
csp and hs-csp;
csp progeny
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp}/+; cspR1) and
(w1118; P{UAS-csp}+;
P{hs-GAL4}, cspR1).
Scanning electron microscope. Three-day-old flies were
collected and washed in 70% ethanol. Whole adult flies were dried and mounted onto scanning electron microscope stubs and gold-coated with a Denton Vacuum Desk II Sputter Coater (100 Å Au coat). Samples were scanned in a JOEL JSM-T330A scanning microscope and photographed.
Immunoprecipitations and immunoblotting. Fresh or frozen fly
heads were homogenized in immunoprecipitation (IP) buffer (50 mM KCl, 0.2% Triton X-100, 1 mM PMSF, 10 mM HEPES, pH 7.0). The homogenate was cleared by
centrifugation at 14,000 rpm in an Eppendorf centrifuge for 10 min at
4°C, and DCSP-1 antibody (Zinsmaier et al., 1994) was added in
sufficient quantity to bind all CSP within 14 hr at 4°C. The extract
was centrifuged four to five times for 10 min at 4°C at 14,000 rpm before sufficient amounts of protein A agarose (Pharmacia) were
added to precipitate all IgGs during a 2 hr incubation at 4°C. The
proteins immobilized to protein A agarose beads were recovered by low
speed centrifugation (5000 rpm). The immunoprecipitate was washed three
times with IP buffer before it was resuspended in SDS sample buffer and
split. Each half of the precipitate was subjected to SDS-PAGE and
immunoblotting as described earlier (Zinsmaier et al., 1990, 1994) and
individually stained for CSP and syntaxin. The following antibody
dilutions were used: monoclonal antibody DCSP-1 at 1:10 and monoclonal
8C3 against syntaxin at 1:40 (gift of S. Benzer, Caltech, Pasadena, CA).
Glutathione S-transferase binding assay.
Drosophila syntaxin-1A and the cytoplasmic part of
Drosophila synaptotagmin were expressed as glutathione
S-transferase (GST) fusion proteins in Escherichia
coli and purified by standard methods (Smith and Johnson, 1988).
Histidine (His)-tagged Drosophila CSP was expressed
in E. coli Bl-21 cells and purified using the pET expression
system (Novagen, Madison, WI) essentially as recommended by the
supplier. Protein concentrations were estimated by Coomassie blue
staining after SDS-PAGE using bovine serum albumin as standard. A
protein binding protocol for immobilized GST fusion proteins was
adapted from Kee et al. (1995). Briefly, GST-agarose beads were
preadsorbed to a protein extract from E. coli cells for 1 hr
at 4°C. Beads were collected by centrifugation, and 10-25
pmol immobilized GST fusion proteins and ~50 pmol soluble
His-tagged proteins were added to a total volume of 200 µl binding
buffer (150 mM KAc, 1 mM
MgCl2, 0.05% Tween-20, 20 mM HEPES, pH 7.4). After rotating for 2 hr at
4° C, the beads were collected by centrifugation and washed twice
with 1 ml of binding buffer containing 1 mg/ml gelatin and three times
with binding buffer containing 5% glycerol. Proteins bound to the
beads were solubilized in SDS sample buffer and subjected to SDS-PAGE
and immunoblotting.
Larval body wall muscle preparation. Climbing third
instar larvae were dissected to expose their body wall muscles. For
dissection, larvae were placed dorsal side up on a small dish with a
thin layer of Sylgard resin in calcium-free hemolymph-like (HL-3)
medium (Stewart et al., 1994). Larvae were pinned down anteriorly and posteriorly and cut along the dorsal midline, and filleted larvae were
pinned out. After removing the viscera, the segmentally repeated larval body wall muscles and the innervating nerve fibers were clearly
visible. The muscles were identified as described previously (Johansen
et al., 1989).
Electrophysiology. Intracellular whole-cell recordings of
miniature excitatory junction potentials (mEJPs) and evoked excitatory junction potentials (EJPs) were made with a single
microelectrode (20-40 M ) filled with 3 M KCl. All
recordings were made from larval muscle 6 of segment 3/4 in the
anterior abdomen of dissected third instar Drosophila larvae
in HL-3 medium containing 1 mM Ca2+. Signals were amplified using an
Axopatch-1D amplifier (Axon Instruments) and filtered at 1 kHz. To
stimulate evoked excitatory junction potentials, nerve fibers were
severed at the base of the ventral ganglion, and EJPs were elicited
with a suction electrode (6-10 µM diameter
tip) connected to an isolated stimulator (A-M Systems) using a
0.1-msec-long stimulus at three times the threshold response. The
temperature of the recording chamber was monitored and controlled using
a perfusion system and an in-line heater (SF-27A, Warner Instruments)
connected to a TC324-A heater controller (Warner Instruments). The data
were digitized at 5 kHz with a Digidata 1200 interface (Axon) and
analyzed using pCLAMP 6.0 software (Axon). Mean evoked EJPs were
obtained by averaging 15 responses per larva for n larvae.
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RESULTS |
Various Drosophila CSP isoforms equally rescue the
loss of evoked neurotransmitter release of csp null
mutations
The csp gene of Drosophila expresses four
different protein isoforms that are derived from at least three
alternatively spliced RNAs (Zinsmaier et al., 1990, 1994; Eberle et
al., 1998). The CSP variants are differentially expressed in various
tissues as revealed by antibodies recognizing only a subset of isoforms
and by immunoblotting showing predominant expression of only one
isoform in non-neuronal tissues (Zinsmaier et al., 1994; Eberle et al., 1998). All CSP isoforms share three typical and conserved domains: the
"J domain" at the N terminus, the centrally located
"cysteine-string domain," and the "linker domain" connecting
the J and the string domains (Fig.
1A). The CSP variants
differ in their C-terminal half, which is poorly conserved between
Drosophila, Caenorhabditis elegans, and
vertebrate CSPs. The C-terminal half of Drosophila CSP1
contains an insertion of 21 amino acids that is absent in CSP2 and
CSP3. CSP2 and CSP3 differ in seven amino acids at the C terminus.
