# 2008 The Authors

Journal compilation # 2008 Blackwell Munksgaard

doi: 10.1111/j.1600-0854.2008.00832.xTraffic 2008; 9: 2190–2205Blackwell Munksgaard

The Drosophila Epsin 1 is Required forUbiquitin-Dependent Synaptic Growth andFunction but Not for Synaptic Vesicle Recycling

Hong Bao1, Noreen E. Reist2 and

Bing Zhang1,3,*

1Department of Zoology, University of Oklahoma,Norman, OK 73019, USA2Department of Biomedical Sciences, Colorado StateUniversity, Fort Collins, CO 80523, USA3Section of Neurobiology, University of Texas, Austin,TX 78712, USA*Corresponding author: Bing Zhang, bing@ou.edu

The ubiquitin-proteasome systemplays an important role

in synaptic development and function. However, many

components of this system, and how they act to affect

synapses, are still not well understood. In this study, we

use the Drosophila neuromuscular junction to study the

in vivo function of Liquid facets (Lqf), a homolog of

mammalian epsin 1. Our data show that Lqf plays a novel

role in synapse development and function. Contrary to

prior models, Lqf is not required for clathrin-mediated

endocytosis of synaptic vesicles. Lqf is required to main-

tain bouton size and shape and to sustain synapse

growth by acting as a specific substrate of the deubiqui-

tinating enzyme Fat facets. However, Lqf is not a sub-

strate of the Highwire (Hiw) E3 ubiquitin ligase; neither is

it required for synapse overgrowth in hiw mutants.

Interestingly, Lqf converges on the Hiw pathway by

negatively regulating transmitter release in the hiw

mutant. These observations demonstrate that Lqf plays

distinct roles in two ubiquitin pathways to regulate

structural and functional plasticity of the synapse.

Key words: neuromuscular junction, synapse develop-

ment, synaptic plasticity, synaptic transmission, ubiquitin

Received 9 October 2007, revised and accepted for publica-

tion 11 September 2008, uncorrected manuscript published

online 15September 2008, publishedonline 15October 2008

The ubiquitin-proteasome system (UPS) serves to not only

degrade misfolded proteins but also dynamically regulate

protein levels and biological processes (1). In neurons, the

UPS has recently emerged as an important cellular mecha-

nism that regulates the structural and functional plasticity

and health of the nervous system in a variety of animals,

including mammals, Drosophila, Caenorhabditis elegans and

Aplysia (reviewed in 2,3). The UPS plays an active role in

synaptic plasticity through modification of synaptic trans-

mission (4,5) and regulation of protein levels in both pre- and

post-synaptic membranes (5–9). During neuronal develop-

ment, the UPS affects axonal outgrowth (10), dendritic

remodeling (11,12) and synapse formation and growth

(13–21). Finally, the UPS is implicated in a number of neu-

rological disorders (4,22–25) andWallerian degeneration (26).

At present, most UPS substrates associated with either

neuronal development or diseases remain unidentified.

Liquid facets (Lqf) possesses ubiquitin interaction motifs

and is a substrate of the deubiquitinating enzyme Fat

facets (Faf) (27,28). Lqf might regulate synaptic develop-

ment as overexpression of Faf in neurons stimulates

synaptic overgrowth at the Drosophila larval neuromuscu-

lar junction (NMJ) (16). Lqf is also a homolog of mammalian

epsin 1(27,29), which has multiple binding sites for

clathrin, adaptor protein (AP)2 and eps15 (reviewed in

30). Lqf appears to be the only homolog of epsin 1 in flies.

Furthermore, epsin 1 shares a highly conserved N-terminal

structure with the clathrin-assembly protein AP180 (30–33).

Importantly, epsin 1 binds to lipid membranes in vitro (34)

where it causes membrane curvature necessary for clathrin-

coated vesicle assembly (32). Both AP180 and epsin 1

promote clathrin cage assembly in vitro (35,36). These

observations highly suggest that epsin 1 plays a key role in

clathrin-mediated endocytosis (CME). Subsequent studies

of mammalian cultured cells, yeasts and fruit flies show that

epsin 1 and its homologs (including Lqf) are involved in the

endocytosis of select ligands (27,29,37–44). Hence, we

expect that deletion of Lqf would significantly block CME

of synaptic vesicles (SVs), homologous to the phenotypes of

the AP180 mutant like-ap180 (lap) (45–47).

Despite these implications from previous studies, it re-

mains untested whether Lqf also acts as a substrate during

Faf-induced synaptic growth and whether Lqf plays a role

in endocytosis of SVs. In this report, we investigated the

in vivo function of Lqf at theDrosophila larval NMJ, amodel

for studying synapse development and function (48,49).

Our results show that Lqf does not play a role in SV

recycling; rather, it regulates synapse development and

function through specific UPS pathways.

Results

Lqf is ubiquitously detected at a low level in both

pre- and post-synaptic membranes

Endocytotic proteins involved in CME of SVs are typically

enriched at presynaptic terminals at these NMJs in third

instar larvae [e.g. Dynamin, (50); LAP, (45); Dap160, (51)].

To reveal the cellular distribution of Lqf at Drosophila

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larval NMJs, we used an Lqf polyclonal antibody for

immunocytochemistry (28). Lqf was uniformly present

at low levels at presynaptic membranes and nerves

marked by the neuronal membrane marker horseradish

peroxidase (HRP) (Figure 1A). While present at presyn-

aptic boutons, Lqf did not show the enrichment typically

seen for the endocytotic protein Dynamin (Figure 1B).

Additionally, Lqf was found at low levels in muscles,

along tracheal tubules and at postsynaptic densities

identified by the postsynaptic marker Discs large (Dlg;

Figure 1C). This pattern of Lqf subcellular localization

suggests that its functions may not be restricted to

endocytosis at nerve terminals.

To ascertain the specificity of the Lqf antibody, we

immunostained lqf mutant NMJs and measured Lqf

protein levels in lqf mutant alleles on western blots.

Several mutations in the lqf locus were generated and

described previously (27,40). Among these alleles, lqfARI

and lqfAG are severe loss-of-function mutations. When

placed over a deficiency in the region uncovering the lqf

locus, flies carrying these alleles grew slowly and died

during late larval stages. A few larvae lived to the third

instar stage with reduced body size and muscle size, but

all died before pupation. For example, the size of muscle

6/7 in abdominal segment 3 was reduced on average by

�32% (Figure S1). Immunoreactivity to the Lqf antibody

at the NMJ (identified by HRP staining) of the lqfARI/Df

mutant larva was nearly undetectable (Figure 1D). Con-

sistent with this immunocytochemistry, western blots

showed a significant reduction of Lqf proteins in third

instar larval brains and ventral nerve cords of lqfARI and

lqfAG alleles (Figure 1E). Hence, the antibody is specific

for Lqf at both larval synapses (this study) and developing

eyes (28,39).

