frequenin/ncs-1 and the ca2+-channel -subunit co- 1 ... · frequenin (frq) and its homologue,...

4109Research Article

IntroductionFrequenin (Frq) and its homologue, neuronal calcium sensor 1(NCS-1), are Ca2+-binding proteins that regulate neurotransmitterrelease and higher functions such as learning and memory (Gomezet al., 2001). In addition, Frq might be involved in several humandiseases, including schizophrenia, bipolar disorder and X-linkedmental retardation (Koh et al., 2003; Bai et al., 2004; Bahi et al.,2003). Frq is highly conserved from yeast to humans, with 100%conservation amongst mammals (Burgoyne et al., 2004). Frqcontains four EF-hand motifs, two of which bind Ca2+ with highaffinity (Pongs et al., 1993), and an N-terminal myristoylationdomain, which allows the protein to be membrane bound. Unlikesome NCS proteins, Frq binds to membranes in the absence of Ca2+

(McFerran et al., 1999) and this localization is not affected bymutations in all functional EF hands (O’Callaghan et al., 2002).Thus, Frq is membrane bound even under resting conditions,although some Frq has also been reported in the cytosol (Koizumiet al., 2002). The crystal structure of Frq revealed a hydrophobiccrevice caused by a C-terminal shift; this crevice is thought tomediate interactions with other proteins (Bourne et al., 2001).

Despite the interest in Frq/NCS-1, a fundamental questionremains unanswered: what are the effects of loss of Frq at thesynapse? The mechanisms by which Frq exerts its effects arecontroversial: modulation of phosphoinositide 4-kinase- (PI4K)(Scalettar et al., 2002; Rajebhosale et al., 2003; Gromada et al.,2005; de Barry et al., 2006) and Ca2+ channels (Wang et al., 2001;

Tsujimoto et al., 2002; Hui et al., 2007; Hui and Feng, 2008) havebeen proposed. A third possibility, which could reconcile theseconflicting reports, is that Frq and NCS-1 increasephosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] levels byregulating PI4K activity, with downstream modulation of Ca2+

channels.Frq interacts with Pik1, the yeast orthologue of PI4K, by binding

to a 13-residue hydrophobic sequence (Huttner et al., 2003; Strahlet al., 2003). Although this binding site is not conserved in otherorganisms, Frq coimmunoprecipitates with mammalian PI4Kfrom COS-7 cells (Zhao et al., 2001), bovine chromaffin cells (Panet al., 2002) and rat brains (Taverna et al., 2002), but not with PI4Kfrom cultured dorsal-root-ganglion neurons (Bartlett et al., 2000).

Evidence for the role of Frq in regulating Ca2+ channels iscontradictory. Overexpression of a dominant-negative form ofNCS-1, in which the third EF hand is inactivated, enhances non-L-type Ca2+ currents in neuroendocrine cells, suggesting that NCS-1 is an inhibitory regulator of Ca2+ channels (Weiss et al., 2000).By contrast, an interfering C-terminal peptide of NCS-1 was shownto abolish activity-dependent facilitation of P/Q-type Ca2+ currentsin the Calyx of Held (Tsujimoto et al., 2002) and to reduce Ca2+

signals in growth cones of cultured primary neurons from Lymnaeastagnalis (Hui et al., 2007), suggesting a stimulatory role.Overexpression of dominant-negative mutations and interferingpeptides can have off-target effects; thus, a null mutant is neededto definitively determine the effects of Frq on Ca2+ channels.

Drosophila Frequenin (Frq) and its mammalian and wormhomologue, NCS-1, are Ca2+-binding proteins involved inneurotransmission. Using site-specific recombination inDrosophila, we created two deletions that removed the entirefrq1 gene and part of the frq2 gene, resulting in no detectableFrq protein. Frq-null mutants were viable, but had defects inlarval locomotion, deficient synaptic transmission, impairedCa2+ entry and enhanced nerve-terminal growth. The impairedCa2+ entry was sufficient to account for reducedneurotransmitter release. We hypothesized that Frq eithermodulates Ca2+ channels, or that it regulates the PI4K pathwayas described in other organisms. To determine whether Frqinteracts with PI4K with consequent effects on Ca2+ channels,we first characterized a PI4K-null mutant and found thatPI4K was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remainedpresent in a PI4K-null background. We conclude that the

effects of Frq are not due to an interaction with PI4K. Usingflies that were trans-heterozygous for a null frq allele and a nullcacophony (encoding the 1-subunit of voltage-gated Ca2+

channels) allele, we show a synergistic effect between theseproteins in neurotransmitter release. Gain-of-function Frqphenotypes were rescued by a hypomorphic cacophonymutation. Overall, Frq modulates Ca2+ entry through afunctional interaction with the 1 voltage-gated Ca2+-channelsubunit; this interaction regulates neurotransmission and nerve-terminal growth.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/22/4109/DC1

Key words: Neuronal calcium sensor 1, Presynaptic, Drosophila,Phosphoinositide 4-kinase, Neuromuscular junction, Synapticboutons, Calcium channels, Quantal content, Nerve growth

Summary

Frequenin/NCS-1 and the Ca2+-channel 1-subunit co-regulate synaptic transmission and nerve-terminalgrowthJeffrey S. Dason1,*, Jesús Romero-Pozuelo2, Leo Marin1, Balaji G. Iyengar1, Markus K. Klose1, Alberto Ferrús2 and Harold L. Atwood1

1Department of Physiology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada2Instituto Cajal, CSIC, Ave. Dr Arce 37, Madrid 28002, Spain*Author for correspondence ([email protected])

Accepted 7 September 2009Journal of Cell Science 122, 4109-4121 Published by The Company of Biologists 2009doi:10.1242/jcs.055095

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Because understanding cellular mechanisms of Frq has beenhampered by the lack of physiological studies on Frq-null orsevere-hypomorph mutants, we created mutant flies lacking Frq.In this study, we performed the first physiological analysis in anyorganism of a mutant with little or no Frq. We found that effectson both neurotransmitter release and nerve-terminal growth derivefrom a functional interaction of Frq with the 1-subunit ofvoltage-gated Ca2+ channels, independently of an interaction withPI4K.

ResultsDeletion of the two frequenin genesPreviously, we examined the effects of overexpression of Frq1,Frq2 and a dominant-negative interfering C-terminal peptide onneurotransmitter release at identified nerve terminals in Drosophila(Romero-Pozuelo et al., 2007). However, a definitive test of theconsequences of the absence of Frq was not available. RNAinterference was used to knock down the expression levels of bothfrq transcripts by 60%, but no phenotype was detected. It ispossible that the remaining 40% of Frq allowed normal synaptictransmission to occur. Alternatively, the reduced synaptictransmission observed in the presence of the interfering C-terminal peptide could be due to off-target effects. In addition,the peptide would only reveal functions that were due to proteininteractions with the C-terminus of Frq, leaving other functionsconcealed. Thus, analysis of null or hypomorphic frq mutants isrequired to define the effects of Frq on synaptic transmission andnerve-terminal growth.

