J Neurophysiol 94: 1888 –1903, 2005.
First published May 11, 2005; doi:10.1152/jn.00080.2005.
AP180 Maintains the Distribution of Synaptic and Vesicle Proteins in the
Nerve Terminal and Indirectly Regulates the Efficacy of
Ca2⫹-Triggered Exocytosis
Hong Bao,1 Richard W. Daniels,1,* Gregory T. MacLeod,2,* Milton P. Charlton,2 Harold L. Atwood,2
and Bing Zhang1
1
Section of Neurobiology, Section of Molecular Cell and Developmental Biology, Institute for Neuroscience, and Institute for Cellular and
Molecular Biology, the University of Texas, Austin, Texas; and 2Department of Physiology, University of Toronto, Toronto, Canada
Submitted 24 January 2005; accepted in final form 3 May 2005
INTRODUCTION
Clathrin-mediated endocytosis is thought to play a major
role in replenishing the synaptic vesicle (SV) pool in nerve
terminals. Along with a large number of accessory proteins,
clathrin recycles SVs through several distinct sequential steps,
including formation of coated vesicles, fission of endocytotic
vesicles from the plasma membrane, and removal of the
clathrin coat (reviewed by Brodin et al. 2000; Brodsky et al.
2001; De Camilli and Takei 1996; Zhang and Ramaswami
1999). Unlike the “kiss-and-run” mode of endocytosis, which
* R. W. Daniels and G. T. Macleod contributed equally to this work.
Address for reprint requests and other correspondence: B. Zhang, Section of
Neurobiology, 1 University Station, The University of Texas at Austin, Austin,
TX 78712 (E-mail: [email protected]).
1888
is believed to recycle SVs that do not intermingle significantly
with the plasma membrane during exocytosis (Fesce et al.
1994; Palfrey and Artalejo 2003), clathrin-mediated endocytosis internalizes SVs that collapse into the plasma membrane
(Heuser and Reese 1973). Hence, an important step in this
pathway is to sort and recruit cargo components unique to SVs
to ensure that newly recycled vesicles are fully competent for
exocytosis.
AP180 is one of the clathrin accessory and assembly proteins promoting the formation of clathrin-coated vesicles (Ahle
and Ungewickell 1986; Murphy et al. 1991; Ye and Lafer
1995) and regulating the size of SVs (Morgan et al. 1999;
Nonet et al. 1999; Zhang et al. 1998). AP180, alone with the
adaptor AP2 and a putative adaptor Stoned, is also thought to
act as an adaptor protein sandwiched between the clathrin coat
and the cargo membrane. Because of their strategic locations
within coated vesicles, these adaptors are also implicated in
assisting the retrieval of SV proteins (De Camilli and Takei
1996; Zhang 2003; Zhang et al. 1999). The results obtained
from recent genetic and biochemical studies appear to support
this notion. Stoned has adaptor-like and clathrin- and eps15binding motifs (Stimson et al. 1998) and directly interacts with
synaptotagmin I (Phillips et al. 2000). Mutations in the stoned
locus mislocalize synaptotagmin I and cysteine-string protein
(CSP) to the axonal membrane and impair synaptic transmission regardless of whether or not the number of vesicles
remains normal (Fergestad and Broadie 2001; Fergestad et al.
1999; Phillips et al. 2000; Stimson et al. 1998; 2001). Biochemical studies reveal that AP2 interacts directly with synaptotagmin I, suggesting that it may help retrieve synaptotagmin
I during endocytosis (Hao et al. 1999; Jarousse et al. 2003;
Zhang et al. 1994). AP180 binds clathrin and AP2 (Ahle and
Ungewickell 1986; Hao et al. 1999), making it likely that
AP180 may act synergistically with AP2 to help retrieve
synaptotagmin I into SVs. AP180 mutants in both Drosophila
(lap) and Caenorhabditis elegans (unc-11) are uncoordinated
in locomotion and defective in synaptic transmission even
though only 1/3 of the total vesicle pool is lost in the lap
mutant (Zhang et al. 1998) and none of the pool in the unc-11
mutant (Nonet et al. 1999). Unexpectedly, synaptobrevin is
mislocalized to axonal membranes in the unc-11 mutant, suggesting that UNC-11 may help recycle synaptobrevin during
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Bao, Hong, Richard W. Daniels, Gregory T. Macleod, Milton P.
Charlton, Harold L. Atwood, and Bing Zhang. AP180 maintains
the distribution of synaptic and vesicle proteins in the nerve terminal
and indirectly regulates the efficacy of Ca2⫹-triggered exocytosis. J
Neurophysiol 94: 1888 –1903, 2005. First published May 11, 2005;
doi:10.1152/jn.00080.2005. AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been
implicated in retrieving SV proteins. In Drosophila, deletion of its
homologue, Like-AP180 (LAP), has been shown to increase the size
of SVs but decrease the number of SVs and transmitter release.
However, it remains elusive whether a reduction in the total vesicle
pool directly affects transmitter release. Further, it is unknown
whether the lap mutation also affects vesicle protein retrieval and
synaptic protein localization and, if so, how it might affect exocytosis.
Using a combination of electrophysiology, optical imaging, electron
microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles
in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle
fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle
docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release
machinery. Consistent with this hypothesis, three vesicle proteins
critical for calcium-mediated exocytosis, synaptotagmin I, cysteinestring protein, and neuronal synaptobrevin, are all mislocalized to the
extrasynaptic axonal regions along with Dap160, an active zone
marker (nc82), and glutamate receptors in the mutant. These results
suggest that AP180 is required for either recycling vesicle proteins
and/or maintaining the distribution of both vesicle and synaptic
proteins in the nerve terminal.
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
METHODS
Fly stocks and genetics
The lap mutant, a deficiency uncovering the lap locus (Df1801),
and the Canton Special (CS) wild-type strain were described previously (Zhang et al. 1998) and used in all our studies presented here.
These flies were raised at either room temperature (20 –23°C) or 19°C
on regular cornmeal-based fly foods. The null or severe loss of
function mutant of the lap gene was obtained as nontubby transheterozygotes (i.e., lap/Df) by crossing the lap mutant with its deficiency,
each of which was balanced with a TM6B balancer (lap/TM6B and
Df1801/TM6B). Previously, we have demonstrated that the lap mutant chromosome harboring an insertion of P element only affects the
lap locus and that the revertant of this line was essentially identical to
the CS wild type (Zhang et al. 1998). We believe that CS can best
serve as a wild type for the lap mutant after losing the revertant line.
Additional alleles of lap identified by us (unpublished) and by others
(Babcock et al. 2003) display similar electrophysiological defects as
the initial lap allele (see RESULTS). This further confirms the specificity
of the lap mutation.
Immunocytochemistry and microscopy
Larval body-wall muscle neuromuscular junction (NMJ) preparations were dissected and fixed in either paraformaldehyde (4%) or
J Neurophysiol • VOL
Bouin’s fixatives. After an hour incubation with a blocker solution
(2–5% bovine serum albumin, 0.2% Tween-20 in PBS), synaptic
proteins were stained overnight at 4°C using primary antisera, followed by 1–2 h incubation with secondary antibodies at room temperature, and visualized using a Nikon fluorescence microscope. The
fluorescence signal in the mutant was generally reduced compared
with that in the wild type (not shown), likely due to loss of SVs. The
final pictures presented here were taken on laser scanning confocal
microscopes with the fluorescence signal adjusted to equal levels in
the mutant and the wild type. Images presented in Fig. 8 were
obtained by using a Leica 4D TCS microscope with a ⫻40 and/or
⫻100 oil objective (1.4 numerical aperture and 0.41 ␮m Z steps).
Images presented in Figs. 9 and 10 were obtained by using a Leica
SP2 AOBS microscope with a ⫻63 oil objective (1.4 numerical
aperture and 0.122 ␮m Z steps). The following primary antibodies
were used in our experiments: antibodies to SV proteins (cysteinestring protein, CSP, mAB49, used at 1:50, courtesy of Dr. Konrad
Zinsmaier; synaptotagmin I, rabbit polyclonal, used at 1:500, courtesy
of Dr. Noreen Reist; n-synaptobrevin, rat polyclonal, used at 1:400,
courtesy of Dr. Hugo Bellen; Dap160, rabbit polyclonal, used at
1:200, courtesy of Drs. Jack Roos, Regis Kelly, and Hugo Bellen, and
glutamate receptor III, rabbit polyclonal, used at 1:300, courtesy of
Dr. Aaron DiAntonio; the active zone mAB nc82, used at 1:50,
courtesy of Dr. Erich Buchner) and an FITC-conjugated antibody to
HRP (goat polyclonal, used at 1:200, Jackson ImmunoResearch Laboratories). Secondary antibodies used in this study include anti-mouse
Texas Red, anti-rat TRITC, and anti-rabbit FITC (Jackson ImmunoResearch Laboratories), all at 1:200.
Electron microscopy
Dissected NMJ preparations were fixed and prepared for transmission electron microscopy according to the methods established by
Atwood et al. 1993. Briefly, dissected samples were fixed for 2 h at
room temperature in a fixative containing 0.1 M phosphate buffer
(PB), 3% glutaraldehyde, 4% sucrose, and 0.5% formaldehyde. Fixed
samples were then washed for 60 min with PB and postfixed for 1 h
with 1% osmium tetroxide in 0.1 M PB. After postfixation, samples
were washed three times in PB. Samples were then dehydrated in an
ethanol series and embedded in an Epon-Araldite mixture. Longitudinal muscles 6 and 7 in segment 3 and 4 were sectioned transversely
to locate nerve terminals on target muscles. Thin sections (75 nm)
were mounted on Formvar-coated single-slot grids, doubled stained
with uranyl acetate and lead citrate, and examined by a Hitachi
H-7000 transmission electron microscopy at 75 kV. Chemical reagents were purchased from Ted Pella (Redding, CA).
Vesicles near synapses in type I boutons were sampled in serial
sections to compare vesicle availability in mutant and wild-type larval
nerve terminals. Morphologically docked vesicles were defined as
those touching or within 20 nm of the presynaptic membrane beneath
or near the electron-dense T-bar, which is thought to be the focal point
and putative active zone for vesicle release in fly synapses (Feeney et
al. 1998; Koenig et al. 1993). Physically, vesicles further than 20 nm
from the presynaptic membrane are probably not releasable by a
single nerve impulse (Parsegian 1977). We also sampled two other
populations of synaptic vesicles: those within 50 nm of the densely
stained presynaptic membrane and those within 500 nm, which
includes almost all vesicles within reasonable range of an individual
synapse. The number of docked vesicles is excluded from these two
vesicle pools. We define a “synapse” as the densely stained, closely
apposed pre- and postsynaptic membranes evident in electron micrographs and an “active zone” as the prominent T-bar structure with its
attendant cluster of synaptic vesicles (Atwood et al. 1993). Limited
freeze-fracture images of fly synapses suggest that the presynaptic
calcium channels at active zones responsible for nerve-impulseevoked release of neurotransmitter are arranged loosely around the
T-bar on the presynaptic membrane (Feeney et al. 1998).
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endocytosis (Nonet et al. 1999). These observations lead to the
hypothesis that vesicle recycling can be decoupled from retrieval of SV proteins and that the “quality” of SVs could
significantly influence exocytosis. In Alzheimer’s patients, the
level of AP180 is significantly reduced prior to the loss of
synapses, implicating a functional role for AP180 in cognitive
integrity (Yao 2004).