lqf mutations slightly reduce transmitter release

at the larval NMJ

To test the hypothesis that Lqf is involved in CME of SVs,

we first characterized the lqf mutant NMJ using electro-

physiology. We examined spontaneous miniature excit-

atory junction potentials (mEJPs or minis) in the wild-type

control and lqfARI/Df larvae by recording from muscles

bathed in HL-3 saline containing 0.1 mM Ca2þ and 1 mm

tetrodotoxin (TTX) (45,47,52,53). The resting potential was

similar between the two genotypes (�65.6� 0.80 mV, n¼12, in the control and �64.3 � 0.97 mV, n ¼ 14, in the lqf

mutant). Minis in lqfARI/Df larvae were reduced in fre-

quency but larger in amplitude (Figure 2A,B). Although

rare, both the mutant and the control larvae displayed

minis with unusually large amplitudes (Figure 2A). Mini

frequency in the lqfARI/Dfmutant was significantly reduced

Figure 1: Lqf is present in both pre-

and post-synaptic membranes at the

larval NMJ. A) Immunoreacivity to Lqf

and the neuronal membrane marker

HRP at the wild-type larval NMJ on

muscle 4 (M4). Lqf (green) is present

in muscles, synaptic boutons and ner-

ves, whereas HRP (red) staining is re-

stricted to neuronalmembranes (nerves

and synaptic boutons). Motor nerves

and tracheal tubules are marked by ‘n’

and ‘t’, respectively. The inset on the

overlay panels shows close-up images

of localization signals (same for panels B

and C). B) Immunoreacivity to Lqf and

the endocytotic protein Dynamin at

the wild-type larval NMJ. Note that

Dynamin (red) is highly enriched at pre-

synaptic boutons. C) Lqf (green) is colo-

calized with Dlg (red) at the postsynaptic

density and at low levels in muscles. D)

Lqf immunoreactivity in the lqfARI/Df mu-

tant larval NMJ is significantly reduced

compared with HRP. The scale is 20 mm

for images in panels (A–C), 5 mm for

insets and 10mm for images in panel (D).

E) Lqf protein levels are nearly absent in

the brain and ventral nerve cords of two

lqf mutants (lqfARI/Df and lqfAG/Df ). The

western blot was reprobed for a-tubulin

to ensure that sample loading was simi-

lar among different genotypes.

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Novel Synaptic Roles for Drosophila Epsin 1

from 5.1 � 0.5 Hz (n ¼ 12) in the wild type to 3.4 � 0.4 Hz

(n ¼ 14) (p < 0.05). In contrast, mini amplitude was

increased from 1.4 � 0.1 mV (n ¼ 12) in the wild type to

2.1 � 0.3 mV (n ¼ 14) in lqfARI/Df larvae (p < 0.05).

Although the mini amplitude was slightly increased, we

failed to detect a change in glutamate receptor levels in

muscle cells (Figure S2).

We next investigated action potential-evoked synaptic

transmission in lqf mutants. Evoked transmitter release

was measured as excitatory junction currents (EJCs)

from muscles voltage clamped at �80 mV in saline

containing 1 mM Ca2þ (Figure 2C). The amplitude of EJCs

was slightly reduced from 54.5 � 2.6 nA (n ¼ 9) in the

control larvae to 38.6 � 3.5 nA (n ¼ 9; p < 0.05) in lqfARI/

Df larvae and to 45.1 � 4.2 nA (n ¼ 21; p > 0.05) in lqfAG/

Df larvae (Figure 2D). Heterozygous lqf mutant alleles or

its deficiency (Df) were essentially similar to wild-type

control larvae and did not have a dominant effect on

evoked EJC amplitudes (Figure S3). It appears that lqfARI

is a more severe allele than lqfAG, consistent with

the conclusion reached by others studying heart de-

velopment in Drosophila (41). For this reason, we con-

ducted most of the remaining experiments with the

lqfARI allele.

Electrophysiological evidence that lqf mutations do

not block SV recycling at the larval NMJ

Given the previously suggested role for Lqf in CME, the

defects we have observed in synaptic transmission could

be caused by an impairment of SV recycling. We ad-

dressed this possibility by measuring the vesicle depletion

rate electrophysiologically. Upon repetitive and prolonged

nerve stimulation, the NMJ depletes SVs first in the readily

releasable pool, then the releasable pool and finally relies

on both translocation of SVs from the reserve pool and

recycling of SVs to sustain transmitter release. Mutations

reducing vesicle recycling have been shown to rapidly and

dramatically reduce transmitter release when challenged

with maintained nerve stimulation (54–58).

We recorded EJCs in HL-3 saline containing high Ca2þ so

that we could maximize the release probability and achieve

a rapid depletion of the vesicle pool. We initially stimulated

the motor nerve at 0.2 Hz to obtain a basal level and then at

10 Hz for 5 min to measure the depletion rate. This was

followed by a short period of nerve stimulation at 0.2 Hz to

measure the EJC recovery rate (Figure 3A,B). The ampli-

tude of EJCs for basal release was 125.2 � 5.72 nA (n ¼ 7

at 1.8 mM Ca2þ) and 124.2 � 4.9 nA (n ¼ 8 at 2 mM Ca2þ)

for the control and lqfARI/Df larvae, respectively. Three

Figure 2: lqf mutations alter synap-

tic transmission. A) A cumulative plot

of spontaneous minis and a histogram

of the average mini amplitude in the

wild-type (wt) and lqfARI/Df larvae. The

average amplitude of minis is signifi-

cantly increased in the lqfmutant (*p <

0.05). B) A histogram of mini frequen-

cies in the wild-type control and mutant

larvae. Mini frequency is significantly

reduced in the mutant compared with

the control larvae (*p < 0.05). C) Rep-

resentative traces of EJCs in the wild-

type control larvae and two lqf mutant

genotypes. D) A histogram of the aver-

age amplitude of EJCs. The lqfARI/Df

mutant significantly reduces the am-

plitude of EJCs compared with the

control larvae (*p < 0.05).

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Bao et al.

consecutive EJCs at the onset of the high frequency

stimulation (marked time 0), at 30 seconds and every

minute thereafter were averaged and then normalized to

the basal amplitude. Our results showed that transmitter

release in the control larvae dropped by�19% at the onset

of 10 Hz stimulation (indicated by time 0 in Figure 3B) and

soon reached �45% of the normal release within 30

seconds. Following this initial and rapid decline, trans-

mitter release was gradually reduced but maintained at

�38% of the normal release near the end of 5 min

stimulation. This steady state of release is thought to

reflect the balance of exocytosis and endocytosis of SVs

(54). EJC amplitude was rapidly recovered to nearly pre-

vious levels within 10–15 seconds.

In lqfAR1/Df mutant larvae, the initial drop of transmitter

release at the onset of stimulation (i.e. time 0) was similar

to that observed in the control larvae. However, trans-

mitter release dropped only to �55% of the basal level at

30 seconds and remained at a higher steady-state level

(�50%) compared with the control larvae. In other words,

the depletion rate in the lqf mutant is significantly slower

than that in the control larva (p < 0.05). Such a lack of

effects on vesicle depletion has also been shown in the

Drosophila amphiphysin mutant (59).