Obtaining a frq-null mutant in Drosophila is complicated bythe presence of two frq genes. The genes are highly homologous(95% at the amino acid level), and data from overexpressersindicate that they are functionally redundant at the larvalneuromuscular junction (NMJ) (Romero-Pozuelo et al., 2007). Thetwo frq genes are separated by only 11.6 kb, and a small genecalled andorra lies between them (Fig. 1A). Searches of severaldatabases uncovered no deficiency lines that remove both frq geneswithout removing a large number of other genes. We used site-specific FLP/FRT recombination of one of two P-elements(d04472 or d06635) inserted before the transcription start site ofthe frq1 gene, and a piggyBac element (f06131) inserted in thefirst intron of the frq2 gene to create two deletions that removethe entire frq1 gene, the andorra gene and part of the frq2 gene.Recombination was confirmed using PCR and sequencing. A 1.5-kb product was detected in both deletion lines (supplementarymaterial Fig. S1), which matched the size of the product expectedfor recombination of the two flanking transposon elements. Noproduct was detected in control lines. The product was purifiedand the resulting sequence confirmed that the two elements hadrecombined (supplementary material Fig. S1). We usedquantitative reverse-transcriptase PCR (QRT-PCR) to determinefrq mRNA levels and found no detectable frq1 mRNA in eitherdeletion (Fig. 1B). There were very low levels of frq2 mRNA inboth deletion lines (frqdel1 and frqdel3), which might be due to thefact that all the coding exons remain in the truncated frq2 gene.To verify these results at the protein level, we performed westernblot analysis. A single band corresponding to the expected sizeof Frq1 and Frq2 was detected in controls (w1118 and D42-GAL4);no Frq protein was detected in either of the deletion lines (Fig.1C). The antibody also detected the overexpression of Frq1 andFrq2, demonstrating that it recognizes both Frq proteins (Fig. 1C).It is clear that, at the very least, these deletion lines represent severe

hypomorphs, and possibly a null for both Frq proteins that canbe used to study the effects of severely reducing Frq expression.

Adult flies from both Frq deletion lines were viable, similar toNCS-1-null worms (Gomez et al., 2001) but unlike yeast, in whichFrq is essential for survival (Hendricks et al., 1999). To determinebehavioural effects of no Frq, we looked at larval locomotion. Frq-null larvae and control (w1118) larvae travelled similar distances(supplementary material Fig. S2C), but Frq-null larvae had astatistically significant reduction in mean contraction frequency(supplementary material Fig. S2D).

Frq negatively regulates the number of 1s boutonsTo better understand the effects of Frq on neurotransmitter releaseand nerve-terminal growth, we examined the third-instar larval NMJ.In previous work (Romero-Pozuelo et al., 2007), we found thatalterations of Frq selectively affected growth of motor-nerve terminalswith small synaptic boutons (type 1s), whereas motor-nerve terminalswith large synaptic boutons (type 1b) were unaffected. The two types

Journal of Cell Science 122 (22)

Fig. 1. Generation of two Frq deletions. (A)Chromosomal arrangement ofariadne, frq1, andorra and frq2 genes. The frq genes are spaced by 11.6 kb.d04472 is a P-element located 21 bp before the transcription start site of theariadne gene. d06635 is a P-element located 6.5 kb upstream of the start siteof frq1. f06131 is a piggyBac element located in the first intron of frq2. In thepresence of FLP recombinase, recombination between d04472 and f06131 ord06635 and f06131 occurred. A schematic of the resulting deletions is shown.Both deletions remove the entire frq1 gene, andorra gene and part of frq2gene. (B)QRT-PCR assays on whole adults of the deletion lines (frqdel1 andfrqdel3), with values normalized to the control genotype (w1118). frq1 mRNA isundetectable; very low levels of frq2 mRNA remain. (C)Western blot analysisof protein extracts from adult flies. No Frq protein was detected in the deletionlines (frqdel1 and frqdel3) by a polyclonal C. elegans anti-NCS-1 antibody; Frq1overexpressers (UAS-frq1;+;D42-GAL4) and Frq2 overexpressers (UAS-frq2;+;D42-GAL4) showed increased Frq protein expression in comparisonwith control (D42-GAL4). A monoclonal anti-Syntaxin-1A antibody was usedas a loading control.

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of terminal differ in physiology and utilization during locomotion(Kurdyak et al., 1994). Such a selective effect on individual motorneurons is unique, and we investigated it in the deletion lines.

Nerve terminals of third-instar larvae double-stained with anti-HRP (a nervous-system-specific marker) and the monoclonal NC82antibody that recognizes a synapse-specific protein (Bruchpilot)(Wagh et al., 2006; Kittel et al., 2006) showed significantly more1s boutons in the Frq deletion lines (Fig. 2C). This phenotype wasrescued by transgenic expression of full-length Frq2, demonstratingthat the enhanced nerve-terminal growth observed in the deletionwas due to the absence of Frq. The number of synapses per bouton,estimated by counting the number of NC82 spots from a sample of1b boutons (about 5 mm in diameter) and 1s boutons (about 2 mmin diameter), was not different in the Frq deletion lines and controls(Fig. 2D). As indicated by the previous study, Frq has a majormorphological effect: negative regulation of the number of 1sboutons.

Additional experiments were performed to rule out the possibilitythat the small andorra gene, removed in the deletion (Fig. 1A),contributed to the nerve-terminal phenotype. No effects could beattributed to alterations of Andorra (supplementary material Fig.S3B,C). Thus, we conclude that the increased number of 1s boutonsin the deletion lines was due to the severe reduction or absence ofFrq expression only.

In this and all subsequent experiments, we used Frq2 as a rescuerfor two reasons. First, the two Frqs are 95% identical in sequenceand their overexpression results in identical phenotypes (Romero-Pozuelo et al., 2007). Second, the Frq deletion lines contain a hybridelement with a UAS site followed by the coding region of thetruncated frq2 gene; when we expressed the ubiquitous LL7-GAL4driver in a Frq deletion background, we found an increase in frq2mRNA levels (data not shown). Thus, it was not possible to do arescue experiment with only Frq1.

Effects of Frq deletion on synaptic transmissionWe assessed the effects of the absence of Frq on synaptictransmission by recording excitatory junction potentials (EJPs) byusing intracellular microelectrodes. Nerve-evoked compound EJPswere significantly smaller than for controls (w1118) in the Frq deletionlines (Fig. 3A,B), and this reduction was rescued by transgenicexpression of full-length Frq2. The frequency and amplitude ofspontaneous release were not affected in the deletion lines (Fig.3C-E). No effects of underexpression or overexpression of Andorracould be demonstrated (supplementary material Fig. S3D-F), so thereduced EJP amplitude observed in the deletion lines is due to thesevere reduction or absence of Frq.

Given the differential effects of Frq on 1b and 1s boutons, itwas essential to record from them selectively with extracellularmacropatch electrodes. The number of quanta released by single1b and 1s boutons was significantly reduced in the deletion linein comparison with controls (w1118), in agreement with whole-cellintracellular recordings (Fig. 4A-D). No differences in amplitudeor time course of spontaneously occurring quantal events wereobserved; thus, Frq does not affect postsynaptic receptors.Transgenic expression of full-length Frq2 rescued the deficit,indicating that Frq regulates evoked neurotransmitter release.

To determine how presynaptic terminals without Frq respond toreduced Ca2+ entry, we measured quantal release from single 1bboutons in low-Ca2+ saline (0.3 mM Ca2+). Quantal content in theFrq deletion line (0.37±0.13) was significantly reduced incomparison with the control (w1118; 1.07±0.25). Thus, the absenceof Frq results in more-severe defects in quantal release in low-Ca2+

saline (65% reduction) than in high-Ca2+ saline (35% reduction).The results for the deletion line confirm the previous observationsobtained with the interfering C-terminal peptide (Romero-Pozueloet al., 2007) and add further evidence for the role of Frq in nerve-evoked Ca2+-mediated neurotransmission.