However, it remains unclear whether LAP also plays a role
in vesicle protein retrieval and how such a defect might affect
exocytosis. In addition, recent studies of the Drosophila endophilin (endo) (Verstreken et al. 2002) and synaptojanin
(synj) (Verstreken et al. 2003) mutants have questioned
whether depletion of SVs readily translates to a reduction in
single action-potential-evoked basal transmitter release. In
these mutants, the vesicle pool is severely depleted, but the
basal transmitter release remains normal, suggesting that factors other than vesicle numbers may play an important role in
transmitter release. To address these questions and to advance
our understanding of AP180, we have further characterized the
lap mutant using electrophysiology, optical imaging, electron
microscopy, and immunocytochemistry. Our results reveal a
novel role for AP180 in maintaining the efficacy of calcium
(Ca2⫹) coupling to exocytosis at steps downstream of calcium
entry and vesicle docking. Furthermore, our studies show that
not only neuronal synaptobrevin (n-Syb), but also CSP, synaptotagmin I, Dap160, the active zone marker nc82, and
glutamate receptors are mislocalized to extrasynaptic regions
along the axon. Finally, the morphology of synapses is changed
from the typical “beads-on-a-string” appearance to “rope”-like
shapes. These findings suggest that AP180 indirectly regulates
the efficacy of Ca2⫹-triggered exocytosis by either helping
recycle SV proteins and/or maintaining the proper subcellular
distribution of synaptic and vesicle proteins in the nerve
terminal. The redistribution of both pre- and postsynaptic
components may in part reflect developmental alterations to
compensate for the impaired synaptic transmission in the lap
mutant.
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BAO, DANIELS, MACLEOD, CHARLTON, ATWOOD, AND ZHANG
Electrophysiology
Calcium imaging
The Ca2⫹ indicator, dextran-conjugated (10 kDa) Oregon Green
488 bis-(o-aminophenoxy)-N,N,N⬘,N⬘-tetraacetic acid 1 (BAPTA-1;
OGB-1, Molecular Probes, Eugene, OR), was loaded into motor nerve
terminals using the forward filling technique described by Macleod et
al. (2002). Briefly, the preparation was scanned through the objective
(⫻40, 0.55 numerical aperture) of a Nikon Optiphot upright microscope fitted with a BioRad 600 scan head (BioRad; Mississauga,
Ontario, Canada) using an Argon ion laser (at 0.5% intensity) in
combination with a BHS filter set (exciter filter: 488 nm DF10;
emission filter: OG 515 nm LP; dichroic reflector: 510 nm LP).
Scanned images were acquired to PC and processed using ImageJ
software (http://rsb.info.nih.gov/ij/). Fluorescence values (F) were
calculated for scan lines through OGB-1-filled boutons by numeriJ Neurophysiol • VOL
RESULTS
Evoked transmitter release is dramatically reduced in the
lap mutant
Synaptic terminals spontaneously release transmitter in the
absence of the nerve impulse (Fatt and Katz 1952). The
frequency of spontaneous release is largely a presynaptic
property, although retrograde signaling can influence it. To
record the mEJP, we used sharp electrodes to impale third
instar larval body-wall muscles bathed in saline containing 1
mM Ca2⫹ and 1 ␮M tetrodotoxin (TTX). The average resting
potential of muscle fibers in the lap mutant was indistinguishable from that in the wild type (CS, – 66.4 ⫾ 0.9 mV, n ⫽ 10;
lap/Df, – 65.9 ⫾ 0.5 mV, n ⫽ 11; P ⬎ 0.5). In addition to the
increased amplitude of minis reported earlier (Zhang et al.
1998), the frequency of mEJPs was 2.5-fold higher in the lap
mutant (9.1 ⫾ 0.4 Hz, n ⫽ 11) than in the wild type (3.7 ⫾ 0.4
Hz, n ⫽ 10) in the presence of Ca2⫹ (Fig. 1, A and B; P ⬍
0.001).
In sharp contrast with the increase in mini size and frequency, evoked transmitter release is dramatically reduced. In
an earlier report, we showed that evoked EJPs were smaller in
amplitude in the lap mutant but did not quantify them or
measure their Ca2⫹ dependence (Zhang et al. 1998). Here, we
confirmed the reduction of quantal release by measuring the
EJC under voltage clamp with the muscle membrane potential
clamped to – 80 mV to maintain a constant driving force (Fig.
1, C and D). The average amplitude of EJCs was significantly
reduced from 67.9 ⫾ 2.7 nA (n ⫽ 13) in the wild type to 4.8 ⫾
0.7 nA (n ⫽ 14) in the mutant (P ⬍ 0.001). The average mEJC
amplitude at the – 80 mV holding potential was determined to
be 0.61 and 0.98 nA for the wild type and the mutant,
respectively (Zhang et al. 1998). Accordingly, the reduction in
quantal content is estimated to be 96%, down from ⬃111
quanta in the wild type to ⬃5 quanta in the lap mutant. It is
important to note that this reduction in quantal content is not a
result of exhaustion of SVs in the nerve terminal caused by
repetitive nerve stimulation. In fact, this small EJP or EJC
amplitude was observed when we stimulated the motor nerve
after a long rest after dissection with either a single stimulus or
five stimuli delivered at a low frequency (ⱕ0.2 Hz). Intracel-
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The third instar body wall muscle-synapse preparation was dissected as described previously (Jan and Jan 1976) and used for both
intracellular recording and two-electrode voltage clamping in longitudinal muscles 6 and 7 bathed in HL-3 saline (Stewart et al. 1994;
Zhang et al. 1998). Membrane potentials or synaptic currents were
amplified through an Axoclamp 2B amplifier (Axon Instruments),
filtered at 2 kHz, digitized and recorded on a Dell PC computer
equipped with pClamp8 software (Axon Instruments). Microelectrodes (filled with 3 M KCl) with an input resistance of 12–18 M⍀
were used for voltage clamping and of 20 –32 M⍀ for intracellular
recording. Muscle input resistance was routinely monitored with a
1-nA current injection and by using the bridge mode on the Axoclamp
2B amplifier to cancel out the series resistance from the microelectrode. Analysis was included only in muscles with an input resistance
⬎5 M⍀. Spontaneous miniature excitatory junction potentials (mEJPs
or minis) were obtained by intracellular recording from muscles
bathed in HL-3 solutions containing 1 mM Ca2⫹ and 1 ␮M tetrodotoxin, which may eliminate spontaneous nerve firing and reduces the
probability for multiquantal release (Zhang et al. 1998). Excitatory
junction currents (EJCs) were evoked by directly stimulating the
innervating motor axons with a suction electrode at 0.2 Hz. The
extracellular Ca2⫹ concentration varied from 0.4 to 10 mM and it is
further specified in RESULTS.
To obtain the amplitude of EJCs at low [Ca] for the Ca2⫹ cooperativity plot, we stimulated the motor nerve 20 times at 0.2 Hz and
measured EJCs as well as the number of failures. Concerned about the
possibility that the high mini frequency in the lap mutant could
contaminate the amplitude of these evoked EJCs and thereby reduce
the slope of Ca2⫹ cooperativity plot, we decided to correct the
measured EJC amplitude for minis that are by chance coincident with
the EJCs. As first suggested by Schwarz and colleagues (Robinson et
al. 2002), we arbitrarily chose a point 110 ms after the stimulus
artifact and collected all minis for which the peak falls at this point.
The average of amplitude of these minis was then subtracted from the
average EJC amplitude. The average EJC amplitude for each muscle
fiber was obtained by dividing the sum of the adjusted EJC amplitude
with the total number of stimuli, including failures. Because mini
frequency is typically higher after nerve stimulation, it is likely that
we may have overcorrected for the number of spontaneous minis.
However, such an over correction would change Ca2⫹ cooperativity
in the opposite direction observed in our studies.
Analysis of the amplitude and frequency of mEJPs or EJCs was
conducted using Mini Analysis (Synaptsoft) and Origin (OriginLab).
Miniature EJPs with a slow rise time arising from electrically coupled
neighboring cells (Gho 1994; Zhang et al. 1998) were excluded from
the final analysis. The unpaired Student’s t-test was used for data
statistics. ANCOVA test was used for treatment of the slope of
Ca2⫹-dependence. Results were considered statistically different
when the P value was ⬍0.05.
cally subtracting the average pixel intensity of the background in the
scan line, containing no OGB-1, from the average pixel intensity of
the entire line. The effect of bleaching was removed by subtracting a
trendline fitted to F when no stimulus pulses had been applied to the
nerve. ⌬F/F is calculated as the change in F (⌬F) divided by the
resting level of F. The data were fit to a first-order exponential in
Sigma Plot (SPSS, Chicago, IL). Unpaired 2-tailed t-tests were used
to test differences in means.
Changes in the plateau level of Ca2⫹ indicator fluorescence in
larval motor nerve terminals, in response to nerve stimulation trains,
have previously been quantified using a frame scan protocol (Macleod
et al. 2002). In this study, we alternated frame scan data and line scan
data acquisition from the same type-1b while stimulating at 20 Hz. A
regression of the estimates of the plateau maximum ⌬F/F attained
from line scan data analysis on the estimates attained from frame scan
data analysis gave an X variable coefficient of 0.96 and a correlation
coefficient of 0.95 (P ⬍ 0.001). This analysis demonstrates that
estimates derived from line scan data will, on average, be the same as
those derived from frame scan data. All Ca2⫹ imaging was performed
in HL6 containing 0.5 mM Ca2⫹ at room temperature (⬃22°C).
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
1891
lular recording from a second lap allele, lapSD3 (Babcock et al.
2003), in trans with the original lap mutant or the lap deficiency also showed a similar reduction in quantal release. In
lap/lapSD3 and lapSD3/Df larvae, the average amplitude of
evoked EJPs obtained in 1 mM [Ca]-containing saline was
10.7 ⫾ 1.6 mV (n ⫽ 5) and 6.2 ⫾ 1.1 mV (n ⫽ 4),
respectively. The amplitude of these EJPs was similar to that in
the lap/Df larva (8.8 mV ⫾1.0 mV, n ⫽ 11). However, all
these lap alleles showed a dramatic reduction in evoked EJP
amplitudes, which was 40.4 ⫾ 1.6 mV (n ⫽ 10) in the wild
type (CS; P ⬍ 0.001). These results provide further genetic
evidence that the lap mutation likely deletes the lap locus. In
following studies, we hence focused on the original lap allele.
We and others have previously shown that quantal size increases concurrently with the enlargement of synaptic vesicles,
suggesting that postsynaptic receptors most likely remain unaffected in the mutant (Karunanithi et al. 2002; Zhang et al.