Delayed rebound of the EJC amplitude, which presumably

reflects impaired SV recycling, is expected for endocytotic

mutants (58). However, this did not occur in the lqf mutant

(Figure 3A,B). The kinetics of EJC amplitude recovery

following the 5-min prolonged depletion period was similar

to that in the wild type or slightly faster in the lqf mutant.

Consistent with these electrophysiological results, our re-

sults demonstrated that lqfmutants readily took up FM 1-43

dyes at the NMJ, similar to the control larvae (Figure S4).

Taken together, these results argue against the possibility

that deletion of lqf blocks SV recycling at the larval NMJ.

Ultrastructural evidence that lqf mutations do not

block SV recycling

To ascertain that indeed SV recycling is not blocked by lqf

mutations, we examined the SV pool at the ultrastructural

level. Previous studies have shown that nerve terminals in

endocytotic mutants typically contain significantly reduced

number of vesicle (45,55–58, 60–62). Although the shape

of boutons was altered (see Discussion below), the SV

pool in type Ib boutons was not reduced in the lqf mutant

compared with the control larvae (Figure 4A,B). Quantifi-

cation of over 10 random transmission electron micro-

scope (TEM) micrographs showed that the average

density of SVs (number of SVs per mm2) inside type Ib

boutons was significantly increased in the lqf mutant

(379.7 � 19.8, n ¼ 12) compared with that in control larvae

(306.4 � 23.8, n ¼ 14) (p < 0.05; Figure 4C). Judging from

the average diameter of SVs, we found no apparent

differences in SV size between these two genotypes

(Figure 4D). Hence, collective evidence from electrophys-

iology, ultrastructure and optical imaging strongly indicates

that contrary to its expected function, Lqf does not play

a detectable role in CME of SVs at Drosophila NMJs.

Synaptic bouton size is increased and bouton

shape is altered in the lqf mutant

To determine whether Lqf plays a role in synapse devel-

opment, we examined synaptic bouton number and mor-

phology using antibodies to the SV protein synaptotagmin I

as well as to HRP. Using antibodies to other SV proteins

[such as cysteine string protein (CSP) and synaptobrevin],

we failed to reveal obvious defects in mislocalization of

Figure 3: lqfmutants do not display a detectable defect in SV

endocytosis. A) The motor nerve is first stimulated at 0.2 Hz

(control), followed by 10 Hz stimulation for 5 min and then by 0.2

Hz until the EJC amplitude is recovered. Examples of EJC traces

from these three stimulation periods are shown here. [Ca2þ] is

1.8 mM for the wild type and 2 mM for the lqf mutant so that

their initial EJC amplitude is similar. B) The depletion rate of the SV

pool by repetitive stimulation of the motor nerve (10 Hz, 5 min)

is shown by normalizing the EJC amplitude to the control EJC

amplitude evoked at 0.2 Hz. The recovery kinetics is revealed by

low rate stimulation (0.2 Hz) following the SV pool depletion. n¼ 7

and 6 for the wild-type and the lqf mutant larvae, respectively.

These results are inconsistent with a role for Lqf in either

endocytosis or recycling of SVs at the NMJ.

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Novel Synaptic Roles for Drosophila Epsin 1

SV proteins to extrasynaptic axons, as previously shown to

the lap mutant [data not shown, (47)]. Our results showed

that synaptic bouton number was reduced in the lqf mutant

(Figure 5A–E). The average number of type Ib and Is

boutons found on muscle 4 was 54.0 � 2.8 (n ¼ 14) and

23.2� 1.7 (n¼ 22) for the control larvae and the lqfmutant

larvae, respectively (p < 0.001) (Figure 5B,E). On muscles

6/7, the total bouton number was also reduced from 104.3

� 11.1 (n ¼ 10) in the control larvae to 65.7 � 6.4 (n ¼ 13)

in the lqfARI/Df mutant (p < 0.001) (Figure 5D,E). This

represents a 57 and 37% reduction from the control larvae

for muscle 4 and muscles 6/7, respectively. Once calibrated

to the surface area of the muscle fiber (63), the difference in

bouton numbers became statistically insignificant except for

muscle 4 on segment 3 (Figure 5F). The results from

muscles 6/7 and 4 on segment 4 showed that the average

bouton number per muscle surface area was similar

between the lqf mutant and the control animal (Figure S5).

Similar findings on bouton numbers were also detected in

the lqfAG/Df allele (data not shown). Hence, lqf mutations

reduce larval body size and muscle growth but do not

significantly alter synaptic bouton numbers on all muscles.

One noticeable defect was that the size and shape of

synaptic boutons were altered. The average size of type Ib

and Is boutons was doubled from 5.68 � 0.27 mm2 (n ¼ 9)

in control to 13.25 � 1.26 mm2 (n ¼ 9) in the lqf mutant on

muscle 4 (p < 0.001) (Figure 5G). Bouton size on muscles

6/7 was also increased from 6.41 � 0.22 mm2 (n ¼ 10) in

control larvae to 9.21 � 0.35 mm2 (n ¼ 13) in the mutant

(p< 0.001) (Figure 5G). It appears that the most significant

change is the enlargement of those small boutons seen

normally between large boutons (compare lower panels in

Figure 5A,B and Figure 5C,D). This increase in bouton size

remained even when type Ib and Is boutons were consid-

ered separately.

Figure 4: Ultrastructural analysis reveals no evidence for a defect in SV pool in the lqfmutant. A and B) Electronmicrographs of the

longitudinal section of type Ib synaptic boutons in the control (wt) and lqfARI/Df larvae. Both synaptic boutons are occupied by a large

number of SVs with apparently similar sizes (see insets). C) The average SV density (number of SVs/mm2) is slightly increased in the lqf

mutant (*p< 0.05). D) The average diameter of SVs does not differ between the wild type and the lqfmutant. These ultrastructural results

are consistent with the electrophysiological observation, indicating that neither SV endocytosis nor recycling is blocked by the lqfmutation.

MTs, microtubules; SVs, synaptic vesicles.

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We next examined the active zone number in the mutant

synapse and found that each bouton contained a slightly

higher number of active zones in the mutant (Figure S2).

As expected for reduced muscle size and bouton number,

the total number of active zones per muscle was signifi-

cantly reduced compared with that in the wild type. These

changes may account for the reduced transmitter release

in the mutant.

Figure 5: lqf mutations increase the size

and alter the shape of synaptic boutons.

A–D) Immunocytochemistry of synaptic mor-

phology shows that the total number of type

Ib and Is synaptic boutons on muscle 4 (M4)

and muscles 6/7 (M6/7) is reduced in the

lqfARI/Df mutant larvae (panels B and D)

compared with that in the control larvae

(wt, panels A and C). Additionally, the size

of synaptic boutons is enlarged on average in

the lqf mutant (see close-up panels). Finally,

some boutons have irregular rather than the

normal oval or round shapes. E) Histograms

of the average number of type Ib and Is

synaptic boutons on muscles 4 (M4) and

muscles 6/7 (M6/7) in segment 3. On aver-

age, the number of synaptic boutons is

significantly reduced in the lqf mutant com-

pared with that in the control larvae (***p <

0.001). F) Histograms of the average number

of synaptic boutons (type Ib and Is) normal-

ized to the surface area of muscles in seg-

ment 3. The difference remains significant in

muscle 4 but not in muscles 6/7 (**p< 0.01).