Fig. 2. The number of 1s boutons is increased in the absence ofFrq. (A,B)Fixed preparations stained with FITC-conjugated anti-HRP antibody (green) and anti-NC82 antibody (red). Panel Brepresents a zoomed-in shot of a few boutons from a w1118

preparation. (C)Total number of boutons was larger in the Frqdeletion lines (frqdel1, frqdel3 and frqdel1/frqdel3) than in controls(w1118; *P<0.01; n5-8; muscle fibres 6-7 segment 3). Theseeffects were specific for 1s boutons (*P<0.01; n5-8). Transgenicexpression of Frq2 (frqdel1;UAS-frq2/+;D42-GAL4/+) rescued thisphenotype. (D)Number of synapses per 1b or 1s boutons was notsignificantly different for Frq deletion lines and controls (P>0.05;n6-8). Error bars represent s.e.m.

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Effects of Frq on short-term synaptic plasticityOverexpression of Frq in Drosophila was previously found toenhance paired-pulse facilitation and cause a large facilitatedresponse (LFR), observed as greatly enhanced, often asynchronous,transmission following the initial stimulus (Mallart et al., 1991;Pongs et al., 1993; Rivosecchi et al., 1994). To test the role of Frqin paired-pulse facilitation and depression, we measured evokedEJCs from single 1b boutons in response to paired pulses withinterstimulus intervals of 20 ms and 100 ms. At external Ca2+

concentrations of 1 mM, control larvae displayed paired-pulsedepression at interstimulus intervals of 20 ms (Fig. 5A,C). Thisdepression was reduced in the Frq deletion line, probably owing tothe reduced amplitude of the first EJC in comparison to its genetic

control (w1118; Fig. 5A,C). This phenotype was rescued by transgenicexpression of Frq2. Conversely, overexpression of Frq1 or Frq2resulted in significantly more depression than in controls (D42-GAL4; Fig. 5A,C), probably owing to the larger amplitude of thefirst EJC. This depression was not observed at interstimulusintervals of 100 ms (Fig. 5B,D).

Because paired-pulse facilitation is inversely related to externalCa2+ concentrations (Mallart, 1993), we repeated the experimentsin low-Ca2+ saline. No differences were found among any of thegenotypes at external Ca2+ concentrations of 0.1 mM (data notshown) or 0.3 mM (Fig. 5E,F); they all showed paired-pulsefacilitation. However, it should be noted that, in the V7 mutant[X-ray-induced chromosomal rearrangement T(X;Y)V7] (Tanouye


Fig. 3. Amplitudes of nerve-evoked EJPs are reduced in Frqdeletion lines. (A)Sample traces of EJPs (50 EJPs averaged)in 1 mM Ca2+. (B)Amplitude of evoked EJPs of the Frqdeletion lines (frqdel1, frqdel3 and frqdel1/frqdel3) decreased incomparison with controls (w1118; P<0.01; n7). Transgenicexpression of Frq2 (frqdel1;UAS-frq2/+;D42-GAL4/+)restored EJP amplitude (Rescue). (C)Sample traces ofmEJPs obtained in 1 mM Ca2+. (D,E)No significantdifferences in frequency or amplitude of mEJPs were found(P>0.05; n6). Error bars represent s.e.m.

Fig. 4. Impaired quantal release at single 1b and 1s boutonsin the absence of Frq. (A)Average of 50 EJCs (2 Hzstimulation) and 10 mEJCs from single 1b boutons (1 mMCa2+). (B)Quantal content of single 1b boutons wassignificantly reduced in the Frq deletion line (frqdel1) incomparison with controls (w1118; *P<0.01; n8-12).(C)Average of 50 EJCs (2 Hz stimulation) and 10 mEJCsfrom single 1s boutons (1 mM Ca2+). (D)Quantal content ofsingle 1s boutons was significantly reduced in the Frqdeletion in comparison with controls (w1118; *P<0.01; n5-6).In both cases, the reduction was rescued by transgenicexpression of Frq2 (frqdel;UAS-frq2/+;D42-GAL4/+). Errorbars represent s.e.m.

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et al., 1981; Mallart et al., 1991), we observed the LFR phenotypetwice in several trials in HL6 saline (data not shown). Thisphenotype was never observed when Frq1 or Frq2 wereoverexpressed using the GAL4/UAS system. It is possible thatthe X-ray-induced translocation that upregulates Frq2 in V7mutants alters the expression of another protein that leads to theLFR.

Frq regulates neurotransmitter release at individual activezonesWe tested the hypothesis that the observed differences inneurotransmitter release of individual boutons could be due todifferences in the number of active zones per bouton. For this,we employed electron microscopy, because it is known that thenumber of active zones per synapse is variable in Drosophila(Atwood et al., 1993; Stewart et al., 1996) and multiple activezones at individual synapses cannot be resolved by countingNC82-positive spots using conventional fluorescence microscopy.With electron microscopy, active zones (where synaptic vesiclesdock to and fuse with the presynaptic membrane and releaseneurotransmitters) can be readily observed as electron-denseregions (Atwood, 2006). We found no change in the number ofactive zones per synapse in 1b boutons of Frq1 overexpressers,Frq2 overexpressers, controls (w1118 and D42-GAL4) or the Frqdeletion line (Fig. 6A). Therefore, Frq enhances release atindividual active zones.

Frq does not alter the number of docked vesiclesThrough its interaction with PI4K, Frq could play a role in vesiclepriming or docking. Overexpression of NCS-1 in pancreatic -cells enhanced glucose-induced insulin secretion, possibly owingto an increase in the number of secretory granules in the readilyreleasable pool (Gromada et al., 2005). We compared the numberof docked vesicles in experimental and control 1b boutons bycounting vesicles touching or within 20 nm of the electron-denseT-bar (active zone). There was no difference in the number ofdocked vesicles in Frq1 overexpressers, Frq2 overexpressers,controls (w1118 and D42-GAL4) or the Frq deletion line (Fig. 6B).In addition, there was no difference in the number of synapticvesicles within 50 nm and 500 nm of the synapse between thesegroups (data not shown).

Frq regulates Ca2+ entry in response to low-frequencystimulationAltered neurotransmitter release in neurons with different Frqexpression could possibly be linked to changes in Ca2+ entry duringstimulation, or to differences in resting Ca2+ levels. We examinedthese possibilities with Ca2+-responsive indicators. IntraterminalCa2+ levels in single 1b boutons at rest were determined with Fura-Dextran forward-filled into presynaptic terminals. Frq1overexpressers, Frq2 overexpressers, Frq deletion lines and controls(w1118 and D42-GAL4) showed no significant differences in theirresting Ca2+ levels (Fig. 7A,B).

Fig. 5. Effects of altered levels of Frq on paired-pulsedepression and facilitation. (A,B)EJCs of single 1b boutonsfor paired-pulse stimulation (0.5 Hz) in 1 mM Ca2+

[interstimulus intervals (ISI) of 20 ms or 100 ms].(C)Overexpression of Frq1 (UAS-frq1;+;D42-GAL4) or Frq2(UAS-frq2;+;D42-GAL4) significantly increased paired-pulsedepression in comparison with controls (D42-GAL4;*P<0.05; n6), whereas the Frq deletion line (frqdel1)exhibited reduced depression in comparison with controls(w1118; *P<0.05; n6-8), which was rescued by transgenicexpression of Frq2 (frqdel1;UAS-frq2/+;D42-GAL4/+). (D)Nosignificant differences among genotypes were found at ISI of100 ms (P>0.05; n6). (E)EJCs for paired-pulse stimulation(0.5 Hz) in 0.3 mM Ca2+, with ISI of 20 ms. (F)Nosignificant differences among genotypes were found in low-Ca2+ saline (P>0.05; n5-8). In all cases, paired-pulsedepression or facilitation is shown as the ratio of the secondEJC to the first EJC. Error bars represent s.e.m.