1998). Immunoreactivity of glutamate receptors shows no
apparent differences in the overall intensity of receptor clusters
for the mutant and the wild type (see Fig. 10). Hence, the great
attenuation of evoked response originates primarily at the
presynaptic side in the mutant neuromuscular junction (NMJ).
lap mutant displays a strikingly low release probability
Calcium not only triggers vesicle fusion, but also controls
the amount of transmitter release (Katz 1969). To probe possible primary defects in presynaptic terminals, we examined
the efficiency of Ca2⫹coupling to transmitter release at the
NMJ by measuring the failure rate defined as the fraction of
stimuli failing to evoke transmitter release at different [Ca]. At
low extracellular [Ca] (ⱕ0.4 mM), we failed to evoke release
from the mutant larvae while succeeding 80% times in the
wild-type larvae (not shown). Further examination and analysis
of failure rates in slightly higher [Ca] are shown in Fig. 2. At
0.6 mM [Ca], the lap mutant showed a high failure rate (32.1 ⫾
5.2%, n ⫽ 7, P ⬍ 0.001), whereas the wild type had no failures
at all (a 100% success rate, n ⫽ 9) after nerve stimulation at 0.2
Hz (Fig. 2A). Most surprisingly, the mutant persistently
showed considerable numbers of failures even when the extracellular [Ca] was further raised ⱕ0.9 mM (Fig. 2B). At these
FIG. 2. The lap mutant displays high rates of synaptic failures in response to low-frequency nerve stimulation. A: representative traces of synaptic currents
evoked by nerve stimulation in 0.6 mM extracellular [Ca]. While the wild-type larvae faithfully respond to each stimulus, the lap mutant displays a significantly
higher rate of failures than the wild type. 3, stimulus artifacts. B: histograms of failure rates for the wild type (CS) and the lap mutant (lap/Df) at [Ca] ranging
from 0.6 and 0.9 mM. At these [Ca]s, the mutant shows significantly higher failure rates compared with the wild type, which has no failures at all (**P ⬍ 0.001;
*P ⬍ 0.05). Synaptic failures observed at 0.8 and 0.9 mM Ca2⫹ are striking and have not been reported in other mutant flies.
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FIG. 1. Evoked transmitter release is dramatically reduced, whereas the frequency of
spontaneous release is increased in the lap
mutant. A and B: frequency of spontaneous
miniature excitatory junction potentials
(mEJPs or minis) is significantly increased in
the lap mutant compared with the CS wild
type (***P ⬍ 0.001). C and D: excitatory
junction currents (EJCs) obtained by 2-electrode voltage clamp from muscles held at
– 80 mV are also significantly reduced in
amplitude in the mutant (***P ⬍ 0.001). The
extracellular [Ca] was 1 mM calcium for all
of the experiments described in this figure.
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BAO, DANIELS, MACLEOD, CHARLTON, ATWOOD, AND ZHANG
Ca2⫹ concentrations, the wild-type larvae did not show any
failures. The failure rate for the mutant was 21.9 ⫾ 7.2% (n ⫽
8) at 0.7 mM [Ca], 16.9 ⫾ 7.1% (n ⫽ 8) at 0.8 mM [Ca] and
to 3.1 ⫾ 1.3% (n ⫽ 8) at 0.9 mM [Ca]. These unusually high
failure rates suggest that the release probability is extremely
low in the lap mutant, which may account for the dramatic
reduction in quantal content.
Calcium sensitivity and cooperativity are both reduced in the
lap mutant
Exocytotic defect occurs at steps downstream of
calcium entry
Our measurements of the failure rate, Ca2⫹ sensitivity and
cooperativity assume no change in Ca2⫹ entry between the
mutant and wild-type nerve terminals. However, with the
exception of Ca2⫹ cooperativity, all other physiological defects
could arise from impaired Ca2⫹ entry to the nerve terminal in
the lap mutant. To investigate this possibility, we examined
synaptic Ca2⫹ influx in larval NMJs using optical imaging
(Macleod et al. 2002). The Ca2⫹-sensitive dye dextran-conjugated Oregon Green 488 BAPTA-1 (OGB-1) was loaded from
the cut end of the segmental nerve and allowed to diffuse and
equilibrate within synaptic boutons for 1 h prior to imaging.
We measured Ca2⫹ influx by line scanning of single boutons
after nerve stimulation with single pulses as well as a short
train of stimulation at 20 Hz in a total of three wild-type and
seven mutant larvae. Our results showed that the relative
change in fluorescence intensity and the decay time constant
after a single stimulation were indistinguishable between the
wild-type control (24.6 ⫾ 5.4%, tau 71.7 ⫾ 9.8 ms, n ⫽ 6
cells) and the mutant (27.1 ⫾ 4.2%, n ⫽ 14; tau 66.5 ⫾ 6.3 ms,
n ⫽ 13; Fig. 4, A–D). In response to a repetitive stimulation (20
Hz, 0.5 s), the rise time, average accumulation, and decay
kinetics of intraterminal Ca2⫹ also remained unaffected by the
lap mutation (Fig. 4, E–I). The average maximal change of
fluorescence was 38.2 ⫾ 4.2 (n ⫽ 5) in the wild type and
41.1 ⫾ 5.3% in the mutant (n ⫽ 12). The decay time constant
was 170 ⫾ 6 ms (n ⫽ 4) in the wild type and 203 ⫾ 32 ms (n ⫽
9) in the mutant, but they are not statistically different (P ⫽
2⫹
FIG. 3. Ca
sensitivity and cooperativity
are both reduced in the lap mutant. A: dosedependence of evoked excitatory junction currents (EJCs) on extracellular [Ca]. In the wild
type, the EJC amplitude increases significantly
when the extracellular [Ca] is increased from
0.4 to 5 mM. At 10 mM Ca2⫹, EJC amplitude
is reduced. In contrast, the lap mutant shows
only a slight increase in the amplitude of EJCs
when [Ca] is raised. B: logarithmic plot of the
EJC amplitude against the extracellular [Ca].
The slope of these lines represents Ca2⫹ cooperativity, which is 3.9 and 2.1 for the wild type
and the mutant, respectively. These slopes are
significantly different (P ⬍ 0.05; ANCOVA
test). In all the experiments shown here, the
nerve was stimulated at 0.2 Hz, and data were
obtained from ⱖ5 animals from each genotype.
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The high failure rate observed in the lap mutant indicates
that Ca2⫹-sensitivity may be reduced. To test this possibility,
we attempted to “rescue” the defect in exocytosis by increasing
the extracellular [Ca]. In the presence of high [Ca] in the saline,
the amplitude of EJCs in the wild type increased dramatically
in ⱕ6 mM saline and then declined at 10 mM for unknown
reasons. In contrast, the EJC amplitude in the lap mutant was
only slightly increased (Fig. 3A). These results and the low
release probability together suggest that Ca2⫹ coupling to
exocytosis, Ca2⫹ entry into the nerve terminal, or the readily
releasable pool of SVs is reduced in the mutant.
To further examine Ca2⫹ sensitivity, we determined Ca2⫹
cooperativity by measuring the slope of the logarithmic plot of
EJC amplitude against [Ca] at low concentration ranges. At
low Ca2⫹ concentrations, it is suggested that at least four Ca2⫹
ions act in a cooperative fashion to trigger exocytosis (Dodge
and Rahamimoff 1967; Smith et al. 1985). As predicted, the
logarithmic plot of EJC amplitude against extracellular [Ca]
ranging from 0.5 to 1 mM shows a linear relationship for both
the wild type and the mutant (Fig. 3B). However, the slope of
these two lines is significantly smaller for the lap mutant
(slope ⫽ 2.1) than for the wild type (slope ⫽ 3.9, P ⬍ 0.05),
demonstrating that Ca2⫹ cooperativity of transmitter release is
significantly reduced in the mutant. As noted in METHODS, we
undertook careful measures to eliminate the potential contamination of EJCs by spontaneous minis that could skew toward
a shallow slope for the mutant. At present, it is unknown how
this change of the slope comes about. Nonetheless, the low
Ca2⫹ cooperativity is consistent with the possibility that Ca2⫹
is less effective in triggering transmitter release, perhaps due to
a change either in Ca2⫹ sensors on the vesicle (Littleton et al.
1994) or the release machinery (Stewart et al. 2000).
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
0.34), suggesting that the mutant clears its Ca2⫹ at a normal
rate. The normal entry and kinetics of terminal Ca2⫹ strengthen
our argument that Ca2⫹ coupling to exocytosis is severely
disrupted in the lap mutant.
Number of morphologically docked SVs is moderately
reduced in the lap mutant
One of the factors influencing transmitter release at steps
downstream of Ca2⫹ entry could be the availability of SVs at
the active zone (Murthy et al. 2001). Because Ca2⫹ entry is
normal, the small EJC amplitude observed in the lap mutant
suggests that the number of SVs in the docked pool is reduced.
We have previously attributed the impairment in release to a
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4. Ca2⫹ influx and decay are both normal at the lap mutant terminal.
A–D: calcium influx and decay kinetics within single boutons (type 1b) after
nerve stimulation by single pulses. Calcium influx and decay were measured as
fluorescence changes of dextran-conjugated Oregon Green 488 bis-(o-aminophenoxy)-N,N,N⬘,N⬘-tetraacetic acid 1 (BAPTA-1; OGB-1). A1 and B1: the
vertical line across the bouton (filled with OGB-1) shows the position of a laser
line scanned at a rate of one line every 4.06 ms by a confocal microscope. A2
and B2: representative traces of calcium fluorescence within single boutons in
the wild type and the mutant. These are serial line scans accumulated from left
to right over a period of 500 ms. The white solid line (arrowhead) indicates the
time at which a single stimulus pulse was delivered to the segmental nerve. A3
and B3: representative plots of relative changes (⌬F/F) of fluorescence within
single synaptic boutons after nerve stimulation. The look-up table, representing
pixel values 0 –255, is shown to the right. Image pixels in A and B have not
been processed or smoothed. C: a histogram comparing the average change of
fluorescence in synaptic boutons between the wild type and the mutant larvae
(P ⫽ 0.71). D: a histogram comparing the average decay time constant (tau) of
fluorescence in synaptic boutons between the wild type and the mutant larvae
(P ⫽ 0.67). The number of terminals boutons sampled (separate muscles, from
3 separate wild type and 7 mutant larvae) is shown on each histogram. E–I:
summary results of calcium rise and decay within single boutons after a brief
train of nerve stimulation (20 Hz, 2 s). E1 and F1: vertical lines mark the
position of laser line across single boutons. E2 and F2: representative traces of
fluorescence change within single boutons in the wild type and the mutant. The
white vertical line indicates the delivery of the 1st pulse in the train, whereas
the horizontal bar indicates the duration of the 20-Hz stimulation. G: representative plots of relative changes of fluorescence within single boutons after
a short train of stimuli at 20 Hz for 2 s. H: a histogram comparing the average
change of fluorescence between boutons in the wild type and the mutant larvae
(P ⫽ 0.71). I: a histogram comparing the average decay time constant (tau) of
fluorescence between boutons in the wild type and the mutant larvae (P ⫽
0.34). Scale bar: 5 ␮m in all panels. Variances are shown as SE of the mean.
The number of terminals boutons sampled (separate muscles, from 3 separate
wild-type and 12 mutant larvae) is shown on each histogram. [Ca2⫹] o ⫽ 0.5
mM. All statistical comparisons are unpaired 2-tailed t-test.
FIG.
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1/3 loss of the SV pool in the lap mutant (Zhang et al. 1998).