G) Histograms of the average size of type Ib

and Is synaptic boutons on muscles 4 (M4)

and muscles 6/7 (M6/7) in segment 3. On

average, the size of synaptic boutons is

significantly larger in the lqf mutant than that

in the control larvae (***p < 0.001).

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Novel Synaptic Roles for Drosophila Epsin 1

Finally, we found that some boutons changed from the

normal round shape to an irregular and elongated ‘potato-

like’ shape (Figure 5B). This change in bouton shape is

similar to those observed in the electron microscope

(Figure 4B). These alterations in bouton size and shape

suggest a novel role for Lqf in synaptic development.

Neuronal expression of the wild-type lqf gene

rescues the developmental and functional defects

in lqf mutants

To ensure that the defects found in the lqf mutant

specifically result from a disruption of the lqf gene, we

used either a complementary DNA rescue construct

[upstream activation sequence (UAS)-Lqf] or a genomic

rescue line carrying the wild-type lqf gene to rescue the

lqfARI/Df mutant phenotype (27). One copy of the geno-

mic lqf gene (Glqfþ) readily rescued the lqfARI/Df mutant

to viable adults. Using the Gal4-UAS-system (64), we

next conducted tissue-specific rescue. Muscle-

specific expression of Lqf driven by the myosin heavy

chain (MHC) Gal4 driver did not rescue the lqf mutant.

Occasionally, a few larvae (estimated to be <1%) lived

to become early pupae. However, all of them died soon

after pupation. In contrast, neuronal expression of Lqf

driven by the pan-neuronal ElavC155 Gal4 driver rescued

the mutant to viable adults when the flies were kept at

18–198C, suggesting a primary role for Lqf in neurons.

At room temperature (�228C), this rescue was not

successful, likely because of gain-of-function effects of

Lqf (Figure 8).

We then further examined the genomically rescued lqf

animals morphologically and physiologically. First, synaptic

boutons in the rescued larvae resumed the oval or round

shape typically seen in the wild-type larvae (Figure 6A,B).

Second, the body size of the rescued larvae was similar to

Figure 6: The wild-type lqf gene rescues

both the morphological and the functional

defects in the lqf mutant. A and B) Repre-

sentative images of NMJs on muscle 4 (M4,

panel A) and muscles 6/7 (M6/7, panel B) in

the lqfARI/Df mutant in which a copy of the

wild-type lqfþ gene has been introduced. The

rescued mutant is viable as adult and does not

display any gross anatomical and behavioral

abnormalities. Note the restoration of well-

spaced round- or oval-shaped boutons in a typ-

ical ‘beads-on-a-string’ arrangement at the

larval NMJ (compare with mutant boutons in

Figure 5). C and D). The amplitude of evoked

EJCs is restored to a level slightly but not

significantly higher than that in the control

larvae. E and F) Representative mini traces

and histograms of mini amplitude and fre-

quency. Recordings were conducted under

the same conditions as those shown in Figure 2.

*p < 0.05. n.s., not significantly different.

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Bao et al.

that of the control larvae. Consequently, the average muscle

size was also restored to near wild-type levels (data not

shown). The number of synaptic boutons was 45.9 � 3.4

(n ¼ 14) in muscle 4. This value is not statistically different

from that in the control larvae (p > 0.05). Third, evoked

synaptic currents were reversed to 64.5 � 7.5 nA (n ¼ 12),

which was slightly above but not statistically significant from

the average amplitude of EJCs in the control larvae (54.5 �2.6 nA, n ¼ 9; p > 0.05) (Figure 6C,D). Fourth, mini ampli-

tude was reversed to near wild-type levels (Figure 6E,F).

The mini frequency, however, was not rescued. Finally, the

rescued mutant flies were fully viable as adults, with no

apparently behavioral or morphological abnormalities. These

results indicate that the morphological and electrophysio-

logical defects detected in the lqf mutant are specifically

caused by the loss of function of Lqf.

Lqf is required for Faf-induced synaptic overgrowth

Neuronal overexpression of Faf promotes overgrowth

of synapses at Drosophila NMJs (16). This overgrowth

seems to depend on Faf’s deubiquitinating activity

because the yeast UBP2 has similar stimulating effects

on synaptic growth (21). The mitogen-activated protein

(MAP) kinase kinase kinase (MAPKKK) Wallenda (Wnd)

is also required downstream of Hiw (21). Lqf has

been demonstrated to be a specific substrate of the

deubiquitinating enzyme Faf (28). Could the overgrowth

induced by Faf overexpression also depend on a Faf–Lqf

interaction?

We tested this hypothesis by overexpressing Faf in the lqf

mutant background (ElavC155 Gal4/þ; lqfARI, EP(3)381/lqf

Df). Consistent with previous observations (16), neuronal

overexpression of Faf induced NMJ overgrowth in a wild-

type background (compare Figure 7A,B). In the absence of

Lqf, overexpression of Faf failed to stimulate synaptic

growth (Figure 7C). The number of synaptic boutons on

muscle 4 was significantly reduced by �78% to a level

similar to that found in the lqf mutant. Bouton numbers

were 99.1 � 10.4 (n ¼ 16) in Faf-overexpressing larvae

and 21.6 � 2.4 (n ¼ 15) when Faf was overexpressed in

the lqf mutant background (p < 0.001) (Figure 7D). After

normalizing to the surface area of respective muscle

fibers, the number of boutons per muscle surface area

remained significantly lower than that in the Faf-over-

expressing larvae (p < 0.01; Figure 7E). These results

suggest that Lqf acts downstream of Faf to promote

synaptic growth.

Figure 7: Lqf is required for Faf-

induced synaptic overgrowth. A)

A representative image of synaptic

morphology onmuscle 4 (M4) in the

wild-type (wt) larva revealed by the

neuronal membrane marker HRP

and the SV protein Syt I. B) Neuronal

overexpression of the deubiquitinat-

ing enzyme Faf induces overgrowth

of synaptic boutons (muscle 4 is

shown here). The arrow indicates

neuronal overexpression of Faf in

ElavC155 Gal4/þ; EP(3)381/þ flies

(same for panels C–E). C) The Faf-

induced synaptic overgrowth is fully

suppressed in the lqfARI/Df mutant

background. D) Histograms of the

average number of synaptic bou-

tons on muscle 4 (segment 3). E)

Histograms of the average number

of synaptic boutons normalized to

the surface area of muscle 4 (seg-

ment 3). ***p < 0.001. n.s., not

significantly different.

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To test whether this suppression of Faf-induced synaptic

overgrowth is mediated by a defect in CME, we overex-

pressed Faf in the lapmutant background in which CME is

severely impaired by deletion of the clathrin assembly

protein LAP (45). In the lap mutant background, over-

expression of Faf consistently induced overgrowth of

synapses (data not shown). Together with our electro-

physiological and ultrastructural data, these results sug-

gest that Lqf mediates Faf-induced synaptic overgrowth

by a mechanism that appears to be independent of CME.