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Nerve-impulse-linked Ca2+ entry was assayed at high temporalresolution with Oregon Green 488 BAPTA-1 conjugated to 10-kDaDextran (OGB-1). After nerve terminals had been forward-filledwith indicator, 2 Hz stimulation was delivered to the motor axonsand signals of single type-1b boutons at the ends of nerve brancheswere analyzed. The Ca2+ indicator is distributed equally throughoutthe cytosol of the bouton and measured changes that report cytosolicCa2+, rather than events at active zones, where most Ca2+ entry isthought to occur.

Peak amplitudes of Ca2+ signals were significantly reduced by15% on average in the Frq deletion line compared with its geneticcontrol (w1118; Fig. 7C,D). This reduction is large enough to accountfor the reduced quantal release observed in the deletion line (seeDiscussion). A significant increase in the peak amplitude of Ca2+

signals in response to 2 Hz stimulation was found in Frq1 and Frq2overexpressers in comparison with controls (D42-GAL4; Fig.

7C,E). This could account for the twofold increase in quantal contentobserved in single 1b boutons of Frq overexpressers (Romero-Pozuelo et al., 2007) (see Discussion).

Acute disruption of Frq reduces Ca2+ entryCa2+-imaging experiments indicate that Frq regulatesneurotransmitter release by regulating Ca2+ entry. Alternatively, Frqmight affect the trafficking of Ca2+-channel subunits duringdevelopment, leading to altered Ca2+ responses. Acute disruptionof the binding of Frq to its target provides a way of bypassingdevelopmental effects and avoiding the potential problem ofcompensation due to the chronic absence of Frq. Chronic transgenicexpression and acute application of an interfering C-terminal Frqpeptide (Romero-Pozuelo et al., 2007) were previously shown toreduce quantal content to a level similar to that of the Frq deletion.To determine whether acute disruption of Frq-target binding impairsCa2+ entry, we forward-filled OGB-1 and 50 mM of an interferingC-terminal Frq peptide. With 2 Hz stimulation, the amplitude ofCa2+ signals in treated 1b boutons decreased significantly incomparison with that of 1b boutons forward-filled with a scrambledpeptide (Fig. 7F). This can account for the 70% reduction in quantalrelease observed in 1b boutons forward-filled with the peptide(Romero-Pozuelo et al., 2007) (Fig. 8H; see Discussion). Chronictransgenic expression of the C-terminal peptide also resulted in asignificant reduction in peak amplitudes of Ca2+ signals (Fig. 7G),which is sufficient to account for the reduced quantal content. Thus,Frq regulates quantal release at individual active zones bymodulating Ca2+ entry.

Effects of Frq on neurotransmitter release are independent ofPI4KWe tested the hypothesis that Frq modulates PI4K activity, therebyaltering PtdIns(4,5)P2 levels, which in turn regulate Ca2+ entry andneurotransmitter release. Given reports in the literature indicatingthe importance of the Frq-PI4K interaction for exocytosis (e.g. deBarry et al., 2006), it was essential to determine whether themechanism is important in Drosophila.

The four wheel drive (fwd) gene encodes the sole predicted PI4Kin the Drosophila genome and provides a means to test thehypothesis. Whereas PI4K is essential for viability in yeast(Hendricks et al., 1999), Drosophila fwd-null mutants are viable.Loss-of-function fwd mutations result in cytokinesis defects andmale sterility. The allele fwd3 is an ethylmethane sulfonate (EMS)-induced mutation that results in a premature stop codon (Brill etal., 2000) and no full-length protein (Julie Brill, University ofToronto, Toronto, Canada, personal communication).

To determine whether the fwd gene modifies synaptictransmission, we compared control [Canton-S (CS)] and PI4K(fwd)-null mutants. No significant differences were found in theamplitude of evoked EJPs, the frequency of miniature EJPs(mEJPs) or amplitude of mEJPs between control and PI4K-nullmutants (Fig. 8A-D); nor were there differences in the number ofquanta released by 2 Hz stimulation (Fig. 8F), or in the amplitudeor the frequency of spontaneously occurring quantal events of 1bboutons (data not shown). Because PI4K-null mutants do notdisplay the reduced quantal release seen in Frq loss-of-functiongenotypes, the two proteins probably function in separatepathways.

The lack of an effect on synaptic transmission in PI4K-nullmutants might be due to the presence of two other predicted PI4Ksin Drosophila. These two PI4Ks might be able to maintain the


Fig. 6. Frq does not regulate the number of active zones per synapse or thenumber of docked vesicles. (A)Electron micrographs showing presynapticelectron-dense regions. Active zones are indicated by arrows. No significantdifference in the number of active zones per synapse were found among Frq1overexpressers (UAS-frq1;+;D42-GAL4; n82), Frq2 overexpressers (UAS-frq2;+;D42-GAL4; n18), D42-GAL4 (control, n60), w1118 (control, n16)and frqdel1 (n19; P>0.05). n represents the number of synapses analyzed.(B)Docked-vesicle counts (vesicles touching or within 20 nm of the activezone) per active zone showed no significant differences among Frq1overexpressers (n13), Frq2 overexpressers (n12), D42-GAL4 (control,n12), w1118 (control, n15) and frqdel1 (n11; P>0.05). n represents thenumber of active zones analyzed. Error bars represent s.e.m.

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necessary levels of phosphoinositides for exocytosis in the absenceof PI4K. However, the only PI4K that has been shown to interactwith Frq in yeast and mammals is PI4K. The putative interactionbetween Frq and PI4K was tested directly by measuring quantalrelease in genotypes that overexpress Frq1 in a PI4K-nullbackground. The enhanced quantal release, reported for Frq1overexpressers, also occurred when Frq1 was overexpressed in aPI4K-null background (Fig. 8F), ruling out a Frq-PI4K interactionfor enhanced quantal content.

It is not clear which of the targets of Frq are prevented frominteracting with Frq by the interfering Frq C-terminal peptide. Todetermine whether a Frq-PI4K interaction is disrupted by thisinterfering peptide, we forward-filled 50 mM of the Frq C-terminalpeptide or the scrambled peptide into motor neurons of control(w1118) and PI4K-null larvae. The Frq C-terminal peptide causeda similar reduction in quantal content at single 1b boutons of w1118

and PI4K-null larvae; no reduction occurred with the scrambledC-terminal peptide (Fig. 8G,H). Therefore, the effects of theinterfering peptide are not due to disruption of an interaction of Frqwith PI4K, and the hypothesis based upon such an interaction canbe rejected.