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Figure 1.
A, Protein domain structure
of Drosophila CSPs. The different protein isoforms CSP1,
CSP2, and CSP3 are derived from alternatively spliced RNA transcripts.
All proteins share three conserved domains, the J domain
(J, residues 19-82), the linker domain
(L, residues 83-113), and the cysteine-string domain
(C, residues 114-138). CSPs differ in their C-terminal
half; CSP1 contains a 21 amino acid insertion termed variable region 1 (V1), which is not present in CSP2 or CSP3. CSP1 and
CSP2 share the same C terminus (V2), which differs by
seven residues in CSP3 (V3). B, Analysis
of CSP overexpression in wild-type driven by the elav promoter.
Immunoblot of fly head protein extracts from wild-type control,
cspR1 null mutants and from flies
overexpressing CSP1-3 with a elav promoter (elav-csp1,
elav-csp2, elav-csp3) or with a csp
promoter contained in a genomic csp transgene
(csp-promoter). Proteins were resolved by 11% SDS-PAGE,
immunoblotted, and stained against CSP with the monoclonal antibody
DCSP1 detecting all CSP isoforms. Each lane contains proteins
equivalent to one adult head from flies raised at 25°C. elav-csp2
expression shows a prominent band at 32 kDa, elav-csp1 at 33-34 kDa,
and elav-csp3 at 36 kDa. Genotypes: elav-csp1
(w1118,
{elav-GAL4}/w1118;
P{UAS-csp1}/+); elav-csp2
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+); elav-csp3 (w1118,
P{elav-GAL4}/w1118;
P{UAS-csp3}/+); csp-promoter
(w1118; P {csp});
csp
(w1118;
cspR1).
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To determine whether different CSP isoforms may have tissue-specific
functions, we individually expressed each isoform in Drosophila using the GAL4/UAS expression system. This system
allows targeted gene expression when transgenic flies expressing the yeast GAL4 transcription factor in a tissue-specific pattern are crossed with flies bearing a transgene containing a GAL4-specific upstream activating sequence (UAS) fused to a target gene (Brand and
Perrimon, 1993). Full-length cDNAs encoding Drosophila CSP1, CSP2, and CSP3 were subcloned into the fly transformation vector pUAST
(Brand and Perrimon, 1993) such that they were expressed under the
transcriptional control of a UAS sequence. We established at least
two independent homozygous viable transgenic lines for each cDNA
(UAS-csp1; UAS-csp2; UAS-csp3). To drive CSP expression we used
transgenic flies expressing the transcription factor GAL4 under the
control of the heat-inducible heat-shock promoter (hs-GAL4), the
neuron-specific elav promoter (elav-GAL4), and the eye-specific glass
promoter (gmr-GAL4). We exclusively investigated flies containing one
copy of a promoter transgene driving GAL4 and one copy of a UAS-csp
transgene that were derived from genetic crosses of strains homozygous
for both the promoter and the csp transgene.
The expression of all UAS-csp transgenes was verified by a
Western blot analysis showing a significant overexpression of each CSP
isoform driven by the elav promoter and the heat-shock promoter. CSPs
overexpressed with the elav promoter show molecular weights ranging
from 32 to 36 kDa, similar to those of endogenous CSPs indicating
normal post-translational palmitoylation of the central string of
cysteines (Fig. 1B). The elav-mediated expression of CSP1 shows a dominant band at 33-34 kDa, of CSP2 at 32 kDa, and of
CSP3 at 36 kDa. Native CSPs exhibit molecular weights of 32, 33, 34, and 36 kDa (Eberle et al., 1998). Thus, CSP1 and CSP2 may mostly
represent the smaller isoforms ranging from 32 to 34 kDa that are
presumed to be specifically expressed in the nervous system. In
contrast, CSP3 correlates with the native 36 kDa isoform, the only CSP
variant expressed in non-neuronal cells at detectable levels (Eberle et
al., 1998). To compare levels of CSP overexpression derived from the
elav promoter with that from a csp promoter, we used a transgenic fly
strain bearing a genomic csp transgene that expresses all CSP isoforms
and fully rescues all genetic phenotypes of csp loss of
function mutations (Umbach et al., 1994; Zinsmaier et al., 1994). Flies
containing one copy of an elav-GAL4 transgene and one copy of a UAS-csp
transgene (elav-GAL4/+; UAS-csp1-3/+) show a
stronger overexpression for a particular CSP isoform than flies
homozygous for the genomic csp transgene (Fig. 1B). A
comparable strong overexpression is also observed with the heat-shock
promoter 3 hr after inducing expression by a heat-shock at 37°C or by
raising flies at a constant temperature of 25°C (data not shown).
Interestingly, most of the heat-shock-induced overexpressed proteins
show lower molecular weights than endogenous CSPs or elav-expressed
CSPs, indicating that the post-translational modification of the
cysteine-string domain is incomplete 3 hr after heat-shock induction.
To determine whether the overexpressed CSP isoforms are indeed
functional and whether they may have alternative functional requirements, we tested the ability of CSP1, CSP2, and CSP3 to rescue
the recessive temperature-sensitive loss of neurotransmitter release at
the larval neuromuscular junction of csp null mutant fly
strains. The cspR1 mutation
deletes the entire csp gene, causing a reduction of evoked
neurotransmitter release at room temperature and a complete block above
29°C without affecting spontaneous neurotransmitter release (Umbach
et al., 1994). We synthesized flies that expressed one copy of each CSP
isoform under the transcriptional control of the elav promoter in a
homozygous cspR1 genetic
background (elav-GAL4/+; UAS-csp/+;
cspR1) and recorded nerve-evoked
excatotory junction potentials (EJPs) and spontaneous miniature
excitatory junction potentials (MEJPs) from neuromuscular
junctions of third instar larvae. Consistent with the normal features
of spontaneous release in csp null mutants, we observed no
significant effects on spontaneous neurotransmitter release by the
individual expression of CSP1, CSP2, and CSP3 at csp mutant
neuromuscular junctions (data not shown). However, each individually
overexpressed isoform rescues the null mutant phenotype of evoked
release. As shown in Figure 2, the mean
EJP amplitude of homozygous
cspR1 larvae is reduced by
~50% at 22°C (21.2 ± 3.3 mV, mean ± SEM, n = 5) when compared with wild-type control (44.0 ± 2.0 mV, n = 4). In contrast, homozygous
cspR1 larvae expressing CSP1
under the control of the elav promoter (elav-csp1;
csp) exhibit normal amounts of evoked
neurotransmitter release (44.3 ± 1.4 mV, n = 4).