Neuronal overexpression of Lqf promotes bouton

budding but does not mimic Faf-induced synapse

overgrowth

If Lqf acts as a substrate of Faf in synaptic overgrowth,

could overexpression of Lqf itself mimic the Faf effect?

To test this idea, we overexpressed Lqf in neurons using

UAS-Lqf and ElavC155 Gal4 and found that this did not

achieve the exuberant overgrowth of NMJs seen in Faf

overexpression larvae. However, we noted a large number

of satellite or miniature boutons, which resemble those

found in larvae overexpressing b-amyloid precursor-like

protein (65) and in a number of endocytotic mutants (66)

(Figure 8B,C). In some NMJs, synaptic boutons were

dramatically enlarged and shaped like ‘growth cones’, with

visible filopodia-like protrusions or tiny budding vesicles

(Figure 8B–D). In rare cases, we also detected giant

synaptic boutons, which had reduced levels of SV markers

(e.g. Syt I, Figure 8E). As with the neuronal rescue

experiment described above, the gain-of-function pheno-

type observed in this study was more pronounced at room

temperature or higher, suggesting that synapse develop-

ment depended on Lqf protein levels. The mechanisms

Figure 8: Neuronal overexpression of Lqf promotes budding of miniature synaptic boutons and the formation of ‘growth cone’-

like boutons. A) A representative image of NMJs onmuscle 4 in the control larvae (ElavC155 Gal4/þ) co-stained for the neuronal membrane

marker HRP and the SV protein synaptotagmin I (Syt I). One area of the NMJ (arrowhead) is enlarged and shown in the far right panel. B–E)

Examples of NMJs from larvae overexpressing Lqf in neurons using the pan-neuronal driver ElavC155 Gal4 (indicated by the arrow). Note the

dramatic changes in synaptic bouton morphology. Panel (B) shows a ‘lobster’-shaped NMJ with one enlarged bouton and numerous small

budding boutons. Particularly, the SV marker is found in these miniature boutons. Panel (C) shows an example where the motor axon

becomes ‘spine’ shaped because of the addition of small boutons along the entire length of the axon. Panel (D) shows that although the

NMJ on muscle 12 appears normal, muscle 13 has a large ‘leaf’-shaped giant synaptic bouton. Note the presence of small ‘filopodia’-like

protrusions. Panel (E) shows a ‘crab’-shaped giant synaptic bouton associated with short and thin protrusions. Note the spatial restriction of

the SV marker within the giant bouton.

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Bao et al.

involved in such dramatic changes in bouton shapes

and sizes are not clear. Nonetheless, the appearance of

mini boutons and filopodia-like protrusions suggests that

overexpression of Lqf stimulates bouton budding but fails

to induce full expansion and maturation of synaptic bou-

tons. These findings imply that Faf has at least one other

substrate that acts synergistically with Lqf in promoting

synaptic growth.

lqf does not suppress synaptic overgrowth

in the hiw mutant

To further explore the role of Lqf in the UPS-mediated

synaptic growth, we next studied the genetic interaction

between lqf and highwire (hiw) mutations. Hiw is a puta-

tive E3 ubiquitin ligase that inhibits synaptic growth in

Drosophila (13,16), C. elegans (14,15) and mammals

(15,18). In Drosophila, hiw and faf mutations interact

genetically (16). Overexpression of Faf mimics the synap-

tic overgrowth seen in the hiw mutant. faf null mutants,

which do not have an apparent phenotype in both synaptic

morphology and function, partially rescue the defect in

transmitter release in hiw mutants. However, faf does not

suppress NMJ overgrowth caused by the loss of hiw (16).

These results suggest that the Hiw-based ubiquitin path-

way may be parallel with the Faf-dependent ubiquitin

pathway in conditioning synaptic growth (67).

To further test this idea, we conducted genetic interactions

between hiw and lqf mutants. hiwND9, a partial loss-

of function allele (68), is adult viable as homozygotes with

minor locomotion defects (13). Our results showed that

the hiwND9; lqfARI/Df double mutant died before reaching

second instar larval stage. In hiwND9; lqfARI/lqfAG double

mutants, we observed mid second instars. lqfFDD9 is

a hypomorphic allele of lqf, which is fully viable as

homozygotes at room temperature, has weakly rough

eyes (27) and retains less than 10% of the Lqf protein

(28) (Figure 9A). hiwND9; lqfFDD9 double mutants showed

partial lethality as some of them died in the pupal case,

whereas others escaped to the adult stage. Even though

hiw and lqf mutations showed lethal genetic interactions,

the hiwND9 mutation did not suppress the rough eye

phenotype in lqfFDD9 homozygotes (data not shown) or

alter the Lqf protein levels measured in adult hiw mutant

head extracts (Figure 9A).

To examine the effect of lqf mutations on synaptic over-

growth in hiw mutants, we generated three double mu-

tants: hiwND9; lqfARI/lqfAG, hiwND9; lqfARI/lqfFDD9 and

hiwND9; lqfAG/lqfFDD9. None of these allelic combinations

significantly inhibited synaptic overgrowth in the hiwND9

mutant background (Figure 9B–G). In second instar larvae,

muscle 4 had 39.5 � 2.4 (n ¼ 11) and 36.8 � 4.1 (n ¼ 12)

boutons in the hiwND9 mutant and hiwND9; lqfARI/lqfAG

double mutants, respectively (Figure 9B,C,H). The synap-

tic bouton number increased to 102 � 4.8 (n ¼ 13) in the

third instar hiwND9 mutant (Figure 9D,E,I). In third instar

Figure 9: lqf mutations do not reduce the synaptic over-

growth in hiw mutants. A) Western blot analyses show that

Lqf levels are significantly reduced in adult head extracts of the

lqfFDD9 homozygote but remain unaltered in the hiwND9 homozy-

gote. The western blot was reprobed for a-tubulin to ensure that

sample loading was similar between the wild-type control flies and

the mutant flies. B, C and H) Synaptic growth in second instar

larval NMJs in the hiwND9 mutant (panel B) is not significantly

affected by lqfARI/lqfAGmutations (panel C). The NMJ is stained for

HRP in these and the following images. A histogram of synaptic

bouton numbers on muscle 4 in these mutants is shown in panel

(H). D, E and I) Synaptic growth in third instar larval NMJs in the

hiwND9 mutant (panel D) is not significantly affected by lqfARI/

lqfFDD9 mutations (panel E). A histogram of synaptic bouton

numbers on muscle 4 is shown in panel I. F and G) Representative

images of exuberant synaptic growth on muscles 12 and 13 (M12

and M13) in third instar larvae of hiwND9 mutants and hiwND9;

lqfAG/lqfFDD9 double mutants.

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Novel Synaptic Roles for Drosophila Epsin 1

larvae of hiwND9; lqfARI/lqfFDD9 double mutants, muscle 4

had 110.4 � 5.7 (n ¼ 12) synaptic boutons. None of these

differences was statistically significant (p > 0.05). Simi-

larly, synaptic overgrowth persisted on muscles 12 and 13

in the hiwND9; lqfAG/lqfFDD9 double mutant (Figure 9F,G).