However, we did not quantify docked vesicles. To this end, we
investigated the distribution of SVs near the active zone with
transmission electron microscopy. We counted vesicles on
three serial sections in which the central one always had an
electron-dense “T” bar, the putative active zone in Drosophila
(see arrows in Fig. 5) (Atwood et al. 1993; Jia et al. 1993;
Koenig et al. 1993). Consistent with our early results (Zhang et
al. 1998), the total number of SVs, including those within 50
and 500 nm to the plasma membrane but excluding docked
vesicles, is significantly reduced in the lap mutant (Fig. 5). We
further focused on the morphologically docked vesicles, which
were defined as the group of vesicles touching or within 20 nm
of the densely stained presynaptic membrane underneath or
near the T-bar. These vesicles could potentially be releasable
during initial stimulation (Parsegian 1977). The wild-type
synapse contained on average 6.5 ⫾ 0.82 morphologically
docked vesicles/active zone (n ⫽ 14 active zones), whereas
this number was 4.2 ⫾ 0.55 (n ⫽ 21) in the lap mutant,
representing a 35% reduction (Fig. 5C).
We next used the active zone marker nc82 monoclonal
antibody to estimate the number of active zones in the wildtype and the mutant larvae (Wucherpfennig et al. 2003; Qin et
al. 2005). Figure 6 shows nc82 immunoreactive puncta on
muscle 4 in segment 3 in the wild type and the lap mutant. A
total of eight muscles from four wild-type and seven muscles
from five mutant larvae were examined. The lap mutant had a
slightly higher average number (381.4 ⫾ 29.4, n ⫽ 7) of
nc82-positive puncta per muscle compared with the wild type
(299.8 ⫾ 27.4, n ⫽ 8). However, this difference was not
statistically significant from each other (P ⫽ 0.06). This is
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BAO, DANIELS, MACLEOD, CHARLTON, ATWOOD, AND ZHANG
would be better for determining the number of active zones.
These studies suggest that the lap mutation does not have a
significant effect on the total number of active zones. However,
the location of the active zone differed in the mutant (see
DISCUSSION later).
Paired-pulse induces short-term facilitation instead of
synaptic fatigue in the lap mutant
consistent with our observation that the number of T-bars
appeared similar between the mutant and the wild type from
our limited sections (data not shown), although serial sections
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FIG. 5. The number of morphologically docked vesicles at the active zone
is mildly reduced at the lap mutant NMJ. A and B: representative electron
micrographs of cross-sections of larval NMJs from the wild type (CS, A) and
the lap mutant (lap/Df, B). It is apparent that the total number of SVs is
significantly reduced in the mutant compared with that in the wild type.
“T”-shaped electron-dense materials (“T”-bars) in the wild type (CS) and the
lap mutant (lap/Df) are putative active zones (3). C: average numbers of 3
populations of vesicles inside the nerve terminal of the wild type (CS) and the
lap mutant (lap/Df). These 3 populations are morphologically “docked vesicles” that are within 20 nm or in contact with the plasma membrane at the
active zone, vesicles within 50 nm of the presynaptic active zone membrane,
and vesicles within 500 nm of the presynaptic active zone membrane, excluding docked vesicles, respectively. On average, the docked-vesicle pool is
mildly but significant statistically (*P ⬍ 0.05). The remaining 2 vesicle
populations are more dramatically reduced in the lap mutant (** P ⬍ 0.01; ***
P ⬍ 0.001).
Ultrastructural reconstruction of mammalian central synapses reveals that the number of morphological docked vesicles and the number of readily releasable pool (RRP) of SVs
are almost identical (Schikorski and Stevens 2001). The rather
mild reduction in the number of morphologically docked vesicles observed in the lap mutant predicts that the RRP at the
active zone may not be significantly reduced. We reasoned that
if the RRP were limited, a brief and repetitive stimulation
would lead to a rapid synaptic fatigue in the lap mutant, as has
been shown in endo (Verstreken et al. 2002), synj (Verstreken
et al. 2003), and dap160 mutants (Marie et al. 2004; Koh et al.
2004). To test this hypothesis, we stimulated the synapse with
paired pulses delivered at 20 and 50 ms apart at different
extracellular [Ca]. As with most synapses, the Drosophila NMJ
also modifies its synaptic strength following the activity pattern
of the motoneuron. Short-term plasticity can be determined
experimentally by monitoring the relative change of EJC amplitude in response to two successive pulses of stimuli. Quantitatively, we define this change as the facilitation index (FI),
which equals (EJC2-EJC1)/EJC1 X100%. A positive plasticity
index represents facilitation, which represents an increased
release of transmitter aided by residual Ca2⫹ following the
initial action potential (Xu-Friedman and Regehr 2004; Zucker
1999). In contrast, a negative plasticity index stands for depression, which is often caused by the depletion of SVs by the
first action potential. Short-term plasticity depends on both the
extracellular calcium concentration and the interval of the two
stimuli. In general, the synapse facilitates at low [Ca] and
depresses at high [Ca]. Furthermore, synaptic facilitation or
depression increases when the interval shortens.
The result of short-term plasticity in the wild type and the
lap mutant is summarized in Fig. 7. At 0.8 mM [Ca], both
wild-type and the mutant larvae showed mostly facilitation.
However, the degree of facilitation differed between these two
animals. The mutant synapse facilitated by 45.3 ⫾ 14.8% (n ⫽
10) and 48.7 ⫾ 7.3% (n ⫽ 10) at the 50- and 20-ms stimulus
intervals, respectively. The wild-type synapse displayed a relatively weak facilitation by 11.1 ⫾ 2.0% (n ⫽ 9) and 5.1 ⫾
5.8% (n ⫽ 9) at the same stimulus intervals. These differences
are mild but significantly different statistically (P ⬍ 0.001 for
20-ms interval, P ⬍ 0.05 for 50-ms interval). At this low [Ca],
synaptic depletion does not occur in both the wild type and the
lap mutant primarily because the release probability is low.
To increase the release probability in both the wild type and
the mutant, we raised extracellular [Ca] to 5 mM. At this [Ca],
the wild-type synapses showed a profound depression with FIs
ranging from ⫺25.5 ⫾ 1.4% (n ⫽ 8) at 50-ms interval to
⫺31.8 ⫾ 2.5% (n ⫽ 9) at 20-ms interval. In contrast, the
mutant synapse did not show depression. Instead, it displayed
2.7 ⫾ 3.3% (n ⫽ 17) and 3.9 ⫾ 3.6% (n ⫽ 12) facilitation at
20- and 50-ms paired-pulse intervals, respectively. The differences in FIs are statistically significant (P ⬍ 0.001). These
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
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FIG. 6. The number of active zones remains unchanged in the lap mutant. A and B: Nc82 antibody-stained NMJs on muscle 4 in segment 3 from the wild
type (CS, A) and the lap mutant (lap, B) are shown here. Insets: enlarged areas identified by 3. C: histograms showing that the average number of nc82-positive
puncta is slightly increased in the lap mutant compared with the wild type. However, this increase is not statistically significant (t-test, P ⫽ 0.06).
SV proteins are abnormally distributed to extrasynaptic
axonal regions
While the mild reduction in docked vesicles may contribute
to the reduced quantal content, it is insufficient to account for
the change in Ca2⫹ sensitivity or cooperativity. What could
explain these exocytotic defects in the lap mutant downstream
of vesicle docking and Ca2⫹ entry? During vesicle recycling,
clathrin-mediated endocytosis accomplishes two major tasks:
to retrieve vesicular components (i.e., lipids and proteins) and
to assemble new vesicles. Even though these two events are
tightly coupled under physiological conditions (Sankaranara-
yanan and Ryan 2000), a partial failure in recycling SV
proteins may occur when clathrin-mediated endocytosis is
impaired. This possibility was first suggested by studies of the
AP180 mutant (unc-11) in C. elegans, in which synaptobrevin
was mislocalized to axonal membranes (Nonet et al. 1999).
Should a loss of synaptobrevin and/or other SV proteins occur
in the lap mutant, transmitter release could also be impaired.
Additionally, perturbation of clathrin-dependent vesicle trafficking may also affect either the localization of synaptic
proteins and/or the development of the synapse. This is suggested by the altered distribution of nc82-positive puncta
shown in Fig. 6.
To test these hypotheses, we examined the distribution of
SV proteins at the NMJ and the morphology of synaptic
boutons using immunocytochemistry. The Drosophila NMJ is
a well-characterized system for synaptic studies due in part to
its large synapse size and stereotyped projection of synaptic
boutons on identifiable muscle surfaces (Atwood et al. 1993;
Hoang and Chiba 2001; Jia et al. 1993; Keshishian et al. 1993).
Here, we used antibodies to three SV proteins (synaptotagmin
I, n-Syb, and CSP) to examine the morphology of the mutant
and wild-type NMJ with a particular focus on the subcellular
FIG. 7. Synaptic facilitation instead of depression is
evoked in the lap mutant by paired pulses. Paired pulses
delivered at 50- and 20-ms intervals are used to induce
short-term synaptic plasticity (facilitation or depression) in
the lap mutant (lap/Df) and wild type (CS) larvae at 0.8 and
5 mM [Ca]. The change in transmitter release in response to
the 2 pulses is quantified by calculating the facilitation index
(FI) using the equation (EJC2-EJC1)/EJC1 X100%. A: representative evoked EJCs by paired pulse at 20-ms interval and
0.8 mM [Ca]. Facilitation is observed in both animals under
these experimental conditions. A1: plots of average FIs induced by paired pulse at 50- and 20-ms intervals and 0.8 mM
[Ca]. Facilitation is more profound in the lap mutant (lap/Df)
than in the wild-type larvae (CS, *P ⬍ 0.05; ***P ⬍ 0.001).
B: representative evoked EJCs by paired pulse at 20-ms
interval and 5 mM [Ca]. At this [Ca], the wild type shows
dramatic depression whereas the lap mutant displays facilitation. B1: plots of average FIs induced by paired-pulse at 50and 20-ms intervals and 5 mM [Ca]. The FIs between the
mutant (lap/Df) and the wild-type (CS) larvae are significantly
different (***P ⬍ 0.001). The lack of depression by paired
pulses at these brief intervals suggest that releasable pool of
vesicles is unlikely the major and immediately limiting factor
for the dramatic reduction in quantal content observed in the
lap mutant.
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results show whether it is at low or high [Ca], the mutant
synapse shows persistent facilitation instead of depression
when paired pulses are delivered. At these brief intervals
(20 –50 ms), vesicle recycling or recruitment is not expected to
play a role to replenish the RRP. Hence, these results suggest
that the limitation of the releasable vesicle pool is unlikely the
major and direct factor contributing to the reduction of release
probability in the lap mutant. This provides further support to
the possibility that the coupling between calcium and secretion
is disrupted.
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BAO, DANIELS, MACLEOD, CHARLTON, ATWOOD, AND ZHANG
The localization of SV proteins to synaptic boutons remains in
the wild type even after extensive stimulation of the motor
axon with high potassium-containing saline (not shown), suggesting that SVs proteins are efficiently internalized from the
plasma membrane.
In the lap mutant, these SV proteins were also found within
synaptic boutons (n ⫽ 12). However, the lap mutant differed
from the wild type in that the extrasynaptic regions were
expanded in 60% of the synaptic branches. Furthermore, the
SV proteins were found in the extrasynaptic axonal regions
between some boutons regardless whether the morphology of
synaptic boutons was altered or not (Fig. 9, B and D, see 3).