An interaction between Frq and Cac regulatesneurotransmitter releaseHaving established presynaptic Ca2+ channels as the most likelysite for the effect of Frq on neurotransmission, we sought additionaldirect evidence for this putative interaction. We screened forgenetic interactions between different frq and cacophony (cac)alleles. cac encodes the 1-subunit of voltage-gated Ca2+ channels.Flies homozygous for l(1) L13HC129, a cac allele, are lethal (Kulkarniand Hall, 1987), so we used l(1) L13HC129/+ larvae for allexperiments. Interestingly, we found that flies trans-heterozygousfor a frq hypomorph allele (PBac{WH}f06131) and a null cac allele(l(1) L13HC129) had a 50% reduction in viability. PBac{WH}f06131is inserted into the first intron of frq2, producing a complete lossof frq2 mRNA expression and a 50% reduction of frq1 mRNA levels(data not shown). To determine whether this interaction hasfunctional consequences for synaptic transmission, we obtainedwhole-cell intracellular recordings from frqdel1/l(1) L13HC129 larvae.We found a significant reduction in EJP amplitude in comparisonwith l(1) L13HC129/w1118 or frqdel1/w1118 larvae (Fig. 9A,B), indicatinga synergistic relationship between Frq and the 1-subunit ofvoltage-gated presynaptic Ca2+ channels. The mEJP frequency and

Fig. 7. Frq regulates Ca2+ entry in response to low-frequencystimulation. (A)Representative 340 nm and 380 nm imagesof a 1b bouton. Intraterminal Ca2+ levels in 1b boutons at restwere determined ratiometrically from ratio images, obtainedby dividing 340 nm images by their corresponding 380 nmimages. (B)No significant differences in resting Ca2+ levelswere found among Frq1 overexpressers (UAS-frq1;+;D42-GAL4), Frq2 overexpressers (UAS-frq2;+;D42-GAL4),controls (w1118 and D42-GAL4) and frqdel1 (P>0.05; n6-9).(C)Changes in fluorescence detected by line scanning of lbboutons loaded with OGB-1. A line-scan synchronizationbox triggered a red-light-emitting diode that was placed inthe optical path of the confocal microscope and lit for 2 mswhen a pulse was initially given. Representative traces ofrelative fluorescence changes in response to a single stimuluspulse are plotted below each image. (D-G)Peak amplitudesof Ca2+ signals were determined in response to 2 Hzstimulation for 2 seconds in 1 mM Ca2+. (D)There was asignificant reduction in the peak amplitude of Ca2+ signals ofthe frqdel1 in comparison with its genetic control (w1118;*P<0.05; n16 for w1118 and n15 for frqdel1). This reductionwas rescued by transgenic expression of Frq2 (frqdel1;UAS-frq2/+;D42-GAL4/+). (E)Peak amplitudes of Ca2+ signals ofFrq1 overexpressers and Frq2 overexpressers weresignificantly larger than for controls (D42-GAL4; *P<0.01;n6-11). (F)Peak amplitudes of Ca2+ signals in the presenceof an interfering C-terminal peptide was significantly smallerthan for controls with a scrambled peptide or no peptide(*P<0.05; n6-16). (G)Chronic expression of a transgenicinterfering C-terminal peptide (+;UAS-DN frq;elav GAL4)throughout development also significantly reduced peakamplitude of Ca2+ signals in comparison to its genetic control(+;+; elav GAL4) (*P<0.05; n6). Error bars represents.e.m.

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amplitudes were not significantly different among any of the lines(data not shown).

Several Ca2+-binding proteins regulate voltage-gated Ca2+

channels by binding to the C-terminal of the 1-subunit. To test thehypothesis that Frq acts on this region, we overexpressed Frq2 ina cacts2 mutant background. cacts2 is a temperature-sensitivemutation in the carboxyl tail of the 1-subunit that results in milddefects in synaptic transmission and Ca2+ entry at room temperature,and severe defects in synaptic transmission and Ca2+ entry atnonpermissive temperatures (37°C) (Macleod et al., 2006). Theenhanced neurotransmitter release seen in Frq2 overexpressers wasnot seen when Frq2 was overexpressed in a cacts2 mutantbackground at permissive or nonpermissive temperatures (Fig.9C,D). At nonpermissive temperatures, channel function is severelyimpaired, making it impossible for Frq to enhance transmitter releaseby enhancing Ca2+ entry. However, it is surprising that the effectsof overexpression of Frq are suppressed in cacts2 mutantbackgrounds at permissive temperatures. To determine whether thiswas due to lack of Frq activation or reduced Ca2+ entry, we recordedEJPs in control (D42-GAL4) and Frq2 overexpressers in HL6 salinewith 0.6 mM external Ca2+ at room temperature. At thisconcentration, EJPs of controls (Fig. 9E,F) were similar to thoseof cacts2 mutants at 1 mM Ca2+ (Fig. 9B). Thus, it is likely thatequivalent amounts of Ca2+ enter the terminal. At 0.6 mM externalCa2+, overexpression of Frq2 resulted in a significant increase in

EJP amplitudes (Fig. 9E,F), demonstrating that the reduced Ca2+

entry is still enough to activate Frq2. Our data strongly suggest thatCa2+ activates Frq, which then acts upon the channel to furtherenhance Ca2+ entry.

Frq regulates nerve-terminal growth through a functionalinteraction with cacTo determine whether the reduction of 1s boutons observed in Frqoverexpressers is due to an interaction between Frq with PI4K orthe 1-subunit of voltage-gated Ca2+ channels, larvae in which Frqwas overexpressed in a fwd or cacts2 background were stained withanti-HRP antibody and the number of synaptic boutons was counted.Frq does not interact with PI4K to regulate nerve-terminal growth,because the reduced number of synaptic boutons observed in Frq1overexpressers is also seen when Frq1 is overexpressed in a fwdbackground (Fig. 10A,B). There were no significant differences inthe number of 1b or 1s boutons in PI4K mutants and control (CS)larvae (Fig. 10A,B), demonstrating that PI4K is dispensable fornerve-terminal growth.

Given that the cac allele l(1) L13HC129 is lethal, we used the cacts2

allele to determine whether cac is required for Frq-dependent nerve-terminal growth. cacts2 mutants have reduced nerve-terminal growthat 25°C (Xing et al., 2005), but have normal growth at 22°C(Macleod et al., 2006). When larvae were grown at 25°C, Frq2overexpressers, cacts2 mutants, and Frq2 overexpressers in a cacts2


Fig. 8. Effects of Frq are not due to an interaction with PI4K.(A)Sample traces of EJPs (50 EJPs averaged) in 1 mM Ca2+.(B)No significant differences in EJP amplitude of fwd mutants[fwd3/Df(3L)7C] and controls (CS) were found (P>0.05; n5-10).(C)Sample traces of mEJPs. (D)Frequency and amplitude ofmEJPs from controls (CS) and fwd mutants are not significantlydifferent (P>0.05; n4-6). (E)Average of 50 EJCs (2 Hzstimulation) and 10 mEJCs from single 1b boutons. (F)Quantalcontent of single 1b boutons was approximately twofold greater inFrq overexpressers (elav-GAL4;UAS-frq1/+) and Frqoverexpressers in a fwd background [elav-GAL4;UAS-frq1/+;fwd3/Df(3L)7C] in comparison with controls (*P<0.01;n6-10). (G)Comparison of EJCs and mEJCs (average of 50EJCs and 10 mEJCs) from single 1b boutons of w1118 and fwdmutants loaded with a scrambled peptide and with a DrosophilaFrq C-terminal peptide. (H)Extracellular recordings (1 mM Ca2+)from single 1b boutons show that interfering Frq1 C-terminalpeptide causes a 70% reduction in quantal content in both w1118

and fwd mutants (*P<0.01; n4-5). Error bars represent s.e.m.Jour

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background, all had reduced nerve-terminal morphology (data notshown). When larvae were grown at 22°C, there was a significantreduction in the number of 1s boutons in Frq2 overexpressers, butno change in the number of boutons in cacts2 mutants (Fig. 10C,D).When Frq2 was overexpressed in a cacts2 background at 22°C, therewas no change in the number of synaptic boutons (Fig. 10C,D).Thus, cac is required for Frq to affect nerve-terminal growth.