Similar EJP responses are obtained for the neural expression of CSP2
(49.0 ± 3.4 mV, n = 5) and CSP3 (37.5 ± 1.2 mV, n = 3) in a homozygous
cspR1 genetic background,
whereas control larvae containing only a UAS-csp transgene show
responses similar to the null mutant (UAS-csp1; csp: 19.8 ± 0.9 mV,
n = 6; UAS-csp2; csp
: 23.7 ± 2.0 mV, n = 3; UAS-csp3;
csp: 28.8 ± 1.5 mV,
n = 5). All mean EJP amplitudes of CSP1-3-expressing flies are statistically different from amplitudes of homozygous cspR1 flies (for CSP1
p = 0.0054; for CSP2 p = 0.0009; for
CSP3 p = 0.039; Student's t test) but not
significantly different from wild-type control white
(p > 0.1). Student's t test also
reveals a statistical difference between mean EJP amplitudes obtained from elav-csp1; csp and UAS-csp1;
csp larvae (p = 0.001), elav-csp2; csp and UAS-csp2;
csp larvae (p = 0.014), and elav-csp3; csp and UAS-csp3;
csp larvae (p = 0.026). The individual elav-driven expression of each CSP isoform also
rescues the slowly developing temperature-sensitive block of
neurotransmitter release of
cspR1 mutants above 29°C
(data not shown). Thus, the similar rescue of the
cspR1 null neurotransmission
phenotype by the neuronal expression of each individual CSP isoform
indicates that CSP1, CSP2, and CSP3 may have strongly overlapping
functions in evoked neurotransmitter release. In particular, the rescue
by CSP3, the presumed non-neuronal CSP isoform in
Drosophila, suggests that structural differences of CSP
isoforms may not account for the different functions of CSP suggested
for evoked neurotransmitter and peptide exocytosis.
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Figure 2.
All CSP isoforms individually rescue the loss of
evoked release in csp null mutants. A,
Representative EJPs are shown for wild-type-like control
white, for larvae expressing CSP1 or CSP3 in a
csp null mutant genetic background
(elav-csp1; csp
and elav-csp3;
csp), and csp null
mutant larvae (csp). EJPs were recorded
from muscle 6 in HL-3 solution at 22°C in the presence of 1 mM Ca2+. Scales for voltage
trace and time sweep are as indicated. B, Mean
amplitudes of evoked EJP responses at 22°C with 1 mM
external Ca2+ from wild-type control
white, from larvae overexpressing CSP1-3 in a csp minus
background (elav-csp1; csp,
elav-csp2; csp, elav-csp3;
csp), from controls containing a UAS-csp
but not a elav-GAL4 transgene in a csp minus background
(UAS-csp1; csp, UAS-csp2;
csp), and from a csp null
mutation (csp). Error bars represent
SEM. Genotypes for A and B: white
(w1118), elav-csp1;
csp
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp1}/+; cspR1), elav-csp2;
csp
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+; cspR1), elav-csp3;
csp
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp3}/+; cspR1), UAS-csp1
(w1118; P{UAS-csp1}/+;
cspR1), and the csp
null mutation (w1118;
cspR1).
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CSP overexpression in Drosophila is semi-lethal and
interferes with wing and eye development
After ensuring functional expression of each UAS-csp transgene, we
determined the effect of CSP overexpression on the synaptic physiology
of wild-type larval motor nerve terminals. The elav-driven overexpression of individual CSPs shows no significant effect on the
amplitude of evoked EJP responses at larval neuromuscular junctions
(Fig. 3). The mean EJP amplitude recorded
from wild-type control is 44.0 ± 2.0 mV (mean ± SEM,
n = 4). Larvae overexpressing CSP1-3 with the elav
promoter show EJP amplitudes similar to wild type (elav-csp1 39.3 ± 1.9 mV, n = 9; elav-csp2 45.0 ± 4.4 mV, n = 4; elav-csp3 49.9 ± 3.4 mV, n = 4). Larvae containing only a silent UAS-csp transgene also show
similar amplitudes (UAS-csp1 39.4 ± 0.2 mV, n = 3; UAS-csp2 40.4 ± 2.1 mV, n = 5; UAS-csp3 44.8 ± 2.3, n = 4). Neither amplitude of
CSP1-3-expressing larvae is statistically different from wild-type
controls (Student's t test, p > 0.1).
Features of spontaneous release are also statistically normal for all
examined transgenic flies (data not shown). We tested further whether
an overexpression of CSP with the heat-shock promoter after heat-shock
induction at 37°C may induce a significant effect on evoked
neurotransmitter release. However, neither evoked release nor
spontaneous release is significantly affected by the heat-shock-induced
overexpression of any CSP isoform (data not shown). Thus,
neurotransmitter release at larval neuromuscular junctions appears
insensitive to increased levels of CSP.
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Figure 3.