These observations suggest that there are at least two

distinct pathways for ubiquitin-dependent synaptic growth

and that Lqf is unlikely to be a substrate for ubiquitination

by Hiw.

lqf partially rescues the synaptic transmission

defect in hiw mutants

Synaptic transmission is severely reduced in hiw mu-

tants (13). Because lqf and hiw mutations are synthetically

lethal, one might predict that lqf mutations could further

reduce transmitter release in the hiw mutant and thus

account for their lethal genetic interactions. However, faf

null alleles partially suppress the synaptic transmission

defect in hiw mutants (16). Lqf levels are reduced in faf

mutants (28), suggesting that lqf mutations may also

partially rescue the hiw physiological defect.

To distinguish these possibilities, we recorded minis and

EJPs in hiw; lqfARI/lqfFDD9 double mutants using lqfARI/

lqfFDD9 and hiw single mutants as controls. With the

exception of increased mini amplitude, other parameters

of synaptic transmission were not significantly altered in

the lqf hypomorphic mutant (Figure 10A–C). Consistent

with earlier observations (16), mini frequency and the

amplitude of evoked EJPs were both reduced in hiw

mutants (Figure 10A–C). This reduction in evoked EJP

amplitude in the hiw mutant was partially suppressed

by the lqf mutation. This suppression was significant as

the EJP amplitude in the hiw mutant was nearly doubled

(p < 0.05). However, lqfmutations had little effect on both

the mini frequency and the amplitude in hiw mutants,

suggesting that this suppression is rather specific to

calcium-evoked exocytosis.

Discussion

Lqf does not play a significant role in SV recycling

One important finding from this study is that Lqf does not

play a detectable role in SV endocytosis. Multiple lines of

evidence obtained from electrophysiological, ultrastruc-

tural and optical imaging studies support this conclusion.

To our knowledge, this is the first in vivo study of Lqf or

epsin 1 on SV recycling. Our finding is also clearly

surprising given that epsin 1 has been highly implicated

to play a key role in the initiation of clathrin-coated vesicle

formation and endocytosis (32,34). Does our observation

reflect the special property of the fly NMJ? Lqf lacking

either the ENTH domain or the clathrin-interacting

C-terminus has been shown to rescue the mutant pheno-

type in the developing eye (39). These rescue results are

intriguing, but they do not readily support a specific role for

Lqf in CME. In particular, there are no clear mechanisms on

how these truncated fragments could fulfill Lqf’s clathrin-

dependent functions. Interestingly, RNA interference and

small interfering RNA-induced knockdown of epsin 1 fails

to block the internalization of EGF receptors in HeLa cells

(69,70). There is also evidence that epsin 1 functions only

in clathrin-independent endocytosis (70,71). Furthermore,

Lqf has been shown to be required for endocytosis of

select receptors but not of all receptors (41–43). More

importantly, Lqf itself is not required for receptor-mediated

endocytosis. Rather, Lqf appears to signal select ligands

(such as Delta/Serrate/Lag2) for internalization or recycling

(42). Hence, these studies lend strong support to our

observations that Lqf does not play a significant role in

CME of SVs.

It should be noted that recent studies reveal that the

epsin 1-interacting protein Eps15 is required for SV

recycling in both C. elegans (72) and Drosophila (73,74).

However, Eps15 is required to maintain the level of

endocytotic proteins in nerve terminals (74). Strikingly,

key endocytotic proteins such as Dynamin and Dap160

are reduced in synaptic boutons by �90 and �80%,

respectively, in eps15mutants. These observations make

it difficult, if not impossible, to assign a direct role for

Eps15 in CME.

Lqf plays a novel role in a specific UPS pathway to

regulate NMJ growth

Synapse development is a highly regulated process

involving a large number of molecules (48,63,75–77).

The first suggestion that Lqf could have a potential role

in synapse development came from studies of its deubi-

quitinating enzyme Faf (16). This notion was further

supported by a direct biochemical demonstration that

Lqf is a specific substrate of Faf (28). Our studies provide

the first experimental test of this hypothesis by showing

that Lqf acts downstream of Faf in promoting synaptic

overgrowth. This effect on NMJ growth appears to be Faf

dependent as lqf mutations alone do not dramatically

affect bouton numbers. It is interesting to note that

neuronal overexpression of Lqf promotes bouton budding

but does not mimic the exuberant synaptic overgrowth

induced by overexpression of Faf. Hence, we suggest

that Lqf is necessary but insufficient for synaptic over-

growth, raising the possibility that Lqf is not the only

substrate of Faf in motoneurons (hence the hypothetical

molecule X in Figure 10D).

Another important finding emerging from this study is that

two distinct UPS pathways may be employed at the

Drosophila larval NMJ to regulate synapse growth. The

Hiw/RPM-1/Phr1 proteins have a conserved role in inhibit-

ing presynaptic development in Drosophila, C. elegans and

mammals (13–15,18,68). In C. elegans (19) and Drosophila

(21), the substrates of RPM-1/Hiw have been shown to be

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Bao et al.

MAP kinases and MAPKKK. Our study indicates that Lqf is

unlikely a substrate of Hiw in conditioning synaptic growth.

In contrast, the Faf pathway is a positive regulator of

synaptic growth at the NMJ (16) in which Lqf is an

essential substrate (this study). Hence, we suggest that

Hiw and Faf/Lqf are two distinct UPS pathways that

regulate synapse development in Drosophila.

However, the relationship between the Faf and Hiw path-

ways in synapse development is rather complex. Intriguingly,

the MAPKKK Wnd is required for synaptic overgrowth

mediated by both Hiw and Faf pathways (21). One possibility

is that Wnd acts downstream of Lqf to fulfill the function of

both the Hiw and the Faf pathways. However, this idea is

inconsistent with the observation that unlike lqfmutants, the

wnd null mutant itself has no morphological or electrophys-

iological defect. More importantly, wnd mutations do not

suppress the transmitter release defect seen in the hiw

mutant (21), whereas the lqf mutant does. Alternatively, we

suggest that Hiw and Faf act through two parallel pathways

and that the suppression of Faf-induced overgrowth by the

wnd mutation may be mediated by Fos/Jun kinase signaling

(Figure 10D). Based on the observation that overexpression

of Ubp2A increases neuronal Wnd levels (21), it is possible

that Faf may also use Wnd as a substrate for synaptic

overgrowth (78). However, this has yet to be tested

experimentally.