DiscussionIn the present study, for the first time in any organism, we presentdata on the physiological and morphological consequences of nodetectable Frq protein by creating two deletions that remove theentire frq1 gene and part of the frq2 gene. Mutants lacking Frq wereviable, but had locomotion defects, reduced quantal releaseprobability, impaired Ca2+ entry, and nerve-terminal overgrowth.We confirmed the effects of disrupting the function of Frq foundin a previous study (Romero-Pozuelo et al., 2007) and provide newevidence for a role of Frq in presynaptic Ca2+ signalling and aninteraction between Frq and the 1-subunit of a presynaptic Ca2+

channel. In addition, we ruled out interactions with PI4K inneurotransmitter modulation and nerve-terminal growth. These newexperiments establish the importance of Frq as a modulator of Ca2+-channel function and consequent physiological processes.

Frq regulates release probability and Ca2+ entryThe role of Frq in exocytosis has been inferred from overexpressionstudies or by use of a dominant-negative approach (Olafsson et al.,1995; Zhao et al., 2001; Pan et al., 2002; Koizumi et al., 2002;Scalettar et al., 2002; Taverna et al., 2002; Rajebhosale et al., 2003;Sippy et al., 2003; Romero-Pozuelo et al., 2007). Frq has beenimplicated in regulating both basal levels of synaptic transmission(Olafsson et al., 1995; Wang et al., 2001; McFerran et al., 1998) andshort-term synaptic plasticity (Mallart et al., 1991; Pongs et al., 1993;Rivosecchi et al., 1994; Sippy et al., 2003). Analysis of a Frq-nullmutant was needed to determine the specific role of Frq in synaptictransmission. In general, synapses that display a higher releaseprobability in response to low-frequency stimulation show lessfacilitation (or more depression) than synapses with lower releaseprobability. Data from this study clearly show that synapses with littleor no Frq have a lower release probability and reduced paired-pulsedepression. By contrast, elevated levels of Frq result in synapses witha higher release probability and enhanced paired-pulse depression.Higher levels of Frq have been observed in crayfish (Jeromin et al.,1999) and frog (Belair et al., 2005) phasic motor neurons, which havesynapses with high release probability exhibiting depression incomparison to their tonic counterparts, which have facilitatingsynapses with low release probability. This raises the possibility that

Fig. 9. Frq regulates neurotransmitter release through afunctional interaction with cac. (A)Sample traces of EJPs (50EJPs averaged) in 1 mM Ca2+. (B)EJP amplitude wassignificantly reduced in l(1)L13HC129/frqdel1 larvae comparedwith controls, l(1)L13HC129/w1118 or frqdel1/w1118 larvae(*P<0.01; n4-9). (C)Sample traces of EJPs (50 EJPsaveraged) in 1 mM Ca2+ from controls (+;+;D42-GAL4), Frq2overexpressers (+;UAS-frq2/+;D42-GAL4/+), Frq2overexpressers/cacts2 (cacts2;UAS-frq2/+;D42-GAL4/+) andcacts2 (cacts2;+;+) larvae at 22 °C and 37°C. Recordings at bothtemperatures were made from the same larvae. (D)At 22°C,EJP amplitudes of cacts2 were significantly reduced comparedwith controls (*P<0.05; n5-8), whereas EJP amplitudes ofFrq2 overexpressers were significantly enhanced (*P<0.01;n5). At 22°C, EJP amplitudes of cacts2 and Frq2overexpressers/cacts2 larvae were not significantly different(P>0.05; n8-10). At 37°C, EJP amplitudes of cacts2 weresignificantly reduced compared with controls (*P<0.01; n5-8),whereas EJP amplitudes of Frq2 overexpressers weresignificantly enhanced (*P<0.05; n5). At 37°C, EJPamplitudes of cacts2 and Frq2 overexpressers/cacts2 larvae werenot significantly different (P>0.05; n8-10). (E)Sample tracesof EJPs (50 EJPs averaged) in 0.6 mM Ca2+. (F)EJP amplitudeswere significantly enhanced in Frq2 overexpressers (+;UAS-frq2/+;D42-GAL4/+) in comparison with controls (+;+;D42-GAL4; *P<0.01; n5). Error bars represent s.e.m.

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the differential expression of Frq might contribute to functionaldifferences at phasic and tonic synapses. In Drosophila, type-1s motor-nerve terminals have physiological and morphological similaritieswith phasic motor-nerve terminals of other organisms, whereas type-1b terminals resemble tonic nerve terminals more closely. The factthat growth of type-1s terminals is selectively affected by Frq raisesthe possibility that physiologically different neurons are dependenton Frq for their differentiation.

Frq has been shown indirectly to regulate Ca2+ entry in Xenopusnerve-muscle co-cultured cells (Wang et al., 2001), neuroendocrinecells (Weiss et al., 2000), the Calyx of Held synapse (Tsujimoto etal., 2002), and growth cones of cultured primary neurons fromLymnaea stagnalis (Hui et al., 2007). However, some of these studiesproposed a stimulatory role, whereas others suggested an inhibitoryrole. Because all of these studies were based on overexpression ordominant-negative approaches, we used a Frq-null mutant to clarifythe role of Frq in Ca2+ entry. We found that, in the absence of Frq,Ca2+ entry was significantly impaired, demonstrating that Frq isrequired for normal Ca2+ entry. However, the frequency ofspontaneous neurotransmitter release was not affected, indicatingthat Frq does not alter the resting level of [Ca2+]i, and this wasconfirmed directly by measuring [Ca2+]i with ratiometric indicators.

Previous studies did not correlate changes in Ca2+ current orsignals with changes in neurotransmitter release. Quantal contentvaries as a fourth-order function of Ca2+ (Dodge and Rahamimoff,

1967; Augustine and Charlton, 1986). This fourth-powerrelationship probably applies to the Ca2+ sensor at the active zone.It was not possible to measure Ca2+ signals from individual activezones, so the Ca2+ signals that we measured were diluted by thevolume of the bouton. Assuming a fourth-power relationship, thedecreased amplitude of Ca2+ signals observed in the absence of Frqor in the presence of the C-terminal peptide was enough to accountfor the 50-70% reduction in release. Similarly, the increasedamplitude of Ca2+ signals in the Frq overexpressers was larger thanthe 15-20% increase required to account for the twofold increasein quantal content.

Effects of Frq are independent of PI4KSeveral studies have indicated that overexpression of Frq increasesexocytosis by enhancing PI4K activity, whereas other studies haveindicated that altered Ca2+ entry is responsible. PtdIns(4,5)P2 hasbeen shown to maintain the activity of Ca2+ channels (Wu et al.,2002). Thus, Frq could regulate Ca2+-channel activity and, in turn,neurotransmitter release, through its effect on PtdIns(4,5)P2 levels.We found that the effects of Frq are not dependent on the PI4Kpathway. First, quantal release was not affected by the absence ofPI4K, and overexpression of Frq resulted in enhanced quantalrelease, whether PI4K was present or not. Second, some studieshave speculated that an interaction between Frq and PI4K regulatesthe size of the readily releasable vesicle pool (Gromada et al., 2005),


Fig. 10. Frq regulates nerve-terminal growth through afunctional interaction with cac. (A,C)Fixed preparationsstained with FITC-conjugated anti-HRP antibody. (B)Thenumber of 1s boutons was significantly reduced when Frqwas overexpressed by itself (elav-GAL4;UAS-frq1/+) or ina fwd background [elav-GAL4;UAS-frq1/+;fwd3/Df(3L)7C] in comparison with controls (*P<0.01;n4-7). There was no difference in the number of boutonsbetween fwd loss-of-function mutants [fwd3/Df(3L)7C] andcontrols (CS; P>0.05; n4-5). (D)There was a significantreduction in the number of 1s boutons when Frq wasoverexpressed by itself (+;UAS-frq2/+;D42-GAL4/+) incomparison with controls (*P<0.01; n5). However, whenFrq was overexpressed in a cacts2 background (cacts2;UAS-frq2/+;D42-GAL4/+), there was no significant differencein the number of boutons in comprison to controls(+;+;D42-GAL4) and cacts2 (cacts2;+;+; P>0.05; n5-6).Error bars represent s.e.m.