Neuronal overexpression of individual CSP isoforms
has no effect on neurotransmitter release at larval neuromuscular
junctions. A, Representative EJPs recorded from larval
neuromuscular junctions of wild-type-like control white,
larvae overexpressing CSP2 in the nervous system with the elav promoter
(elav-csp2), and control larvae containing only the
UAS-csp2 or the elav-GAL4 transgene. EJPs were recorded from muscle 6 in HL-3 solution at 22°C in the presence of 1 mM
Ca2+. Scales for voltage trace and time sweep are as
indicated. B, Mean EJP amplitudes from wild-type-like
control white, larvae overexpressing CSP1-3 with the
elav promoter (elav-csp1, elav-csp2,
elav-csp3), and control larvae containing either an
elav-GAL4 or a UAS-csp transgene in trans
(UAS-csp1, UAS-csp2,
UAS-csp3, elav-GAL4). Fifteen EJPs
were averaged for n larvae. Recordings were in the
presence of 1 mM Ca2+ at 22°C. Error
bars represent SEM. Genotypes for A and
B: white (w1118),
elav-csp1 (w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+), elav-csp2
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+), elav-csp3
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp3}/+), UAS-csp1
(w1118; P{UAS-csp1}/+),
UAS-csp2 (w1118;
P{UAS-csp2}/+), UAS-csp3
(w1118; P{UAS-csp3}/+),
elav-GAL4 (w1118,
P{elav-GAL4}/w1118).
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Although overexpression of CSP has no effect on evoked release at
larval neuromuscular junctions, it has significant deleterious effects
on normal development of Drosophila. Independent UAS-csp lines for each CSP isoform all show similar phenotypes when placed in
trans to any of the examined GAL4 expressing lines. The overexpression of individual CSP variants is semi-lethal in a temperature-dependent manner (Table 1). For flies raised at
29°C, the elav-driven overexpression of each CSP isoform in the
nervous system is completely lethal. At 25°C, the overexpression is
semi-lethal: 1% of CSP1-, 70% of CSP2-, and 23% of CSP3-expressing
flies survive. Raising flies at 18°C significantly increases the
survival rate. The continuous overexpression of CSP with the
ubiquitously expressing heat-shock promoter is lethal at 29°C and at
25°C but allows a few flies to survive at 18°C. We also noticed
that the lethality induced by the elav-driven overexpression of CSP is
more severe in male than in female flies. At 18°C, the ratio of male
to females overexpressing CSP2 is normal, but for males expressing CSP1
the ratio is only 27%, and for males expressing CSP3 it is 68%.
First, we speculated that this may be attributable to the X-chromosomal
insertion of the elav-GAL4 transgene. However, the sex-linked lethality
is also apparent when CSP is overexpressed with the heat-shock promoter transgene, which is linked to an autosome. Only 25% of the expected number of males survive when either CSP isoform is ubiquitously expressed with the heat-shock promoter.
Flies escaping from the semi-lethal developmental phenotype caused by
the elav-driven overexpression of CSP exhibit motor defects, short
adult life spans, and morphological defects. At 25°C, adult wild-type
flies live longer than 55 d, but adult escapers expressing CSP1
die within the first 5 d of adulthood (Fig.
4). Flies overexpressing CSP2 show
life-spans not longer than 51 d, whereas CSP3-overexpressing flies
die within 10-21 d. All adult flies escaping the lethal overexpression
of either isoform are extremely sluggish, show severely uncoordinated
movements, and slip frequently. Morphologically, overexpression of all
CSP isoforms reduces body size and impairs wing inflation after pupal
eclosion such that the wings appear severely crumpled (Fig.
5B-D).
Interestingly, overexpression of CSP from the weaker csp promoter using
the genomic csp transgene shows no crumpled wing phenotype (Fig.
5F), indicating that a high level threshold of
overexpressed CSP may be critical for the expression of the wing
phenotype. Alternatively, it is possible that the elav promoter may
ectopically misexpress CSP. We tested these possibilities and reduced
the levels of overexpressed CSP by removing the endogenous
csp gene. Removal of one copy of endogenous csp in flies
heterozygous for the cspR1
deletion (elav/+; UAS-csp2/+;
cspR1/+) has little effect
on the crumpled wing phenotype (Fig. 5G). However,
homozygous cspRI null mutant
flies overexpressing CSP2 (elav/+; UAS-csp2/+;
cspR1/cspR1)
exhibit normal wings (Fig. 5H), suggesting that a
critical threshold level of overexpressed CSP is necessary for the
expression of the wing phenotype.
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Figure 4.
Overexpression of CSP reduces adult life span.
Survival curves of adult flies overexpressing CSP1-3 and wild-type
controls. Adult elav-csp1 flies die prematurely after 4-5 d, elav-csp3
within 10-25 d, and elav-csp2 within 51 d. Controls exhibit life
spans longer than 55 d. Flies were raised at 25°C. Newly emerged
flies were collected within 12 hr, kept at 25°C, and dead flies were
counted every 12 hr.
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Figure 5.
Wing defects caused by the elav-driven
overexpression of individual CSP isoforms. A-L, Light
microscopic images of wings from 2- to 5-d-old flies raised at 25°C
unless otherwise indicated. Flies overexpressing CSP1-3 in the nervous
system by the elav promoter fail to inflate their wings after eclosion
such that wings appear "crumpled" (B-D).
This phenotype is not observed in flies overexpressing CSP from a csp
promoter contained in a genomic csp transgene, csp-csp
(F). Reduction of CSP levels by removing one copy
of endogenous csp (elav-csp2; csp/+) partially
suppresses the crumpled wing overexpression phenotype
(G), and abolishment of endogenous csp
(elav-csp2; csp) fully suppresses the
overexpression phenotype (H). Simultaneous
overexpression of syntaxin-1A driven by a heat-shock promoter
(elav-csp; hs-syx) partially suppresses the elav-CSP wing phenotype in
flies raised at 25°C (K). Higher levels of
syntaxin induced by the application of a daily heat-shock during late
larval and pupal development fully suppresses the wing phenotype for
flies raised otherwise at 25°C (L). Genotypes:
(A) wild type; (B)
elav-csp2 (w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+); (C) elav-csp1
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp1}/+); (D) elav-csp3
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp3}/+); (E) elav-GAL4
(w1118,
P{elav-Gal4}); (F) csp-csp
(w1118; P{csp} a
genomic csp transgene expressing all isoforms);
(G) elav-csp3; csp/+
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+; cspR1/TM3
Sb); (H) elav-csp3;
csp
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+; cspR1);
(I) elav-csp2 raised at 18°C -compare to
(B); (J) hs-syx control
(w1118, P{hs-syx}/+;
P{UAS-csp2}/+); (K) elav-csp2; hs-syx at
25°C (w1118,
P{elav-GAL4}/w1118;
P{hs-syx}/+; P{UAS-csp2}/+); (L)
elav-csp2; hs-syx raised at 25°C and heat-shocked for 1 hr at 37°C
per day.