Dissociation of synaptic growth from function

Recent genetic studies have revealed an interesting feature

of synapse growth and function that closely depends on

protein turnover by specific UPS pathways. InDrosophila, faf

or lqf mutations are capable of partially suppressing the

defect in transmitter release in hiw mutants. This partial

suppression is specific and should not be viewed simply as

a reduction of transmitter release in faf or lqf mutant back-

grounds by hiw mutations. If there were no partial suppres-

sion by faf or lqfmutations, the amplitude of EJPs would be

similar to that in hiw single mutants. Because faf null

mutations reduce Lqf levels (28), it is reasonable to suggest

that Lqf acts downstream of Faf to inhibit synaptic trans-

mission in hiw mutants. Unlike the functional interactions

with hiw, however, faf or lqfmutations do not affect synaptic

overgrowth in hiw mutants [(16); this study]. Differing from

lqf and fafmutations,wndmutations fully suppress synaptic

overgrowth but do not affect synaptic physiology in the hiw

mutant (21). Hence, different ubiquitin pathways can specif-

ically dissociate synapse growth from function.

The physiological stimuli involved in such selective modu-

lation of synapse growth and function remain to be

Figure 10: lqf mutations partially suppress the synaptic

release defect in hiwmutants. A) lqfmutations partially rescues

the EJP amplitude in hiw mutants. The top panel shows the

representative traces of EJPs from control larvae (wt), a lqf

hypomorph (lqfARI/lqfFDD9), hiwND9; lqfARI/lqfFDD9 double mutants

and hiwND9. The bottom panel shows the average amplitude of

EJPs in these genotypes. Note that the EJP amplitude in the hiw

mutant is nearly double by the lqf mutation. The [Ca] was 0.8 mM.

B) Histograms of the average mini amplitude in the above four

genotypes. C) Histograms of the average mini frequency in the

above four genotypes. D) A proposed model of the regulation of

synaptic growth and function by Faf/Lqf- and Hiw-dependent

ubiquitin-proteasome pathways, based on our results and those

of DiAntonio et al. (16, 21, 68). 1). We propose that Lqf acts

downstream of Faf to stimulate synaptic growth and maintain

synaptic transmission. However, there appears to be least one

other substrate (marked X) of Faf that acts synergistically with Lqf.

2). Hiw downregulates the MAPKKK Wnd, which in turn activates

the Jun kinase (JNK) and fos to stimulate synaptic growth by

transcriptional regulation (21). Because wnd neither alters synap-

tic transmission by itself nor suppresses synaptic release defects

in hiwmutants (21), the Hiw pathway appears to regulate synaptic

function and growth by two different mechanisms. 3). Faf, but not

Lqf, converges on the Hiw pathway to regulate Wnd-dependent

synaptic growth. 4). Lqf converges on the Hiw pathway to

negatively regulate transmitter release in the hiw mutant. See

Discussion. Filled arrows represent stimulatory or enhancing

effects. ‘T-stops’ represent inhibitory effects. *p < 0.05; ***p <

0.01. n.s., not significantly different.

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Novel Synaptic Roles for Drosophila Epsin 1

identified. Given the conserved role of the UPS in synaptic

plasticity across animal species (4,22,79–83), the findings

reported in this study may have general neurobiological

implications. In particular, we note that the Faf homolog in

mouse, Usp9x or Fam is differentially expressed in differ-

ent regions of the brain (20). Such spatial distribution

patterns may provide a means for Usp9x to locally regulate

synaptic function. Importantly, Usp9x is localized at syn-

apses, where calcium influx rapidly regulates its enzymatic

activity and deubiquitination of epsin 1 (84). Hence, Faf and

Lqf/epsin 1 are good candidate mediators of activity-

dependent synaptic plasticity.

Materials and Methods

Fly strains and geneticsThree alleles of the lqfmutant, lqfARI, lqfAG and lqfFDD9, a Df uncovering the

lqf locus, Df(3L) pbl-X1 (85), and a genomic rescue line (P{wþ, genomic

lqfþ}) were described previously (27). In this study, we generated lqfmutant

larvae in w and w/þ background. To minimize possible effects of genetic

background, we also used w or w/þ as our ‘wild-type’ control animals. The

lqfmutant alleles and the Df were balanced with TM6B, Hu, Tb. lqf alleles in

trans to the Df chromosome were selected as non-Tubby larvae. Post-

synaptic rescue was conducted by crossing w; UAS-lqf; lqfARI/TM6B, Hu,

Tb flies with þ; lqf Df, MHC-Gal4/TM6B, Hu, Tb. Neuronal rescue was

achieved by driving UAS-lqf expression with the pan neuronal driver

ElavC155 Gal4 in the lqfARI/Df background. Genomic rescue was conducted

by introducing one copy of P{wþ, genomic lqfþ} in lqfARI/Df mutants.

EP(3)381 was used for Faf overexpression (16). Other strains used in this

study include hiwND9 (a hypomorphic allele of hiw) (13,68) andMHC-Gal4 (63).

BiochemistryTo determine Lqf protein levels in wild-type control and lqfmutants, at least

five larval brains and ventral nerve cords were dissected in ice-cold PBS

solution and transferred directly to 20–30 mL sample buffers in an

Eppendorf tube. Larval brains and ventral nerve cords were ‘ground’

carefully using a straightened paper clip, spun down and the supernatant

equivalent to one larval brain and ventral nerve cord was loaded on a

SDS–PAGE gel (10–12%), followed by standard western blotting. Blots

were probed with antibodies to Lqf (guinea pig, 1:4000) (28) and a-tubulin

(mouse, 1:4000; Sigma). To determine Lqf levels in hiw flies, adult fly heads

were used instead of larval brains and ventral nerve cords.

Immunocytochemistry and analysisThird instar larvae at wandering stage were dissected as described (52),

fixed with Bouin’s fixative for 15–20 min at room temperature, rinsed with

phosphate buffer plus Tween (PBT), blocked with 2%BSA and stained with

primary antibodies overnight at 48C, washed and incubated with secondary

antibodies (45,47). The primary antibodies included guinea pig anti-Lqf

[1:150; (28)], rabbit anti-Shi [1:300; (50)], goat anti-HRP (1:200; Sigma), goat

anti-HRP conjugated with fluorescein isothiocyanate (FITC) (1:400; Jackson

ImmunoResearch Laboratories), rabbit anti-synaptotagmin I [1:500; (86)],

mouse monoclonal antibody (mAB) 22C10 [1:50; (87); the Developmental

Studies of Hybridoma Bank at the University of Iowa], mAb for Dlg [4F3,

1:50; (88)], mAb nc82 for Bruchpilot [Brp, 1:20; (89)] and rabbit against

glutamate receptor III [GluRIII, 1:500; (90)]. Secondary antibodies included

donkey anti-goat immunoglobulin G (IgG) conjugated with FITC (1:100),

donkey anti-guinea pig IgG conjugated with FITC (1:100) and donkey anti-

rabbit IgG conjugated with tetramethylrhodamine isothiocyanate (TRITC)

(1:100) (Jackson ImmunoResearch Laboratories). Stained preparations

were mounted on standard glass slides with Vectashield medium (Vector

Laboratories) and examined on a fluorescence microscope (Nikon or Zeiss).

The final images were taken on a confocal microscope (Leica 4D TCS).

Unless otherwise noted, all morphological analyses of the NMJ (i.e., bouton

size and number) were conducted on muscles 6/7 or muscle 4 in abdominal

segments 3 and 4. Data from the same segment were averaged and

compared to ensure minimal variations from segment to segment. At least

five different larvae were used for the control ‘wild-type’ group, and

typically, more larvae were used for the mutant groups.