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but we showed that Frq does not regulate the number of dockedvesicles. Third, we found a similar reduction in quantal release inPI4K-null mutants and controls in the presence of the C-terminalpeptide, demonstrating that it binds to something other than PI4K.Also, yeast-two-hybrid assays failed to show an interaction inDrosophila between Frq and PI4K (Julie Brill, personalcommunication).

Effects of Frq are dependent on cacSynaptic transmission was significantly impaired in frq and cactrans-heterozygotes. This indicates a synergistic relationshipbetween Frq and the 1-subunit of voltage-gated Ca2+ channels.We also used a hypomorphic cacts2 mutation to show a geneticinteraction between Frq and the Ca2+ channel. The temperature-sensitive cacts2 mutation occurs in the carboxyl tail of the 1-subunitand has been shown to be crucial for Ca2+-channel inactivation atboth permissive and nonpermissive temperatures (Macleod et al.,2006). Given that gain-of-function Frq phenotypes are not presentin a cacts2 background, Frq might negatively regulate Ca2+-channelinactivation. Interestingly, another member of the NCS family,visinin-like protein-2, binds to the carboxyl tail of the 1-subunitof Ca2+ channels, slowing inactivation (Lautermilch et al., 2005).It is tempting to speculate that Frq also binds to the carboxyl tailof the 1-subunit. However, coimmunoprecipitation experimentshave thus far been inconclusive. Thus, we cannot entirely rule outthe possibility that Frq is acting through an intermediary protein toregulate the channel.

Frq negatively regulates nerve-terminal growthIn the absence of Frq, the number of 1s boutons was selectivelyincreased. Consistent with this, we previously found thatoverexpression of Frq1 or Frq2 selectively reduced the number of1s boutons (Romero-Pozuelo et al., 2007). The effects of Frq onnerve-terminal growth are not due to an interaction with PI4K,because PI4K-null mutants did not show the enhanced nerve-terminal growth seen in the Frq deletion, whereas overexpressionof Frq resulted in reduced nerve-terminal growth, whether PI4Kwas present or not. Interfering with or reducing the level of NCS-1 results in reduced Ca2+ levels and enhanced neurite outgrowth inPC12 cells and cultured primary neurons from Lymnaea stagnalis(Hui et al., 2006; Hui et al., 2007). The enhanced nerve-terminalgrowth we observed in Frq loss-of-function genotypes might resultfrom reduced Ca2+ entry.

The role of Ca2+ in nerve-terminal growth at the larval NMJ iscontroversial. Nerve-terminal growth is very susceptible to changesin Ca2+ level. For instance, the cacts2 mutant shows reduced nerve-terminal growth when grown at 25°C, but normal growth whengrown at 22°C (Xing et al., 2005; Macleod et al., 2006) (and thisstudy). Knockdown of Ca2+ channels by RNA interference yieldsreduced nerve-terminal growth (Rieckhof et al., 2003). Conversely,loss-of-function mutations in the 2 voltage-gated Ca2+-channelsubunit cause enhanced nerve-terminal growth (Dickman et al.,2008; Ly et al., 2008). However, nerve-terminal growth was notaltered in fuseless mutants, which probably have defects inintraterminal Ca2+ levels, because defects in 1-subunit traffickingwere reported (Long et al., 2008). Our data demonstrate that theeffects of Frq are highly dependent on Ca2+. When Ca2+ entry ismildly impaired using a cacts2 mutation (larvae grown at 22°C),overexpression of Frq no longer reduces nerve-terminal growth.However, when grown at 25°C, at which Ca2+ entry is more severelyimpaired, nerve-terminal growth is reduced when Frq is

overexpressed in a cacts2 background, probably because the cacts2

phenotype predominates. Clearly, the specific effects of geneticmanipulations that affect Ca2+ entry depend on the quantitativeeffects of these manipulations.

In summary, this is the first study to determine the physiologicalconsequences of loss of Frq protein in any organism. We found thatFrq1 and Frq2 regulate release probability by modulating Ca2+ entry,independently of an interaction with PI4K. We also found thatPI4K is dispensable for neurotransmitter release and nerve-terminal growth, and we provide evidence for a functionalinteraction between Frq and the 1-subunit of voltage-gated Ca2+

channels.

Materials and MethodsFly stocks and geneticsAll fly stocks were grown in uncrowded conditions at 25°C on cornmeal agar withdry yeast, unless otherwise indicated. Wandering third-instar larvae were used for allexperiments, unless otherwise stated. Canton-S (CS) and w1118 flies were used ascontrols.

Frq deletion lines (frqdel1 and frqdel3) were generated using site-specificrecombination (Thibault et al., 2004; Parks et al., 2004). A P-element insertion(P{XP}d06635 or P{XP}d04472) upstream of the transcription start site of the frq1gene and a piggyBac element insertion (PBac{WH}f06131) in the first intron of thefrq2 gene were used to create FLP/FRT-mediated chromosomal deletions. The twotransposons (d06635 and f06131 or d04472 and f06131) were placed in trans in aFLP recombinase background. Larvae were heat shocked at 32°C daily for 1 hourover 4 days. Females of w1118, P{XP}d06635/PBac{WH}f06131;P{ry+t7.2FLP};+or w1118, P{XP}d04472/PBac{WH}f06131;P{ry+t7.2FLP};+ genotypes were crossedto FM7h males. In the next generation, white-eyed male flies were isolated as putativerecombinants and maintained as stable lines over a FM7 balancer.

The GAL4/UAS system was used for rescue experiments and to overexpress aninterfering C-terminal peptide, full-length Frq1, or Frq2. Flies carrying UAS-DN frq,UAS-frq1 or UAS-frq2 were previously described by Romero-Pozuelo et al. (Romero-Pozuelo et al., 2007). Flies carrying the UAS-andorra-RNAi transgene on the thirdchromosome were obtained from the Vienna Drosophila RNAi Center (Dietzl et al.,2007). Flies carrying the UAS-andorra transgene were generated using an andorracDNA IP07934 clone, obtained from the Drosophila Genomics Resource Center(Bloomington, IN). The cDNA was subcloned from a pOT2 vector into a pUAStvector using EcoRI and XhoI restriction sites. We used D42-Gal4 and elav-Gal4 (Linand Goodman, 1994; Parkes et al., 1998) to drive the expression of UAS constructsselectively in motor neurons and throughout the nervous system, respectively.

fwd3/Df(3L)7C flies were previously described (Brill et al., 2000). elav-GAL4;+;Df(3L)7C/TM6,Tb females were mated with w–;UAS-frq1/TS;fwd3/TL orw–;UAS-frq1/CyO,GFP;fwd3/TM3,Sb males to generate Frq1 overexpressers in a fwd-null background [elav-GAL4;UAS-frq1/+; fwd3/Df(3L)7C].

l(1)L13HC129 and cacts2 flies have been previously described (Kawasaki et al., 2002;Brooks et al., 2003). To generate Frq2 overexpressers in a cacts2 mutant background,male +;UAS-frq2;D42-GAL4 flies were crossed to female cacts2 flies. Male larvaefrom the resulting progeny were selected for experiments (cacts2;UAS-frq2/+;D42-GAL4/+).