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Overexpression of CSP also interferes with eye development, causing a
significantly misshaped eye. Externally, the surface of the fly
compound eye forms a smooth and regular surface of ~800 facets, or
ommatidia. Small mechanosensory bristles project from alternate facet
vertices over most of the eye's surface (Fig. 6A). The overexpression
of all CSP isoforms disrupts this regular ommatidial pattern such that
the surface of the eye appears rough (Fig.
6B-D). A closer examination of the rough
eye reveals a slightly elliptical shape of the compound eye and the
loss of some bristles; a fusion of several ommatidial units is
occasionally observed (data not shown). As for the wing and the lethal
phenotypes, overexpression of CSP from the csp promoter causes no eye
phenotype. However, a reduction of CSP levels caused by removing
endogenous CSP reduces the severity of the rough eye phenotype caused
by the elav-driven CSP overexpression (Fig. 6F),
indicating that the phenotype requires a critical threshold of
overexpressed CSP. In addition, we examined CSP overexpression mediated
by the glass promoter driving expression in photoreceptors and pigment
cells (Ellis et al., 1993). Glass-mediated expression causes a more
severe eye phenotype than the overexpression with the elav promoter,
showing a very elliptical shape, lacking most bristle cells, and
appearing glassy (data not shown).
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Figure 6.
Eye defects caused by the elav-driven
overexpression of individual CSP isoforms. A-I,
Scanning electron microscopic images of adult eyes from 2- to 5-d-old
flies raised at 25°C unless otherwise indicated. The elav-GAL4 driven
overexpression of all three CSP isoforms in the nervous system disrupts
eye development, causing a rough surface of the eye
(B-D). Flies overexpressing CSP from a genomic
csp gene fragment with the csp promoter (csp-csp) show normal eyes
(E). The rough eye phenotype of elav-CSP2 is
partially suppressed by the removal of endogenous CSP
(F). Simultaneous overexpression of syntaxin-1A
(hs-syx; elav-csp2) driven by the heat-shock promoter at 25°C
partially suppresses the rough eye phenotype of CSP overexpressing
flies (H). Higher levels of syntaxin-1A
coexpression induced by the application of a daily heat-shock during
late larval development completely suppress the rough eye phenotype
(I). Genotypes for
(A) wild type; (B)
elav-csp1 (w1118,
P{elav-GAL4}/w1118;
P{UAS-csp1}/+); (C) elav-csp2
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+); (D) elav-csp3
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp3}/+); (E) csp-csp
(w1118; P{csp} - a genomic
csp transgene expressing all isoforms); (F)
elav-csp2; csp
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+; cspR1);
(G) hs-syx control
(w1118, P{hs-syx}/+;
P{UAS-csp2}/+); (H) hs-syx; elav-csp2
raised at 25°C (w1118,
P{elav-GAL4/w1118; P{hs-syx}/+;
P{UAS-csp2}/+); (I) hs-syx;
elav-csp2 raised at 25°C and heat-shocked for 1 hr at 37°C per day.
Scale bar, 100 µm.
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Simultaneous overexpression of syntaxin-1A and CSP suppresses the
effects of their individual overexpression
Because the overexpression of CSP may cause a "titration"
effect of CSP interacting proteins, one may consequently expect that
the simultaneous expression of an interacting protein suppresses the
effects of CSP overexpression. Thus, we tested whether the coexpression
of syntaxin suppresses the CSP overexpression phenotypes because
syntaxin has been suggested as a possible target of CSP chaperone
action to indirectly modulate presynaptic calcium channel activity
(Seagar et al., 1999). To overexpress syntaxin, we used a transgenic
line expressing syntaxin-1A (hs-syx) under the direct transcriptional
control of a heat-shock promoter (Wu et al., 1998). To exclude
nonspecific effects by the overexpression of a synaptic protein, we
used a transgenic line expressing SNAP-25 with the heat-shock promoter
as a control (Wu et al., 1998). Both fly lines are homozygous viable
and show normal wings and eyes when raised at 25°C. Flies were
synthesized containing a hs-syx (or hs-SNAP-25), an elav-GAL4, and a
UAS-csp transgene in trans to simultaneously overexpress CSP
from an elav promoter and syntaxin or SNAP-25 from a heat-shock
promoter. At 25°C, coexpression of syntaxin partially suppresses the
crumpled wing (Fig. 5K) and the rough eye phenotype
(Fig. 6H) induced by the overexpression of CSP. Both
the wing and the eye phenotypes are completely rescued by the
application of daily heat-shocks during late larval and pupal development to elevate syntaxin expression (Figs. 5L,
6I). In contrast, flies coexpressing SNAP-25 show no
effect on the mutant wing or eye phenotype induced by the
overexpression of CSP (data not shown). This suggests that CSP and
syntaxin may specifically interact in vivo.
As shown, CSP overexpression itself does not significantly interfere
with evoked neurotransmitter release at motor nerve terminals. However,
heat-shock-induced overexpression of syntaxin-1A reduces evoked
neurotransmitter release at Drosophila neuromuscular
junctions (Wu et al., 1998), raising the possibility that CSP
overexpression may suppress the syntaxin overexpression phenotype.
Thus, we analyzed evoked release at neuromuscular junctions of larvae
overexpressing CSP and syntaxin-1A (Fig.