Synaptotagmin I displayed a similar mislocalization pattern to
those found for CSP and n-Syb in the lap mutant (n ⫽ 10, not
shown). In rare cases, the motor axon bundle innervating
muscles 12 and 13 was also stained with a polyclonal antibody
to n-Syb in the lap mutant (data not shown). The mislocalization of these SV proteins occurred in nearly all synapses
examined. Taken together, these results are consistent with the
possibility that either these SV proteins or intact SVs are
mislocalized to the extrasynaptic regions.
Other synaptic components are also mislocalized to
extrasynaptic regions
The alterations in SV protein distribution and synapse morphology are similar to the finding that clathrin heavy chain and
dynamin are diffusely distributed at the NMJ (Zhang et al.
1998). This raises the possibility that lap mutations may also
affect the distribution of other synaptic proteins due to its
effects on either SV recycling or general vesicle trafficking.
FIG. 8. Synaptic vesicle proteins are mislocalized at 3rd instar larval NMJs. Fluorescent images of immunoreactive NMJs on muscle 4 show the localization
of 3 SV proteins (Syt, synaptotagmin I; CSP, cysteine string protein; and n-Syb, neuronal synaptobrevin) in synaptic boutons and presynaptic membranes. The
muscles on which these synapses are formed are not labeled and thus invisible. A–a1: Syt (green) and CSP (red) are colocalized to synaptic boutons in the wild
type (CS) forming a “beads-on-a-string” appearance. Note that these synaptic vesicle proteins are restricted to the bouton area and absent in the extrasynaptic
axonal region between boutons (see 3 in the enlarged images shown in a and a1). B– b1: Syt (green) and n-Syb (red) are also colocalized to synaptic boutons
in the wild type (CS) forming the beads-on-a-string appearance. Note that these synaptic vesicle proteins are restricted to the bouton area and absent in the
extrasynaptic region between boutons (see enlarged images in b and b1, 3). C– c1: Syt (green) and CSP (red) are colocalized to synaptic boutons in the lap
mutant (lap/Df). However, they also appear in the axonal region between individual boutons forming a “rope”-like morphology. This mislocalization of vesicle
proteins is more apparent in the enlarged images (c and c1, 3). D– d1: Syt (green) and n-Syb (red) are colocalized to synaptic boutons in the lap mutant (lap/Df).
However, they also appear in the axonal region between individual boutons (see the enlarged images in d and d1, 3).
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localization of these SV proteins in extrasynaptic regions
between synaptic boutons. As shown in Fig. 8, synaptotagmin
I, n-Syb, and CSP were generally well localized within synaptic boutons at the NMJ in the wild type (n ⫽ 10 larvae).
Importantly, these immunoreactive boutons were discrete and
separated from each other giving a beads-on-a-string appearance. This typical pattern of SV protein distribution is well
established for the fly NMJ (e.g., Estes et al. 1996), suggesting
that SV proteins are usually absent from extrasynaptic regions
that connect two neighboring boutons (Fig. 8, A and B; also see
Figs. 9A and 10A). These SV proteins were also found within
synaptic boutons in the lap mutant (Fig. 8, C and D; n ⫽ 20).
However, the morphology of most synapses was altered to a
rope-like appearance instead their normal shape. Importantly,
these SV proteins were also detected in contiguous and tubular
structures that fill the extrasynaptic regions between individual
synaptic boutons (compare a, a1, and b1 with c, c1, and d1).
These results show that the synaptic morphology is altered to
ropes and that these SV proteins or SVs are mislocalized to
extrasynaptic axonal regions in the lap mutant.
To confirm that these SV proteins are indeed mislocalized to
axonal regions, we used an antibody to the neuronal membrane
protein Nervana/HRP (Jan and Jan 1982; Sun and Salvaterra
1995) to mark the axonal membrane. In doubly stained NMJs
with antibodies to HRP and CSP or n-Syb, we confirmed that
CSP and n-Syb were localized primarily to synaptic boutons in
the wild type (n ⫽ 8). As expected, the HRP antibody labeling
is found in the extrasynaptic areas between boutons that are
usually devoid of SV proteins (Fig. 9, A and C, see 3). A small
number of NMJs in the wild type occasionally show a “spillover” of SVs proteins from synaptic boutons to the axonal area.
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
1897
We therefore examined the subcellular localization of the
endocytotic protein Dap160 (Koh et al. 2004; Marie et al.
2004; Roos and Kelly 1998, 1999) in synaptic terminals and
type III glutamate receptors (GlutRIII) (Marrus et al. 2004) in
the postsynaptic density using specific antibodies (Fig. 10).
Dap160 was restricted within synaptic boutons in the wild
type, but it was redistributed into regions between synaptic
boutons in the mutant (Fig. 10, A and B; n ⫽ 8). Although we
did not examine all the presynaptic components, our results
suggest that presynaptic proteins as well as SV proteins are
both present in extrasynaptic regions. As one would expect,
glutamate receptors were highly enriched in the muscle membrane opposing synaptic boutons (Marrus et al. 2004; Petersen
et al. 1997) (Fig. 10C). Unlike most presynaptic and SV
proteins, however, a small number of the receptor is also found
in extrasynaptic regions between synaptic boutons in the wild
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type (Fig. 10C, 3; n ⫽ 8) (Marrus et al. 2004). Nonetheless,
the usual beads-on-a-string morphology of synapses remains in
the wild type. In the lap mutant, no apparent change in
GlutRIII and DAP160 immunoreactivity was detected (n ⫽
16). As in the wild-type larvae, GlutRIII also remained in the
postsynaptic density opposing synaptic boutons. However,
they were redistributed to adapt to the rope-like pattern of
NMJs in the mutant, indicating that the receptor was also
mislocalized at the NMJ. The redistribution of receptors is
consistent with the altered distribution of active zones in the
lap mutant (Fig. 6).
DISCUSSION
In the present study, we have examined the role of the
endocytotic protein AP180 (LAP) in synaptic transmission
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FIG. 9. Co-localization of cysteine-string
protein (CSP) and neuronal synaptobrevin
(n-syb) with HRP in the extrasynaptic region.
A: localization of CSP (red) and horseradish
peroxidase (HRP, green) at the wild type
(CS) NMJ. While CSP is localized to synaptic boutons, HRP marks the axonal membrane as well as the bouton membrane. Both
CSP and HRP are found in synaptic boutons.
Note that CSP is absent from the extrasynaptic axonal region between synaptic boutons
(see 3). B: localization of CSP (red) and
HRP (green) at the lap mutant NMJ. Note
that CSP is now mislocalized to the extrasynaptic region between synaptic boutons
marked by HRP (see 3). C: localization of
n-Syb (red) and HRP (green) at the wild-type
(CS) NMJ. Similar to CSP, n-Syb is also
localized to synaptic boutons. Note that nSyb is absent from the extrasynaptic region
between synaptic boutons (see 3). D: localization of n-Syb (red) and HRP (green) at the
lap mutant NMJ. Unlike in the wild type,
n-Syb is found within synaptic boutons and
in the extrasynaptic region between synaptic
boutons (see 3).
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BAO, DANIELS, MACLEOD, CHARLTON, ATWOOD, AND ZHANG
and synaptic protein localization at the Drosophila NMJ.
Our results are consistent with a primary role for AP180 in
vesicle recycling revealed in our previous work. In addition,
we have uncovered several features of exocytotic defects in
the lap mutant that are more perplexing and cannot be
explained simply by the reduction in the SV pool alone. Our
studies show that the exocytotic defects occur at steps
downstream of calcium influx. Contrary to previous assumptions, the reduction in vesicle docking (34%) is rather mild
compared with the 96% reduction in quantal content. The
exocytotic defect cannot be explained entirely by the change
in vesicle docking, as calcium sensitivity and cooperativity
and failure rate are also altered in the lap mutant. We have
further shown that n-Syb, CSP, and synaptotagmin I, three
vesicle proteins critical for calcium-evoked release, are
mislocalized to the extrasynaptic axonal regions in the lap
mutant. The endocytotic protein DAP160, the active zone
maker nc82, and glutamate receptors are also found in the
extrasynaptic regions at the mutant NMJ. These results
suggest that both vesicle recycling and the subcellular
distribution of pre- and postsynaptic proteins are impaired.
Hence, possibilities remain that LAP is involved in not only
vesicle recycling but also synaptic development.
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AP180 is required for calcium-evoked transmitter release at
steps downstream of calcium influx and vesicle docking
Our studies have revealed three lines of evidence supporting
the hypothesis that LAP/AP180 regulates the efficacy of Ca2⫹
coupling to transmitter release at the Drosophila NMJ. First
lap mutations significantly reduce Ca2⫹-evoked release but
leave spontaneous fusion relatively intact. In fact, the rate for
spontaneously release, indicated by the frequency of minis, is
actually increased, perhaps due to developmental homeostasis
that compensates for the loss of synaptic release evoked by
action potentials (Davis and Goodman 1998; Turrigiano and
Nelson 2004). Second, Ca2⫹ sensitivity is reduced in the lap
mutant, evidenced by the high failure rate and inability by high
[Ca] to rescue transmitter release in the mutant. Third, we show
a reduction in Ca2⫹ cooperativity in the lap mutant, providing
further support that Ca2⫹ is less efficient in triggering exocytosis. We have definitely shown that these exocytotic defects
occur at steps downstream of calcium influx in the nerve
terminal. A key question remaining is whether these physiological defects can be explained simply by the increase in
vesicle size and/or by the reduction in vesicle numbers, two
prominent defects revealed in our initial characterization of the
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FIG. 10. Localization of Dap160 and glutamate receptors in
the lap mutant. A and B: localization of Dap160 (a presynaptic
marker, green) and cysteine-string protein (CSP, red) at the wild
type (CS) and the lap mutant NMJ. As with other presynaptic
components, both Dap160 and CSP are localized to synaptic
boutons in the wild type and in the mutant. However, both
Dap160 and CSP are absent in the wild type (see A, 3) but
found in the lap mutant (see B 3), in intersynaptic bouton
regions. C and D: localization of glutamate receptor III (a
postsynaptic marker, green) and CSP (red) at the wild-type (CS)
and the lap mutant NMJ. Glutamate receptor III is primarily
found in postsynaptic density closely opposing the presynaptic
terminal identified with CSP (C). Small amount of the receptor
is also found along the intersynaptic bouton regions (see C2,
3). Similar patterns of glutamate receptor III distribution are
found in the lap mutant. However, more receptors are found in
the intersynaptic bouton regions to match the shape of the
rope-like synapse in the mutant (D2, 3).
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
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has reduced numbers of SVs similar to that observed in the lap
mutant. It reduces quantal content but keeps the amplitude of
EJPs normal (Koh et al. 2004; Marie et al. 2004). Based on
these observations, we favor the possibility that the moderate
change in docked vesicles unlikely accounts substantially for
the reduction in quantal release in the lap mutant.
The precise cause for the mild reduction in docked vesicle
pool is unknown. One possibility is the high frequency of
spontaneous fusion of SVs may deplete the size of docked
vesicle pool. Other factors, such as the size of SVs and active
zones could influence the quantification of morphologically
docked vesicles. We do not have data to quantitatively compare the size of active zones between the wild type and the lap
mutant. However, it is known that the average size of SVs is
increased in the lap mutant (Karunanithi et al. 2002; Zhang et
al. 1998). This may lead to fewer vesicles being able to fit
around the T-bar at the active zone. A third possibility is that
this reduction in the number of docked vesicles could be
related to possible loss of synaptotagmin I on vesicles. Reist
and colleagues showed previously that vesicle docking is
reduced in synaptotagmin I mutants (Reist et al. 1998).