PCRGenomic DNA from candidate deletion lines and controls were screened for thepresence of the resulting hybrid element by PCR detection using P-element-specificsense primer, 5�-AATGATTCGCAGTGGAAGGCT-3�, and piggyBac element-specific antisense primer, 5�-GACGCATGATTATCTTTTACGTGAC-3�. PCRproducts were purified using an Invitrogen Purelink Purification kit and sequencedat The Centre for Applied Genomics (Toronto, Ontario).

QRT-PCRmRNA extraction, RT reaction and quantification were performed as describedpreviously (Romero-Pozuelo et al., 2007). Quantitative RT-PCR was done on an AbiPrism 7000 instrument (Applied Biosystems) using Taqman Universal PCR MasterMix (Applied Biosystems) following the manufacturer’s instructions. Probes andprimers were from TaqMan gene expression assay reagents (Applied Biosystems) ordesigned. The following TaqMan gene expression assays were used: Frq1(Dm01844112), Frq2 (Dm01799640). Specific upper 5�-TAAACGAAG-GAGCAGTGCAG-3� and lower 5�-GGAATTTGCCAAAGATGCG-3� primers wereused to amplify a 95 bp of andorra 3�UTR region. Primers for frq1 and frq2 aredescribed by Romero-Pozuelo et al. (Romero-Pozuelo et al., 2007). To correct forpossible differences in the efficiency of mRNA extraction or RT reaction, we designeda Taqman probe overlapping exon 1-2 of the housekeeping gene RpolII (encoding 140-kDa RNApolII subunit). In order to determine the efficiency of each Taqman gene-expression assay, we generated standard curves by serial dilution of cDNA; quanti-

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tative evaluations of target and housekeeping gene levels were obtained by measuringthreshold cycle numbers (Ct). Because the differences among efficiencies of eachTaqman gene-expression assay were <0.1, we used the relative quantification methodCts to quantify gene expression.

Western blotsSamples were resolved by SDS-PAGE and blots were incubated with a rabbitpolyclonal Caenorhabditis elegans anti-NCS-1 antibody (1:500) (Gomez et al., 2001)or a mouse monoclonal Drosophila anti-Syntaxin-1A antibody (1:1000; IowaHybridoma Bank) overnight at 4°C. The following day, the membrane was washed,incubated with the secondary antibody for 2 hours at room temperature, washed again,and immunoreactivity was then detected using Immobilon Western ChemiluminescentHRP substrate (Millipore, MA). Blots were imaged on a Kodak Image Station 2000R(Guelph, Ontario).

ImmunohistochemistrySelective immunostaining of nerve terminals and of synapses was carried out aspreviously described (Romero-Pozuelo et al., 2007). Dissected larvae were fixed inBouin’s solution for 5 minutes, washed in phosphate-buffered saline containing 0.2%Triton X-100 (PBT) and then incubated in the blocking solution [5% goat serum andBSA (1 g/50 ml) in PBT] for 30 minutes. The preparations were incubated overnightat 4°C with FITC-conjugated anti-horseradish peroxidase (HRP) antibody (1:100dilution; Jackson ImmunoResearch, USA) to visualize neurons and the mousemonoclonal NC82 antibody (1:10 dilution; Iowa Hybridoma Bank) to visualizesynapses. All primary antibodies were diluted in blocking solution. The followingday, the preparations were washed in PBT and then incubated with the secondaryantibody (1:400 dilution; Alexa-Fluor-594 goat anti-mouse) in PBT for 2 hours atroom temperature, followed by washing in PBT. Preparations were mounted in adrop of Permafluor (Immunon, Pittsburgh, PA) on a glass slide and viewed under aLeica TCS LS confocal laser-scanning microscope (Heidelberg, Germany) with 40�or 63� oil-immersion lenses.

ElectrophysiologyWhole-cell intracellular recordings and extracellular focal recordings were made fromthe ventral longitudinal muscle fibre 6 (abdominal segment 3) in HL6 saline (Macleodet al., 2002) as described previously (Romero-Pozuelo et al., 2007). Intracellularrecordings of the compound EJP showed no significant differences among w1118, CS,D42-GAL4, elav-GAL4, and all UAS lines (Romero-Pozuelo et al., 2007). Accordingly,w1118, CS, D42-GAL4 and elav-GAL4 were used as controls for this study. Forextracellular recordings, we identified 1b and 1s boutons, which are morphologicallyand physiologically distinct (Kurdyak et al., 1994), with Nomarski optics and recordedfrom single distal boutons at the ends of nerve terminals.

Electron microscopySpecimens were prepared and processed as described previously (Atwood et al., 1993).Serial sections were photographed, synapses were reconstructed and quantitative datafor number of active zones per synapse were obtained. To quantify the number ofdocked vesicles, we counted vesicles touching or within 20 nm of the electron-denseT-bar (active zone). Synaptic vesicles from two other pools were also counted: thosewithin 50 nm of the densely stained presynaptic membrane and those within 500 nm.

Ca2+ imagingMotor neurons were forward-filled with the Ca2+ indicator Oregon green BAPTA-1Dextran (OGB-1) or Fura-Dextran (Molecular Probes, Eugene, OR) through cut axonsusing the method described by Macleod et al. (Macleod et al., 2002). The relativelyfast Ca2+-binding and -unbinding kinetics of OGB-1 allowed us to measure Ca2+

signals in response to single pulses. A line-scanning method (4 milliseconds per scan)was used to follow Ca2+ responses to single pulses in a single bouton with hightemporal resolution. Fura-Dextran is a ratiometric indicator and was used to measureabsolute resting levels. Ratio images were created by dividing images obtained at340 nm by their corresponding 380 nm images. The detailed procedures for Ca2+

imaging and analysis are described in Macleod et al. (Macleod et al., 2004).

Interfering C-terminal peptidesSynthesis of Frq1 C-terminal peptide (DKNHDGKLTLEEFREGSKADPRIVQAL-SLGGG) and scrambled Frq1 C-terminal peptide (DIDGDGQVNGEEFRGT-LASLSKLHKGLKAPER) are described in Romero-Pozuelo et al. (Romero-Pozueloet al., 2007).

Larval-locomotion data acquisition and analysisForaging larvae of approximately 96 hours were used for locomotory tests. Imagingof larval locomotion was conducted using a Pixelink Firewire camera (PL-A642)connected to a PowerMac G4 computer. Movies were acquired using the Pixelinksoftware at ten frames per second. The camera settings allowed the capture of blackand white movies, depicting black larval silhouette against white background. Movieswere pre-processed in ImageJ to remove background noise using the ‘despeckle’option and any remaining holes within the larval silhouette was filled using the ‘fillholes’ option. A gentle tail touch with a fine brush was applied to induce linear

locomotory episodes. Feret length and area measurements were performed by usingthe morphology macro for ImageJ written by Gabriel Landini (School of Dentistry,University of Birmingham, England, UK). Distance travelled by the larva wasmeasured by tracking the tail x, y coordinate (the posterior x, y coordinate of the feretline).

This work was supported by grants from CIHR, Canada MOP-37774(H.L.A.), CIHR, Canada MGP-37773 and MOP-82827 (Milton P.Charlton), and MEC, Spain BFU2006-10180 and MYORES EuropeanNetwork CE: 511978 (A.F.). We are especially grateful to MiltonCharlton for valuable advice and use of laboratory equipment and space.We thank Julie Brill, Howard Lipshitz, Zhong-Ping Feng, Patrick Nef,Ernst Niggli and Marianne Hegström-Wojtowicz for helpful discussions,reagents and/or technical assistance. We also thank Maisam Makarem,Katrina Choe, Harmandeep Virk, Gavasker Sivaskandarajah and WardaIqbal for analysis of electron micrographs.

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