7). Although wild-type larvae exhibit mean EJP amplitudes of 44.0 ± 2.0 mV (mean ± SEM,
n = 4), larvae continuously expressing syntaxin from
one copy of the hs-syx transgene at 25°C show reduced EJP amplitudes
of 29.2 ± 2.2 mV (n = 6). This 34% decrease of
evoked release is statistically significant (p = 0.0086, Student's t test) and comparable with the
originally described 67% decrease of evoked release where syntaxin-1A
overexpression has been induced from two copies of the transgene by a
heat-shock at 37°C (Wu et al., 1998). The effect of syntaxin
overexpression is not modulated by the presence of either an elav-GAL4
(data not shown) or a UAS-csp2 transgene (30.4 ± 3.2 mV,
n = 5). However, larvae containing all transgenes and
simultaneously overexpressing syntaxin and CSP (hs-syx; elav-csp2) show
normal EJP amplitudes (42.7 ± 3.1 mV, n = 6) at
25°C. Statistical comparison of hs-syx; elav-csp2 with hs-syx or
hs-syx; UAS-csp2 responses shows that they are significantly different
(p = 0.029 and 0.0031, respectively), whereas
hs-syx; elav-csp2 responses are not significantly different from
wild-type control (p > 0.05, Student's
t test). A similar suppression of syntaxin overexpression
phenotypes has also been observed for the coexpression of CSP3 (data
not shown). Thus, CSP overexpression fully suppresses the 34% decrease
of evoked release induced by the overexpression of syntaxin-1A,
indicating that both proteins interact through a common protein complex
at synaptic terminals.
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Figure 7.
Overexpression of CSP suppresses the decrease of
evoked release induced by the overexpression of syntaxin-1A. Continuous
overexpression of syntaxin-1A from the heat-shock promoter at 25°C
reduces neurotransmitter release in a similar manner as the induction
of syntaxin expression by a heat-shock. This loss of evoked release is
suppressed by the simultaneous overexpression of CSP2.
A, Representative EJP recordings from larval
neuromuscular junctions of wild-type-like control white,
larvae overexpressing CSP2 (elav-csp2), larvae
overexpressing syntaxin-1A and containing a csp transgene but not an
elav transgene (hs-syx; UAS-csp2), and for larvae
overexpressing syntaxin-1A and CSP2 (hs-syx; elav-csp2).
All larvae were raised at 25°C and recordings are from muscle 6 at 1 mM Ca2+ at 25°C. B,
Mean amplitudes of evoked EJP responses at 25°C with 1 mM
external Ca2+ from control white,
larvae overexpressing CSP2 (elav-csp2), larvae
overexpressing syntaxin-1A (hs-syx and hs-syx;
UAS-csp2), and larvae overexpressing CSP and syntaxin-1A
(hs-syx; elav-csp2). Recordings were as in
A. Bars indicate SEM. Genotypes: white
(w1118); elav-csp2
(w1118,
P{elav-GAL4}/w1118;
P{UAS-csp2}/+); hs-syx
(w1118; P{ hs-syx}/+);
hs-syx; UAS-csp2 (w1118; P{
hs-syx}/+; P{UAS-csp2}/+); and hs-syx; elav-csp2
(w1118,
P{elav-GAL4/w1118; P{hs-syx}/+;
P{UAS-csp2}/+).
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CSP directly binds syntaxin-1A
To gather independent biochemical evidence for a protein
interaction of CSP with syntaxin, we analyzed whether syntaxin
coimmunopurifies with CSP from Drosophila protein extracts
and compared CSP immunoprecipitations from wild-type extracts with
those from cspX1 null mutations
where CSP is absent because of a partial gene deletion (Zinsmaier et
al., 1994; Eberle et al., 1998). Proteins of wild-type and homozygous
cspX1 extracts were
solubilized, and CSP was immunoprecipitated with a monoclonal antibody
detecting all CSP isoforms (Zinsmaier et al., 1994; Eberle et al.,
1998). Each immunoprecipitate was split and analyzed by immunoblotting
for the presence of CSP and syntaxin-1A. CSP antibodies
immunoprecipitated CSP and coimmunoprecipitated syntaxin from wild-type
protein extracts but not from controls where the antibody, protein A
agarose, or the fly extract were omitted (Fig.
8A). Consistently,
immunoprecipitations from protein extracts of
cspX1 deletion mutants showed
no coimmunopurification of syntaxin (Fig. 8B),
suggesting that the coimmunopurification of syntaxin specifically depends on the presence of CSP. Controls showed no coimmunopurification of synaptotagmin or synapsin with CSP (data not shown).
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Figure 8.
In vitro binding of CSP and
syntaxin -1A. A, Coimmunoprecipitation of syntaxin-1A
from Drosophila wild-type extracts.
Drosophila head protein extracts were solubilized with
Triton X-100 and immunoprecipitated with DCSP1 antibodies detecting all
CSP isoforms (IP CSP). Control experiments omitted the
monoclonal antibody (no Ab), the fly extract (no
extract), or protein A-coupled beads (no protein
A). Bound proteins were recovered by centrifugation, washed,
and resuspended in SDS buffer. Three percent of the soluble fraction
(S) of the immunoprecipitation and one-half of
the immunoprecipitated pellet fraction (P) were
analyzed on separate immunoblots for the presence of CSP and
syntaxin-1A (Syx). CSP antibodies immunoprecipitate CSPs
and coimmunoprecipitate syntaxin. Controls show neither a CSP- nor a
syntaxin-specific signal. B, Similar
coimmunoprecipitation of syntaxin-1A from protein extracts of wild-type
Drosophila and cspX1
deletion mutants where CSP is absent shows copurification of syntaxin
from wild-type extracts but not from mutant extracts. C,
Recombinant protein binding of syntaxin-1A with CSP. Soluble His-CSP
was incubated in a 2:1 molar ratio with immobilized GST-syntaxin-1A
(GST-Syx), with immobilized GST-synaptotagmin
(GST-Syt), or with immobilized GST protein (GST
control) for 2 hr at 4°C. The affinity precipitate was
recovered by centrifugation, washed, and resuspended in SDS-PAGE
buffer. Recombinant His-CSP (1/25) used for the binding assay and all
of the affinity precipitate was analyzed by immunoblotting for the
presence of recombinant CSP. Note that <4% of total His-CSP binds by
GST-syntaxin (GST-Syx + His-CSP) but not to
GST-synaptotagmin (GST-Syt + His-CSP) or the GST
control.