AP180 indirectly maintains the efficacy of calcium-evoked
transmitter release
Although AP180 might directly participate in calcium coupling to exocytosis, we deem it very unlikely because of its
biochemical properties as a clathrin-assembly protein and the
endocytotic phenotypes revealed in the lap and unc-11 mutant.
Therefore we favor the idea that AP180 indirectly regulates
synaptic transmission through endocytosis. At present, the
change in the distribution of both synaptic and vesicle proteins
at the NMJ stands out as the most striking morphological
phenotype in the mutant. The questions are then how this
subcellular change in protein distribution might come about
and whether it contributes to the physiological defects in the
mutant. A simple and straightforward interpretation of the
aberrant protein localization at the NMJ is that it results
naturally from SV depletion in endocytotic mutants. Although
an easily acceptable and logical assumption, it may not be
supported by current experimental data. In endo and synj
mutants, a severe depletion of SV occurs such that the reserve
pool is abolished and the remaining SVs are found near the
active zone. Consequently, SV proteins are only found in the
peripheral areas within the synaptic bouton (Verstreken et al.
2002, 2003). However, SV proteins remain inside synaptic
boutons and are absent from the extrasynaptic axonal regions
in these mutant animals. An absence of synaptotagmin I and
CSP in extrasynaptic regions in endo and synj mutants has been
recently confirmed by others (D. Dickman and T. Schwarz,
personal communications). Similarly, a partial depletion of
SVs in dap160 mutants does not result in an accumulation of
SV proteins in extrasynaptic regions (Koh et al. 2004; Marie et
al. 2004). In contrast, SV proteins are mislocalized in unc-11
and stn mutant animals despite the fact that the SV pool
remains normal in these animals. Our work confirms the
observation of synaptobrevin mislocalization first made in the
unc-11 mutant (Nonet et al. 1999) and further shows that CSP
and synaptotagmin I are also mislocalized to extrasynaptic
regions in the lap mutant. We note that the phenotype of SV
protein mislocalization in the lap mutant is reminiscent of the
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lap mutant (Zhang et al. 1998). Quantal size and vesicle size
increase concurrently (Karunanithi et al. 2002; Zhang et al.
1998), consistent with a normal density of postsynaptic receptors. Furthermore, spontaneous release appears normal and
even occurs at a higher rate. These results indicate that the
defect in exocytosis is primarily presynaptic and that the
change in vesicle size has minimal effects on vesicle fusion per
se. The change in vesicle size, however, may influence its
response to calcium should the vesicle protein or lipid compositions be altered.
One might argue, as we previously assumed (Zhang et al.
1998), that these synaptic physiological defects are simply
caused by a reduction in the total number of SVs and docked
vesicles. However, insights from our current results and from
recent genetic studies of other endocytotic mutants now force
us to take a fresh look at the relationship between SV pools and
synaptic transmission. First, the reduction in calcium sensitivity and cooperativity cannot be easily explained by changes in
the number of docked vesicles at the active zone. Second, the
number of docked vesicles is only mildly reduced compared
with the reduction in quantal content. At the morphological
level, each larval muscle is estimated to have on average 50
synaptic boutons and a total of ⬎500 active zones (Atwood et
al. 1993; Jia et al. 1993). Assuming some of these active zones
are “silent” due to the lack of docked vesicles (Atwood and
Wojtowicz 1999), 400 – 450 active zones are estimated to be
functional. At 1 mM [Ca], the typical quantal content is
estimated to be 110 (see Fig. 3), suggesting that each active
zone releases transmitter from 0.24 to 0.28 SVs. In the lap
mutant, the number of active zones does not appear to change
based on our limited random EM sections. The average number
of docked vesicles (4.2 SVs) per active zone in the mutant
should be sufficient to support a normal level of basal exocytosis. Our estimated number of active zones using the nc82
antibody was ⬃300 in the wild type and 381 in the mutant.
They are slightly below the estimated numbers of active zone
by electron microscopy. One likely reason for this could be the
difficulty counting and resolving all nc82-positive puncta at
light microscopic levels. Nonetheless, our results suggest that
there is no statistical difference in the number of active zones
per muscle fiber between the mutant and the wild type. Unless
some of the active zones in the extrasynaptic region are
“inactive,” this finding further supports the notion that the
exocytotic defect occurs downstream of vesicle docking. Third,
paired-pulse stimulation induces facilitation rather than depression in the lap mutant, suggesting that the RRP of SVs is not
a direct and limiting factor for the dramatic reduction in basal
release. In comparison, other endocytotic mutations, such as
endo (Verstreken et al. 2002), synj (Verstreken et al. 2003), and
dap160 (Koh et al. 2004; Marie et al. 2004), show an immediate synaptic depression on repetitive stimulation. Fourth, the
C. elegans AP180 mutant unc-11 (Nonet et al. 1999) and the
Drosophila stn mutant (Fergestad et al. 1999; Stimson et al.
1998, 2001) are also defective in synaptic transmission even
though the number of SVs remains normal in the terminal. In
fact, the number of docked SVs is increased in the unc-11
mutant (Nonet et al. 1999). Finally, other endocytotic mutants
in Drosophila, such as endo (Verstreken et al. 2002) and synj
(Verstreken et al. 2003), do not alter basal transmitter release
even though they are all nearly depleted of SVs, displaying far
fewer SVs than the lap mutant does. The dap160 mutant also
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J Neurophysiol • VOL
affecting calcium-evoked release. Both synaptotagmin I
(Broadie et al. 1994; Fernandez-Chacon et al. 2001; Geppert et
al. 1994; Mackler et al. 2002; Sudhof 2004) and CSP (DawsonScully et al. 2000) have been shown to play important roles in
coupling Ca2⫹ with release. Interestingly, the lap mutant phenocopies the increase in mini frequency and reduction in
evoked release observed in some alleles of the syt I mutant
(Broadie et al. 1994; DiAntonio and Schwarz 1994; Littleton et
al. 1994; Reist et al. 1998). Although synaptotagmin I may not
play a role in calcium cooperativity (Broadie et al. 1994;
Fernandez-Chacon et al. 2001; but see Littleton et al. 1994),
soluble N-ethylmaleimide-sensitive factor attachment protein
receptor (SNARE) proteins (Bevan and Wendon 1984; CullCandy et al. 1976; Stewart et al. 2000) have been shown to
contribute to Ca2⫹ cooperativity. According to the model
proposed by Stewart and colleagues (2000), Ca2⫹ cooperativity reflects the cooperative formation of ‘tight’ trans-SNARE
complexes from “loose” trans-SNARE complexes (Chen et al.
1999). SNARE complexes have been suggested to directly
respond to Ca2⫹ during exocytosis (Sorensen et al. 2002). We
have observed that the change in Ca2⫹ cooperativity in the lap
mutant is similar to those reported in n-syb hypomorphic
alleles (Stewart et al. 2000). Together with the observation that
n-Syb is mislocalized in the lap mutant, our electrophysiological data implies that the reduction in Ca2⫹ cooperativity might
result from a partial loss of n-Syb on SVs.
Potential mechanisms and implications of AP180 action
in synapses
A role for AP180 in protein retrieval would be in good
agreement with both the biochemical properties of AP180 and
its strategic location within coated vesicles. AP180 might help
retrieve synaptotagmin I during endocytosis through its interaction with the clathrin-AP2-synaptotagmin complex (De Camilli and Takei 1996; Zhang et al. 1999). Our observation that
synaptotagmin I is mislocalized in the lap mutant may support
this notion. However, we are puzzled that synaptotagmin I is
localized normally in the unc-11 mutant (Nonet et al. 1999).
Such a difference could reflect differences in synapse types
(GABAergic and cholinergic in C. elegans vs. glutamatergic in
Drosophila). It is also known that LAP differs from UNC-11
dramatically in the size and sequence at the C-terminus (Nonet
et al. 1999; Zhang et al. 1998), although the sequence responsible for this different function is not apparently clear. In
comparison with synaptotagmin I, the molecular mechanism
by which CSP and n-Syb are recycled remains elusive. It
remains to be determined whether AP180 plays a role (direct or
indirect) in facilitating the recycling of CSP and n-Syb. A
likely possibility is that CSP and n-Syb may be recruited into
SVs through their interaction with other vesicle proteins. For
example, CSP may be indirectly recycled into SVs by its direct
interaction with synaptotagmin I (Evans and Morgan 2002).
Further studies are needed to reach a better understanding of
CSP and n-Syb recruitment during endocytosis.
It should be noted that SV proteins traditionally thought to
be exocytotic are increasingly intertwined with vesicle recycling. Specific AP2-synaptotagmin I interactions have been
demonstrated experimentally important for synaptotagmin I
recycling (Jarrousse et al. 2003). Similarly, deletion or perturbation of synaptotagmin I (Fukuda et al. 1995; Jorgensen et al.
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mislocalization of CSP and synaptotagmin I in the stn mutant
(Fergestad and Broadie 2001; Fergestad et al. 1999; Stimson et
al. 1998, 2001). However, the axonal regions between synaptic
boutons are expanded in the lap mutant but not in the stn
mutant. Furthermore, the lap phenotype differs from the complete translocation of vesicle proteins to the plasma membrane
observed in shits1 flies after the depletion of SVs at the
restrictive temperature (Estes et al. 1996; van de Goor et al.
1995). In shits1 flies, vesicle proteins are depleted inside the
synaptic bouton and reside exclusively in the plasma membrane. In the lap mutant, the vesicle protein is more diffusely
and evenly distributed within synaptic boutons as well as the
extrasynaptic membrane. We realize that a true co-localization
of vesicle proteins and the plasma membrane will be difficult
to resolve under light microscopic levels. Nonetheless, the
close overlap with the membrane marker HRP suggests that
some of the vesicle proteins are present in the plasma membrane. Hence, a reasonable conclusion is that mislocalization of
SV proteins in nerve terminals in the lap mutant may not be a
general consequence of defective vesicle recycling and depletion of SV pools.
The change in SV protein localization in the lap mutant
could be explained by at least two possibilities. One possibility
is that these changes result from a general defect in synaptic
development. This is supported by the observation that synapse
morphology is dramatically altered in the mutant to rope-like
boutons. With this change in synapse shape, not only are SV
proteins but also other presynaptic proteins, including putative
active zones marked by nc82 antibody, as well as postsynaptic
receptors redistributed along the altered NMJ in the mutant.
This developmental reorganization of synaptic morphology
may be a result of reduced endocytosis of surface molecules
such as receptors or other signaling molecules. It is noted that
synaptic morphology is also altered in other endocytotic mutants, such as endo (Verstreken et al. 2002) and dap160 (Koh
et al. 2004; Marie et al. 2004). However, these mutants have
different effects on synaptic morphology. For example, endo
mutations enlarge synaptic boutons and do not display the
rope-like synapse seen in the lap mutant (Verstreken et al.
2002). This suggests that AP180 may have additional roles in
synapse development.