ElectrophysiologyA standard third instar larval body wall preparation was used for electro-

physiological recordings and two-electrode voltage clamp recordings

(45,47,52). The preparation was bathed in HL-3 solution (53). The concen-

tration of calcium contained in the HL-3 solution is specified in the Text

describing the experiments for Figures 2, 3, 6 and 10. For mini recordings,

TTX (1 mm) was added to prevent unwanted evoked release (45). The input

resistance of each muscle was monitored and those with 5 MV or higher

were retained for final data analysis. The input resistance of the recording

microelectrode (backfilled with 3 M KCl) was 20–25 MV. The current

injection electrode (also filled with 3 M KCl) used in voltage clamp experi-

ments had an input resistance of 15–20 MV. Muscle synaptic potentials or

currents were recorded using an Axon Clamp 2B amplifier (Axon Instru-

ments) and acquired by a Dell PC computer equipped with pClamp

software. The MINI ANALYSIS program (Synaptosoft) was used to measure

the amplitude of individual mEJPs or minis. Minis with a slow rise time

arising from neighboring electrically coupled muscle cells were excluded

from analysis (45). The final figures were prepared using ORIGIN (OriginLab)

and PHOTOSHOP (Adobe).

Electron microscopy and analysisThird instar tissues were processed according to standard procedures

(91). Briefly, they were dissected in ice-cold, Ca2þ-free HL-3 saline, fixed

for 1 h in ice-cold 1% acrolein, 2.5% glutaraldehyde in 0.1 M cacodylate

(Cac) buffer, pH 7.2, post-fixed in 0.5% OsO4, 0.8% KFeCn in 0.1 M Cac

for 1 h, incubated in 5% uranyl acetate for 1 h to overnight, dehydrated

and embedded in EmBed 812 araldite. Seventy nanometer sections were

post-stained with uranyl acetate and Reynold’s lead citrate. Boutons

were imaged at 12 K magnification using a JEOL JEM 2000 EX-II TEM

operated at 100 kV. Negatives were scanned at 1500 dpi into a Macintosh

G4 computer using an AGFA Duoscan T2500. Images were adjusted for

brightness, contrast, sharpness and evenness of illumination using

PHOTOSHOP.

To evaluate SV density, a small square box (250 � 250 pixel) was placed

manually at four to five different locations on the electron micrograph of

synaptic boutons. The number of SVs was counted, averaged and

converted into an average number of SVs per square micrometer area

(which is the SV density presented in Figure 4D). The central ‘core’ regions

(Figure 4B,C) were avoided because they often do not have SVs. Vesicle

diameter reported here is the outer diameter.

FM 1-43 uptakeTo measure FM 1-43 dye uptake at the larval NMJ, we adapted the

methods described previously (45,58,92). Briefly, third instar larval prep

was dissected and pinned down on a Sylgard dish, incubated with 5 mmFM

1-43 dye (Invitrogen) for 5 min in Jan’s standard fly saline (52) containing

2 mM Ca2þ and 90 mM KCl. The NMJ preparation was then washed three to

five times with 0 Ca2þ saline and examined on a Zeiss AxioImager

microscope equipped with a �63 water immersion objective (Achroplan,

0.9 NA) and AXIOVISION software (Zeiss Inc.). Four different larvae from each

genotype (wild type and the lqf mutant) and at least three NMJs from each

larva were examined.

StatisticsUnless otherwise specified, data are presented as mean � SEM and

considered significantly different from each other when p value is smaller

than 0.05 in unpaired Student’s t-tests using the ORIGIN software. In

addition, the Kolmogorov–Smirnov test was administrated when comparing

mini sizes between preparations using MINI ANALYSIS program.

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Bao et al.

Acknowledgments

We thank Richard Daniels and Mingshan Xue for their initial contributions to

this project, Suzanne Royer for technical assistance with electron micros-

copy, and Erin Overstreet and Janis Fischer for providing the lqf mutant

alleles, UAS-lqf and the genomic rescue lines. We also wish to thank our

colleagues, Mani Ramaswami (University of Arizona) for the Shi antibody

and Brian McCabe (Columbia) and Corey Goodman (University of California

Berkeley/HHMI) for providing the mAb to Dlg and hiw alleles, Aaeron

DiAntonio (Washington University) for the GluRIII antibody, Erich Buchner

(Universitat Wurzburg) for the Bruchpilot nc82 mAb, Wes Thompson

(University of Texas), Randy Hewes (University of Oklahoma), Rudolf Bohm

(OU) and two anonymous reviewers for helpful comments on the manu-

script. This research was supported by a startup fund from OU, in part by

National Science Foundation (NSF) grants (CAREER Award IBN-0093170

and IOS-0822236) and National Institutes of Health (NIH) grant (ES014441)

to B. Z. and in part by NIH grant R01-NS045865 to N. E. R., and H. B. thanks

T. H. Lee Williams, Vice President for Research at OU, for her salary

support.

Supporting Information

Additional Supporting Information may be found in the online version of

this article:

Figure S1: Muscle size is significantly reduced in lqf mutant. Panel A

shows the average muscle size of muscles 6/7 in abdominal segments 3

whereas panel B shows the average muscle size of muscles 6/7 in

abdominal segments 4. In both segments, the lqf mutant muscle size is

significantly reduced compared to that in the wild type. ***p < 0.001.

Figure S2: Immunoreactivity to the active zone protein Bruchpilot

(Brp; panels A and C) and glutamate receptor III (GluRIII; panels B and

D) at the larval NMJ. There are no apparent differences in the level of

these two proteins between the wild type and lqf mutant. The total active

zone number (defined by Brp puncta) on muscle 4 (segment 3) is

significantly reduced in the lqf mutant (panel E). **p < 0.05. However,

the normalized active zone number per synaptic bouton in the lqf mutant is

similar to or slightly higher than that in the wild type (panel F).

Figure S3: Lqf mutations or its deficiency do not have a dominant

effect on evoked synaptic transmission. Top panels show the represen-

tative traces of EJCs, whereas the bottom panels show the average

amplitude of EJCs obtained from wt, lqfARI/þ, lqfARI/Df, Df/þ and lqfþ;

lqfARI/Df (lqf rescue) larvae.

Figure S4: FM 1-43 dye uptake is normal at the lqf mutant

NMJ. Examples of FM 1-43-stained NMJs from muscles 4 and 12 are

shown here for the wild type (panels A and C) and the lqf mutant (panels B

and D).

Figure S5: Synaptic bouton number is not significantly reduced in the

lqf mutant when normalized to the muscle surface area. The average

number of synaptic boutons is significantly reduced on muscles 6/7 in

segment 4 in the lqf mutant (panel A). **p < 0.05. However, the average

number of boutons normalized to the muscle surface area is unaffected by

the lqf mutation (panel B).

Please note: Wiley-Blackwell are not responsible for the content or

functionality of any supporting materials supplied by the authors. Any

queries (other than missing material) should be directed to the correspond-

ing author for the article.

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