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Because coimmunopurifications do not indicate a direct protein-protein
interaction, we performed protein binding assays with recombinant CSP
and syntaxin-1A fusion proteins. For these experiments, we used His-CSP
and GST-tagged syntaxin-1A. GST-syntaxin fusion protein was immobilized
to glutathione agarose beads and incubated with defined amounts of
purified His-tagged CSP fusion protein. Controls contained equal
amounts of immobilized GST-synaptotagmin fusion protein or immobilized
GST protein. As shown in Figure 8C, His-CSP fusion protein
binds to GST-syntaxin but not to GST-synaptotagmin or to GST protein
alone, indicating that CSP directly binds to syntaxin. Comparing the
amount of His-CSP retained by GST-syntaxin with the total amount of
His-CSP used reveals that <4% of the incubated His-CSP is bound to
syntaxin. Together, the genetic interactions and the in
vitro binding of CSP and syntaxin support the speculation that
syntaxin may be a target of CSP chaperone function.
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DISCUSSION |
It has been suggested that CSP modulates depolarization-dependent
calcium fluxes in neurotransmitter exocytosis (Gundersen and Umbach,
1992; Mastrogiacomo et al., 1994) and mediates a direct step in
peptidergic exocytosis independent of calcium transmembrane fluxes
(Brown et al., 1998; Chamberlain and Burgoyne, 1998; Zhang et al.,
1998; Morales et al., 1999). Because several slightly different CSP
variants are differentially expressed in invertebrates and mammals
(Zinsmaier et al., 1994; Chamberlain and Burgoyne, 1996; Brown et al.,
1998; Eberle et al., 1998), it appears possible that these different
protein isoforms may mediate the contrasting functions of CSP for the
secretion of neurotransmitters and peptides. To test whether the
differentially expressed Drosophila CSP isoforms may equally
mediate neurotransmitter release, we individually expressed each
isoform in the nervous system of csp null mutant Drosophila. As shown here, the individual expression of each
CSP variant is able to restore the loss of evoked neurotransmitter release at larval motor nerve terminals of csp null
mutants. The full rescue of evoked release by CSP3, which we identified
to encode a 36 kDa isoform abundantly expressed in non-neuronal tissues but not at neuromuscular junctions (Eberle et al., 1998), especially indicates that the three examined CSP variants may have largely overlapping functions in neuronal and non-neuronal cells despite their
differential expression patterns. Because CSP has been shown to mediate
a direct step of exocytosis independent of transmembrane calcium fluxes
in PC12 and insulin-secreting cells, it now appears possible that CSP
may mediate a similar step in neurotransmitter exocytosis in addition
to the suggested modulation of calcium channels.
We further investigated the effects of CSP overexpression for regulated
neurotransmitter release in Drosophila. Although the overexpression of CSP potentiates dopamine release in permeabilized neuroendocrine PC12 cells (Chamberlain and Burgoyne, 1998), it depresses stimulated insulin release in insulin-secreting cell lines
(Brown et al., 1998; Zhang et al., 1999). In contrast to the opposing
effects of CSP overexpression in endocrine and neuroendocrine secretion, the overexpression of CSP at Drosophila motor
nerve terminals has no significant effect on evoked neurotransmitter release as shown by normal EJP amplitudes in larvae overexpressing CSP1-3. This difference between PC12,  -cells, and fly motor nerve terminals may be attributable to the different approaches used, or it
may be influenced by different levels of endogenous CSP. Alternatively,
the different observations may reflect different functional
requirements of CSP for stimulated exocytosis in endocrine, neuroendocrine, and nerve terminals.
Although CSP overexpression does not modulate neurotransmission at fly
motor nerve terminals, it has otherwise severe effects that reduce
viability during development and adult life. Adult escapers exhibit
motor defects such as uncoordinated walking and impaired balance
control, which may indicate that synaptic transmission at synapses
of the CNS is impaired, whereas the robust larval neuromuscular
junctions are less sensitive to increased levels of CSP. Escapers also
exhibit morphological defects in wing and eye development that may be a
result of improper secretion of protein factors that influence
cell-cell communication in the developing eye or wing disk.
Alternatively, the defects may arise from anomalies in membrane
biogenesis. As for the overexpression of mammalian CSPs in
insulin-secreting cell lines (Zhang et al., 1999), the overexpression
of individual Drosophila CSP isoforms causes similar
effects, although some slight quantitative differences are observed.
These could be caused by position effects at different insertion sites
of individual transgenes (Spradling and Rubin, 1983), but this is
unlikely because we examined at least two independent lines for each
transgene. Thus, it may indicate that the three CSP isoforms may indeed
somewhat differ in their function, although this difference is
apparently too subtle or not relevant for evoked neurotransmitter
release at motor nerve terminals.