Alternatively, the change in synaptic morphology as well as
protein localization may be closely linked to the role of AP180
in SV recycling or vesicle trafficking. Consistent with studies
of the unc-11 and stn mutants, our results may suggest that SV
protein retrieval is partially failed during endocytosis in the lap
mutant resulting in the mislocalization of SV proteins in the
extrasynaptic region. The “lost” SV components would then
diffuse into the extrasynaptic membranes causing them to
expand the surface area such that the usual “strings” between
synaptic boutons become ropes. To compensate for this change
as well as the reduction in synaptic transmission, the synapse
may expand both the pre- and postsynaptic regions by translocating pre- and postsynaptic proteins into extrasynaptic regions.
Although both possibilities may co-exist and account for the
phenotypes observed in the lap mutant, we favor the second
working model because it better explains the electrophysiological defect in the lap mutant. A failure to fully recycle synaptotagmin I, CSP, and n-Syb could have significantly negative
consequences on the amount of these proteins on SVs thereby
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
ACKNOWLEDGMENTS
We are grateful to W. Thompson for use of the fluorescence microscope and
for constructive comments on the manuscript, to L. Marin (University of
Toronto) for conducting the electron microscopic work, to A. Bardo and J.
Mendenhall at UT’s Imaging Center for assistance with confocal imaging. We
thank A. DiAntonio (Glutamate receptor III), H. Bellen (Synaptobrevin,
DAP160), E. Buchner (Nc82 mAb), R. Kelly (DAP160), N. Reist (Synaptotagmin I), J. Roos (DAP160), and K. Zinsmaier (CSP) for the gift of antibodies
and L. Pallanck and M. Babcock for the SD3 allele of lap. We also thank D.
Dickman and T. Schwarz for personal communication of their unpublished
work on SV protein localization in endo and synj mutants. We thank two
anonymous reviewers for constructive comments.
Present addresses: R. W. Daniels, Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and
G. T. Macleod, Div. of Neurobiology, University of Arizona, Tucson, AZ
85721.
GRANTS
This work was supported by a CARRER Award (IBN-0093170) from the
National Science Foundation and a startup fund from UT-Austin to B. Zhang,
by an Undergraduate Research Award at UT-Austin to R. W. Daniels, and by
grants from the Canadian Institutes of Health Research to H. L. Atwood, M. P.
Charlton, and G. T. Macleod.
REFERENCES
Ahle S and Ungewickell E. Purification and properties of a new clathrin
assembly protein. EMBO J 5: 3143–3149, 1986.
Atwood HL, Govind CK, and Wu CF. Differential ultrastructure of synaptic
terminals on ventral longitudinal abdominal muscles in Drosophila larvae.
J Neurobiol 24: 1008 –1024, 1993.
Atwood HL and Wojtowicz JM. Silent synapses in neural plasticity: current
evidence. Learn Mem 6: 542–571, 1999.
Babcock MC, Stowers RS, Leither J, Goodman CS, and Pallanck LJ. A
genetic screen for synaptic transmission mutants mapping to the right arm of
chromosome 3 in Drosophila. Genetics 165: 171–183, 2003.
Bevan S and Wendon LM. A study of the action of tetanus toxin at rat soleus
neuromuscular junctions. J Physiol 348: 1–17, 1984.
J Neurophysiol • VOL
Broadie K, Bellen HJ, DiAntonio A, Littleton JT, and Schwarz TL.
Absence of synaptotagmin disrupts excitation-secretion coupling during
synaptic transmission. Proc Natl Acad Sci USA 91: 10727–10731, 1994.
Brodin L, Low P, and Shupliakov O. Sequential steps in clathrin-mediated
synaptic vesicle endocytosis. Curr Opin Neurobiol 10: 312–320, 2000.
Brodsky FM, Chen CY, Knuehl C, Towler MC, and Wakeham DE.
Biological basket weaving: formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol 17: 517–568, 2001.
Chen YA, Scales SJ, Patel SM, Doung YC, and Scheller RH. SNARE
complex formation is triggered by Ca2⫹ and drives membrane fusion. Cell
97: 165–174, 1999.
Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell
L, Nemoto Y, Shears SB, Flavell RA, McCormick DA, and De Camilli
P. Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99: 179 –188, 1999.
Cull-Candy SG, Lundh H, and Thesleff S. Effects of botulinum toxin on
neuromuscular transmission in the rat. J Physiol 260: 177–203, 1976.
Davis GW and Goodman CS. Genetic analysis of synaptic development and
plasticity: homeostatic regulation of synaptic efficacy. Curr Opin Neurobiol
8: 149 –156, 1998.
Dawson-Scully K, Bronk P, Atwood HL, and Zinsmaier KE. Cysteinestring protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila. J Neurosci 20: 6039 – 6047, 2000.
Deak F, Schoch S, Liu X, Sudhof TC, and Kavalali ET. Synaptobrevin is
essential for fast synaptic-vesicle endocytosis. Nat Cell Biol 6: 1102–1108,
2004.
De Camilli P and Takei K. Molecular mechanisms in synaptic vesicle
endocytosis and recycling. Neuron 16: 481– 486, 1996.
DiAntonio A and Schwarz TL. The effect on synaptic physiology of synaptotagmin mutations in Drosophila. Neuron 12: 909 –920, 1994.
Dodge FA Jr and Rahamimoff R. Co-operative action of calcium ions in
transmitter release at the neuromuscular junction. J Physiol 193: 419 – 432,
1967.
Estes PS, Roos J, van der Bliek A, Kelly RB, Krishnan KS, and Ramaswami M. Traffic of dynamin within individual Drosophila synaptic
boutons relative to compartment-specific markers. J Neurosci 16: 5443–
5456, 1996.
Evans GJ and Morgan A. Phosphorylation-dependent interaction of the
synaptic vesicle proteins cysteine string protein and synaptotagmin I. Biochem J 364: 343–347, 2002.
Fatt P and Katz B. Spontaneous subthreshold activity at motor nerve endings.
J Physiol 117: 109 –128, 1952.
Feeney CJ, Karunanithi S, Pearce J, Govind CK, and Atwood HL. Motor
nerve terminals on abdominal muscles in larval flesh flies, Sarcophaga
bullata: comparisons with Drosophila. J Comp Neurol 402: 197–209, 1998.
Fergestad T and Broadie K. Interaction of stoned and synaptotagmin in
synaptic vesicle endocytosis. J Neurosci 21: 1218 –1227, 2001.
Fergestad T, Davis WS, and Broadie K. The stoned proteins regulate
synaptic vesicle recycling in the presynaptic terminal. J Neurosci 19:
5847–5860, 1999.
Fernandez-Chacon R, Konigstorfer A, Gerber SH, Garcia J, Matos MF,
Stevens CF, Brose N, Rizo J, Rosenmund C, and Sudhof TC. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410:
41– 49, 2001.
Fesce R, Grohovaz R, Valtorta F, and Meldolesi J. Neurotransmitter
release: fusion or “kiss-and-run.” Trend Cell Biol 4: 1– 4, 1994.
Fukuda M, Moreira JE, Lewis FM, Sugimori M, Niinobe M, Mikoshiba K,
and Llinas R. Role of the C2B domain of synaptotagmin in vesicular
release and recycling as determined by specific antibody injection into the
squid giant synapse preterminal. Proc Natl Acad Sci USA 92: 10708 –10712,
1995.
Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, and
Sudhof TC. Synaptotagmin I: a major Ca2⫹ sensor for transmitter release at
a central synapse. Cell 79: 717–727, 1994.
Gho M. Voltage-clamp analysis of gap junctions between embryonic muscles
in Drosophila. J Physiol 481: 371–383, 1994.
Grunwald ME, Mellem JE, Strutz N, Maricq AV, and Kaplan JM.
Clathrin-mediated endocytosis is required for compensatory regulation of
GLR-1 glutamate receptors after activity blockade. Proc Natl Acad Sci USA
101: 3190 –3195, 2004.
Hao W, Luo Z, Zheng L, Prasad K, and Lafer EM. AP180 and AP-2
interact directly in a complex that cooperatively assembles clathrin. J Biol
Chem 274: 22785–22794, 1999.
94 • SEPTEMBER 2005 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 17, 2017
1995; Littleton et al. 2001; Poskanzer et al. 2003; but see Reist
et al. 1998) and synaptobrevin (Deak et al. 2004) leads to
partial depletion of SVs. These observations raise the possibility that defects in protein retrieval may further exacerbate the
impairment of endocytosis as well as exocytosis through a
negative cycle. It is interesting to note that AP180, synaptobrevin, and synaptotagmin, but not all synaptic proteins, are
significantly reduced prior to the loss of synapses in the
neocortex of early and mild Alzheimer’s patients (Masliah et
al. 2001; Sze et al. 2000; Yao 2004; Yao and Coleman 1998;
Yao et al. 2003). It remains to be determined whether similar
synaptic dysfunctions are also found in these patients. Our
studies have advanced the understanding of AP180 in clathrinmediated endocytosis and its likely indirect roles in exocytosis
by retrieving vesicle proteins. Our studies also raise an important question on whether SV composition is indeed altered. A
direct demonstration for a change in vesicle proteins would be
a definitive proof. However, it has been hampered by the
difficulty obtaining sufficient amount of adult brains from the
lap mutant, which dies mostly as larvae. A conditional mutation, such as a temperature-sensitive paralytic allele of the lap
mutant, would be valuable for such biochemical studies.
RNAi-mediated knockdown techniques could also be used to
reduce LAP levels in viable adult flies. These flies would be
valuable for studying not only the precise mode for AP180 in
SV protein retrieval but also the potential role for LAP in
synaptic development.
1901
1902
BAO, DANIELS, MACLEOD, CHARLTON, ATWOOD, AND ZHANG
J Neurophysiol • VOL
ogy of Neurons, edited by Cowan WM and Ferrendelli JA. Bethesda, MD:
Soc. Neurosci. Symp., 1977, vol. 2, p. 161–171.
Petersen SA, Fetter RD, Noordermeer JN, Goodman CS, and DiAntonio
A. Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron 19: 1237–
48, 1997.
Phillips AM, Smith M, Ramaswami M, and Kelly LE. The products of the
Drosophila stoned locus interact with synaptic vesicles via synaptotagmin.
J Neurosci 20: 8254 – 8261, 2000.
Poskanzer KE, Marek KW, Sweeney ST, and Davis GW. Synaptotagmin I
is necessary for compensatory synaptic vesicle endocytosis in vivo. Nature
426: 559 –563, 2003.
Qin G, Schwarz T, Kittel RJ, Schmid A, Rasse TM, Kappei D, Ponimaskin
E, Heckmann M, and Sigrist SJ. Four different subunits are essential for
expressing the synaptic glutamate receptor at neuromuscular junctions of
Drosophila. J Neurosci 25: 3209 –3218, 2005.
Reist NE, Buchanan J, Li J, DiAntonio A, Buxton EM, and Schwarz TL.
Morphologically docked synaptic vesicles are reduced in synaptotagmin
mutants of Drosophila. J Neurosci 18: 7662–7673, 1998.
Robinson IM, Ranjan R, and Schwarz TL. Synaptotagmins I and IV
promote transmitter release independently of Ca(2⫹) binding in the C(2)A
domain. Nature 418: 336 –340, 2002.
Roos J and Kelly RB. Dap160, a neural-specific Eps15 homology and
multiple SH3 domain-containing protein that interacts with Drosophila
dynamin. J Biol Chem 273: 19108 –19119, 1998.
Roos J and Kelly RB. The endocytic machinery in nerve terminals surrounds
sites of exocytosis. Curr Biol 9: 1411–1414, 1999.