We tested further whether the effects of CSP overexpression may be
suppressed by the simultaneous overexpression of syntaxin, which has
earlier been speculated to interact with CSP (Seagar et al., 1999). We
provide genetic and biochemical evidence that supports an interaction
of CSP with syntaxin. Coimmunoprecipitation of syntaxin with CSP from
Drosophila protein extracts and further protein binding
assays using recombinant proteins demonstrate a direct protein-protein
interaction between CSP and syntaxin-1A. The weak nature of this
interaction is not unexpected because transient protein-protein
interactions are often a critical feature among molecular chaperones
and their substrates (Gething and Sambrook, 1992; Silver and Way,
1993). This biochemical evidence is further strengthened by genetic
interactions because the coexpression of syntaxin-1A, but not that of
the SNAP-25 control, suppresses the wing and eye phenotypes caused by
the overexpression of CSP, indicating that syntaxin specifically
interacts with CSP. The suppression of the eye phenotype by syntaxin is
consistent with our speculation that overexpression of CSP may titrate
its binding partners, because loss-of-function mutations of syntaxin
also show a rough eye phenotype (Schulze and Bellen, 1996). Although the overexpression of CSP itself does not modulate evoked
neurotransmitter release, it fully suppresses the decrease of
neurotransmitter release at Drosophila neuromuscular
junctions induced by the overexpression of syntaxin-1A alone. Together,
the genetic interactions and the in vitro binding of CSP and
syntaxin-1A suggest that CSP and syntaxin-1A are associated with a
common protein complex, possibly to mediate evoked neurotransmitter
release and other steps of membrane traffic.
Two alternative models of CSP function have been suggested. For
peptidergic secretion, CSP has been shown to mediate a direct step of
stimulated exocytosis independent of calcium transmembrane fluxes
(Brown et al., 1998; Chamberlain and Burgoyne, 1998; Zhang et al.,
1998; Morales et al., 1999). An increase or decrease of CSP levels in
PC12 and insulin-secreting cells severely reduced exocytosis without
affecting transmembrane calcium fluxes. Because these effects persisted
in permeabilized cells, any regulation via soluble second messengers
can be excluded, suggesting that CSP may function directly in
exocytosis (Chamberlain and Burgoyne, 1998; Zhang et al., 1998, 1999).
In contrast, for fast neurotransmitter release, CSP has been suggested
to link synaptic vesicles with calcium channels and to modulate channel
activity because CSP is associated with synaptic vesicle membranes
(Mastrogiacomo et al., 1994) and modulated N-type calcium channel
currents when ectopically coexpressed in frog oocytes (Gundersen and
Umbach, 1992). This idea has been further supported by the in
vitro binding of CSP to P/Q-type calcium channels (Leveque et al.,
1998) and by the consistent defects observed in csp mutant
Drosophila like the reduction of evoked but not spontaneous
neurotransmitter release (Umbach et al., 1994; Zinsmaier et al., 1994)
and reduced presynaptic cytosolic calcium levels after repetitive
stimulation (Umbach et al., 1998).
It has been further speculated that CSP may coordinate sequential
protein-protein interactions between calcium channels and their
associated synaptic proteins (Seagar et al., 1999) because CSP is
presumably a vesicular, membrane-bound cofactor of the molecular
chaperone Hsc70 (Braun et al., 1996; Chamberlain and Burgoyne,
1997a,b) and because CSP binds to the same cytoplasmic loopII-III of calcium channels that contains the
synprint site mediating channel interactions with SNAP-25,
synaptotagmin, and syntaxin (Leveque et al., 1998). Interestingly,
disruption of these interactions by the presynaptic injection of the
synprint binding peptide in rat neurons (Mochida et al., 1996) causes
strikingly similar defects of evoked neurotransmitter release as the
deletion of the Drosophila csp gene (Umbach et al., 1994;
Heckmann et al., 1997): both reduce synchronous release but increase
asynchronous release and paired pulse facilitation. Because the
syntaxin/channel interaction reduces channel activity by prolonging an
inactivated state (Bezprozvanny et al., 1995; Wiser et al., 1996), it
has been speculated that CSP could promote the dissociation of a
syntaxin/channel complex (Seagar et al., 1999), if CSP promotes calcium
channel activity as originally suggested (Gundersen and Umbach,
1992).
Here we provide experimental evidence for this speculation showing that
CSP interacts with syntaxin in vitro and in vivo. This interaction may modulate the dissociation of syntaxin from calcium
channels as previously speculated (Seagar et al., 1999). Alternatively,
CSP may modulate protein interactions with other syntaxin-interacting
proteins to mediate a calcium-dependent step of exocytosis as implied
by the direct role of CSP in peptidergic exocytosis (Chamberlain and
Burgoyne, 1998; Zhang et al., 1998, 1999). An obvious candidate for
this interaction is synaptobrevin, which has been shown to bind CSP
in vitro (Leveque et al., 1998). Both speculations are
consistent with our results and with the recent characterization of a
mutation in Drosophila syntaxin-1A deleting a multiple
protein binding domain that reduces CSP, synaptotagmin, and calcium
channel binding (Wu et al., 1999). A third possible role for the
CSP/syntaxin interaction is that CSP may simply chaperone protein
folding or protein transport of syntaxin. Such a function should cause
reduced levels of syntaxin in csp null mutants and proportionally reduce evoked and spontaneous release as observed in
Drosophila syntaxin mutants (Schulze et al., 1995; Wu et
al., 1998). However, this possibility is unlikely because only evoked release but not spontaneous release is abolished in csp null
mutants at restrictive temperatures (Umbach et al., 1994). To finally determine whether the CSP/syntaxin interaction is required for a
modulation of presynaptic calcium channel activity or for a step of
evoked exocytosis independent of calcium transmembrane fluxes, or both,
will require the analysis of mutations in CSP that specifically
interrupt the interaction with syntaxin.
| |
FOOTNOTES |
Received July 26, 1999; revised Sept. 3, 1999; accepted Sept. 17, 1999.
This work was supported by grants to K.E.Z. from the National Science
Foundation, National Institutes of Health, the March of Dimes Birth
Defects Foundation, and the Whitehall Foundation. We thank Drs. T. L. Schwarz, M. Wu, and H. Bellen for sharing cDNA constructs and fly
stocks, and S. Benzer for the gifts of antibodies used in this investigation.
Z.N. and R.R. contributed equally to this work.
Correspondence should be addressed to Konrad E. Zinsmaier, Department
of Neuroscience, 234d Stemmler Hall, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104-6974. E-mail:
zinsmaie{at}mail.med.upenn.edu.
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ABSTRACT
INTRODUCTION