Sankaranarayanan S and Ryan TA. Real-time measurements of vesicleSNARE recycling in synapses of the central nervous system. Nat Cell Biol
2: 197–204, 2000.
Schikorski T and Stevens CF. Morphological correlates of functionally
defined synaptic vesicle populations. Nat Neurosci 4: 391–395, 2001.
Smith SJ, Augustine GJ, and Charlton MP. Transmission at voltageclamped giant synapse of the squid: evidence for cooperativity of presynaptic calcium action. Proc Natl Acad Sci USA 82: 622– 625, 1985.
Song W, Ranjan R, Dawson-Scully K, Bronk P, Marin L, Seroude L, Lin
YJ, Nie Z, Atwood HL, Benzer S, and Zinsmaier KE. Presynaptic
regulation of neurotransmission in Drosophila by the g protein-coupled
receptor methuselah. Neuron 36: 105–119, 2002.
Sorensen JB, Matti U, Wei SH, Nehring RB, Voets T, Ashery U, Binz T,
Neher E, and Rettig J. The SNARE protein SNAP-25 is linked to fast
calcium triggering of exocytosis. Proc Natl Acad Sci USA 99: 1627–1632,
2002.
Stewart BA, Atwood HL, Renger JJ, Wang J, and Wu CF. Improved
stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J Comp Physiol [A] 175: 179 –191,
1994.
Stewart BA, Mohtashami M, Trimble WS, and Boulianne GL. SNARE
proteins contribute to calcium cooperativity of synaptic transmission. Proc
Natl Acad Sci USA 97: 13955–13960, 2000.
Stimson DT, Estes PS, Rao S, Krishnan KS, Kelly LE, and Ramaswami M.
Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle
internalization. J Neurosci 21: 3034 –3044, 2001.
Stimson DT, Estes PS, Smith M, Kelly LE, and Ramaswami M. A product
of the Drosophila stoned locus regulates neurotransmitter release. J Neurosci 18: 9638 –9649, 1998.
Sudhof TC. The synaptic vesicle cycle. Annu Rev Neurosci 27: 509 – 47, 2004.
Sun B and Salvaterra PM. Characterization of nervana, a Drosophila melanogaster neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase antibodies. J Neurochem 65: 434 – 443, 1995.
Sze CI, Bi H, Kleinschmidt-DeMasters BK, Filley CM, and Martin LJ.
Selective regional loss of exocytotic presynaptic vesicle proteins in Alzheimer’s disease brains. J Neurol Sci 175: 81–90, 2000.
Takei K, Mundigl O, Daniell L, and De Camilli P. The synaptic vesicle
cycle: a single vesicle budding step involving clathrin and dynamin. J Cell
Biol 133: 1237–1250, 1996.
Turrigiano GG and Nelson SB. Homeostatic plasticity in the developing
nervous system. Nat Rev Neurosci 5: 97–107, 2004.
van de Goor J, Ramaswami M, and Kelly R. Redistribution of synaptic
vesicles and their proteins in temperature-sensitive shibire(ts1) mutant
Drosophila. Proc Natl Acad Sci USA 92: 5739 –5743, 1995.
94 • SEPTEMBER 2005 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 17, 2017
Heuser JE and Reese TS. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J Cell
Biol 57: 315–344, 1973.
Hoang B and Chiba A. Single-cell analysis of Drosophila larval neuromuscular synapses. Dev Biol 229: 55–70, 2001.
Jan LY and Jan YN. Properties of the larval neuromuscular junction in
Drosophila melanogaster. J Physiol 262: 189 –214, 1976.
Jan LY and Jan YN. Antibodies to horseradish peroxidase as specific
neuronal markers in Drosophila and in grasshopper embryos. Proc Natl
Acad Sci USA 79: 2700 –2704, 1982.
Jarousse N, Wilson JD, Arac D, Rizo J, and Kelly RB. Endocytosis of
synaptotagmin 1 is mediated by a novel, tryptophan-containing motif.
Traffic 4: 468 – 478, 2003.
Jia XX, Gorczyca M, and Budnik V. Ultrastructure of neuromuscular
junctions in Drosophila: comparison of wild type and mutants with increased excitability. J Neurobiol 24: 1025–1044, 1993.
Jorgensen EM, Hartwieg E, Schuske K, Nonet ML, Jin Y, and Horvitz
HR. Defective recycling of synaptic vesicles in synaptotagmin mutants of
Caenorhabditis elegans. Nature 378: 196 –199, 1995.
Karunanithi S, Marin L, Wong K, and Atwood HL. Quantal size and
variation determined by vesicle size in normal and mutant Drosophila
glutamatergic synapses. J Neurosci 22: 10267–10276, 2002.
Katz B. The Release of Neural Transmitter Substances. Liverpool, UK:
Liverpool University Press, 1969.
Keshishian H, Chiba A, Chang TN, Halfon MS, Harkins EW, Jarecki J,
Wang L, Anderson M, Cash S, Halpern ME, and Johansen J. Cellular
mechanisms governing synaptic development in Drosophila melanogaster.
J Neurobiol 24: 757–787, 1993.
Koenig JH, Yamaoka K, and Ikeda K. Calcium-induced translocation of
synaptic vesicles to the active site. J Neurosci 13: 2313–2322, 1993.
Koh TW, Verstreken P, and Bellen HJ. Dap160/intersectin acts as a
stabilizing scaffold required for synaptic development and vesicle endocytosis. Neuron 43: 193–205, 2004.
Littleton JT, Bai J, Vyas B, Desai R, Baltus AE, Garment MB, Carlson
SD, Ganetzky B, and Chapman ER. Synaptotagmin mutants reveal essential functions for the C2B domain in Ca2⫹-triggered fusion and recycling
of synaptic vesicles in vivo. J Neurosci 21: 1421–1433, 2001.
Littleton JT, Stern M, Perin M, and Bellen HJ. Calcium dependence of
neurotransmitter release and rate of spontaneous vesicle fusions are altered
in Drosophila synaptotagmin mutants. Proc Natl Acad Sci USA 91: 10888 –
10892, 1994.
Macleod GT, Hegstrom-Wojtowicz M, Charlton MP, and Atwood HL.
Fast calcium signals in Drosophila motor neuron terminals. J Neurophysiol
88: 2659 –2663, 2002.
Mackler JM, Drummond JA, Loewen CA, Robinson IM, and Reist NE.
The C(2)B Ca(2⫹)-binding motif of synaptotagmin is required for synaptic
transmission in vivo. Nature 418: 340 –344, 2002.
Marie B, Sweeney ST, Poskanzer KE, Roos J, Kelly RB, and Davis GW.
Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity
endocytosis and normal synaptic growth. Neuron 43: 207–219, 2004.
Marrus SB, Portman SL, Allen MJ, Moffat KG, and DiAntonio A.
Differential localization of glutamate receptor subunits at the Drosophila
neuromuscular junction. J Neurosci 24: 1406 –1415, 2004.
Masliah E, Mallory M, Alford M, DeTeresa R, Hansen LA, McKeel DW
Jr, and Morris JC. Altered expression of synaptic proteins occurs early
during progression of Alzheimer’s disease. Neurology 56: 127–129, 2001.
Morgan JR, Zhao X, Womack M, Prasad K, Augustine GJ, and Lafer
EM. A role for the clathrin assembly domain of AP180 in synaptic vesicle
endocytosis. J Neurosci 19: 10201–10212, 1999.
Murphy JE, Pleasure IT, Puszkin S, Prasad K, and Keen JH. Clathrin
assembly protein AP-3. The identity of the 155K protein, AP 180, and
NP185 and demonstration of a clathrin binding domain. J Biol Chem 266:
4401– 4408, 1991.
Murthy VN, Schikorski T, Stevens CF, and Zhu Y. Inactivity produces
increases in neurotransmitter release and synapse size. Neuron 32: 673– 682,
2001.
Nonet ML, Holgado AM, Brewer F, Serpe CJ, Norbeck BA, Holleran J,
Wei L, Hartwieg E, Jorgensen EM, and Alfonso A. UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol Biol Cell 10: 2343–2360, 1999.
Palfrey HC and Artalejo CR. Secretion: kiss and run caught on film. Curr
Biol 13: R397–399, 2003.
Parsegian VA. Considerations in determining the mode of influence of
calcium on vesicle-membrane interaction. In: Approaches to the Cell Biol-
IMPACTS OF SYNAPTIC VESICLE RECYCLING ON EXOCYTOSIS
Verstreken P, Kjaerulff O, Lloyd TE, Atkinson R, Zhou Y, Meinertzhagen
IA, and Bellen HJ. Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109: 101–112, 2002.
Verstreken P, Koh TW, Schulze KL, Zhai RG, Hiesinger PR, Zhou Y, Mehta
SQ, Cao Y, Roos J, and Bellen HJ. Synaptojanin is recruited by endophilin to
promote synaptic vesicle uncoating. Neuron 40: 733–748, 2003.
Wucherpfennig T, Wilsch-Brauninger M, and Gonzalez-Gaitan M. Role
of Drosophila Rab5 during endosomal trafficking at the synapse and evoked
neurotransmitter release. J Cell Biol 161: 609 – 624, 2003.
Xu-Friedman MA, and Regehr WG. Structural contributions to short-term
synaptic plasticity. Physiol Rev 84: 69 – 85, 2004.
Yao PJ. Synaptic frailty and clathrin-mediated synaptic vesicle trafficking in
Alzheimer’s disease. Trends Neurosci 27: 24 –29, 2004.
Yao PJ and Coleman PD. Reduced O-glycosylated clathrin assembly protein
AP180: implication for synaptic vesicle recycling dysfunction in Alzheimer’s disease. Neurosci Lett 252: 33–36, 1998.
Yao PJ, Zhu M, Pyun EI, Brooks AI, Therianos S, Meyers VE, and Coleman
PD. Defects in expression of genes related to synaptic vesicle trafficking in
frontal cortex of Alzheimer’s disease. Neurobiol Dis 12: 97–109, 2003.
1903
Ye W and Lafer EM. Clathrin binding and assembly activities of expressed
domains of the synapse-specific clathrin assembly protein AP-3. J Biol
Chem 270: 10933–10939, 1995.
Zhang B. Genetic and molecular analysis of synaptic vesicle recycling in
Drosophila. J Neurocytol 32: 567–589, 2003.
Zhang B, Ganetzky B, Bellen HJ, and Murthy VN. Tailoring uniform coats
for synaptic vesicles during endocytosis. Neuron 23: 419 – 422, 1999.
Zhang B, Koh YH, Beckstead RB, Budnik V, Ganetzky B, and Bellen HJ.
Synaptic vesicle size and number are regulated by a clathrin adaptor protein
required for endocytosis. Neuron 21: 1465–1475, 1998.
Zhang B and Ramaswami M. Synaptic vesicle endocytosis and recycling. In:
Frontiers in Molecular Biology: Neurotransmitter Release, edited by
Bellen, H. Oxford, London, 1999, p. 389 – 431.
Zhang JZ, Davletov BA, Sudhof TC, and Anderson RG. Synaptotagmin I
is a high affinity receptor for clathrin AP-2: implications for membrane
recycling. Cell 78: 751–760, 1994.
Zucker RS. Calcium- and activity-dependent synaptic plasticity. Curr Opin
Neurobiol 9: 305–313, 1999.
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