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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2010 Sep 15;107(40):17379–17384. doi: 10.1073/pnas.1001794107

WASP is activated by phosphatidylinositol-4,5-bisphosphate to restrict synapse growth in a pathway parallel to bone morphogenetic protein signaling

Thang Manh Khuong a,b,1,, Ron L P Habets a,b,1, Jan R Slabbaert a,b, Patrik Verstreken a,b,2
PMCID: PMC2951409  PMID: 20844206

Abstract

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is a membrane lipid involved in several signaling pathways. However, the role of this lipid in the regulation of synapse growth is ill-defined. Here we identify PI(4,5)P2 as a gatekeeper of neuromuscular junction (NMJ) size. We show that PI(4,5)P2 levels in neurons are critical in restricting synaptic growth by localizing and activating presynaptic Wiscott-Aldrich syndrome protein/WASP (WSP). This function of WSP is independent of bone morphogenetic protein (BMP) signaling but is dependent on Tweek, a neuronally expressed protein. Loss of PI(4,5)P2-mediated WSP activation results in increased formation of membrane-organizing extension spike protein (Moesin)-GFP patches that concentrate at sites of bouton growth. Based on pharmacological and genetic studies, Moesin patches mark polymerized actin accumulations and correlate well with NMJ size. We propose a model in which PI(4,5)P2- and WSP-mediated signaling at presynaptic termini controls actin-dependent synapse growth in a pathway at least in part in parallel to synaptic BMP signaling.

Keywords: Nervous Wreck, synapse formation, Tweek, Wishful Thinking, neuromuscular junction


Proper control of synapse development underlies neuronal connectivity and function (1). In the Drosophila larval stages, neuromuscular junctions (NMJs) grow rapidly to accommodate the more than 100-fold increase in body wall muscle area (2). Although growth factors and signaling pathways are implicated in NMJ growth control, local signaling that directly affects cytoskeletal elements probably plays a role also (35). However, how these different signaling systems cooperate and result in local changes that control NMJ morphology remains poorly understood.

Transsynaptic signaling, mediated by the bone morphogenetic protein (BMP) receptor Wishful Thinking (WIT), facilitates NMJ growth (611). Recently, the adaptor Nervous Wreck (NWK) was shown to inhibit WIT receptor activity (12). Interestingly, NWK also interacts with Wiscott-Aldrich syndrome protein (WSP), an actin-binding protein (13, 14). In vitro, NWK stimulates actin related proteins 2/3 (ARP2/3)-mediated actin polymerization in a WSP-dependent manner, and nwk and wsp mutants both show longer NMJs with more boutons than controls. Although these data suggest that NWK cooperates with WSP to link BMP signaling to actin dynamics at the synapse (13, 14), NMJ length in wsp;nwk double mutants is significantly increased compared with either null mutant alone (13). Hence, genetic data indicate NWK and WSP harbor separate functions to restrict NMJ growth.

Here we provide evidence that presynaptic WSP is localized autonomously in the cell by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], thereby restricting NMJ growth. Our data indicate that reduced PI(4,5)P2-mediated WSP activation correlates with increased membrane-organizing extension spike protein (Moesin)-GFP patch formation. Moesin-GFP concentrates at sites of bouton growth, and pharmacological studies using actin-polymerizing and -depolymerizing drugs indicate that Moesin patches correlate positively with NMJ size. We also implicate another protein, Tweek, that regulates synaptic PI(4,5)P2 levels and vesicle recycling (15) in both WSP- and NWK-dependent restriction of NMJ growth. Our data suggest that Tweek controls both an NWK- and a WSP/PI(4,5)P2-dependent pathway and that these pathways, at least in part, operate in parallel to restrict NMJ growth.

Results and Discussion

PI(4,5)P2 Restricts Synaptic Growth.

To explore a role for PI(4,5)P2 in synaptic development, we used a fusion of the phospholipase Cδ1 pleckstrin homology domain (PLCδ-PH) and EGFP (PLCδ1-PH-GFP) (15, 16). PLCδ1-PH binds PI(4,5)P2, thereby shielding it from endogenous binding partners resulting in dominant negative effects (1719). We tested the effect of PLCδ1-PH-GFP expression on PI(4,5)P2-availability at NMJ boutons using antibodies that label α-adaptin, an adaptor that binds PI(4,5)P2. We find significantly less α-adaptin labeling at Drosophila larval NMJ boutons than in controls, suggesting reduced PI(4,5)P2 availability (Fig S1).

Next we quantified NMJ morphology in third-instar larvae (Fig. 1 AC). Expression of PLCδ1-PH-GFP, but not PLCδ1-PHS39R-GFP that cannot bind PI(4,5)P2, shows NMJ overgrowth, including increased total branch length and satellite boutons (Table S1 and Fig. S2). Neuronal expression of an independent construct PLCδ1-PH-Cherry shows similar defects (Fig. 1C), and this effect is dosage sensitive as gauged by different neuronal Gal4 expression levels (C155-Gal4 < D42-Gal4 < neuronal synaptobrevin (nSyb)-Gal4) (Fig. 1C). Our data indicate that neuronal expression of PLCδ1-PH leads to NMJ overgrowth.

Fig. 1.

Fig. 1.

PI(4,5)P2 inhibits synaptic growth. (A and B) NMJs of animals neuronally expressing (A) PLCδ1-PHS39R-GFP (green; nSyb-Gal4 > PLCδ1-PHS39R-GFP) or (B) PLCδ1-PH-GFP (green; nSyb-Gal4 > PLCδ1-PH-GFP). NMJs are labeled with anti-HRP, marking neuronal membranes (red) and anti-DLG, a mostly postsynaptic marker (blue). (Scale bar in A: 50 μm.) (C) Quantification of NMJ branch length and synaptic GFP or Cherry fluorescence of PLCδ1-PH-GFP (green), PLCδ1-PH-Cherry (red), and PLCδ1-PHS39R-GFP (black) expressed using C155-Gal4, D42-Gal4, or nSyb-Gal4. Control: w1118. Fluorescence was normalized to GFP/Cherry expressed by nSyb-Gal4. (DI) mEJCs (D and E) and EJCs (F and G) from controls (nSyb-Gal4 > PLCδ1-PHS39R-GFP; D and F) and nSyb-Gal4 > PLCδ1-PH-GFP (E and G) and quantification of their amplitudes (H and I) and mEJC frequency (I). Error bars indicate SEM; n is indicated within the bars. (JL) FM 1–43 dye uptake in nSyb-Gal4 > PLCδ1-PHS39R-GFP (K) and nSyb-Gal4 > PLCδ1-PH-GFP (L) using 1-min stimulation with 90 mM KCl in 4 μM FM 1–43, and quantification of fluorescence following loading, and unloading (90 mM KCl for 5 min) (J). Error bars indicate SEM; **P < 0.01; n is indicated in the bars. (MR) NMJs labeled with anti-HRP in controls (nSyb/+) (M), in larvae expressing RNAi in neurons to PI4KinaseIIIα (N) or to PI4P5K (O), in tweek mutants rescued with a genomic tweek+ fragment (P), in tweek mutants (Q), and in animals expressing PLCδ1-PH-GFP (nSyb-Gal4 > PLCδ1-PH-GFP) that are heterozygous for synj1 (R). (S) Quantification of total branch length in these genotypes as well as in additional controls: RNAi transgene insertions to PI4KinaseIIIα and PI4P5K, as well as animals expressing a PI(3,4,5)P3 (GRP1-GFP)- or a PI(3)P (2XFYVE-GFP)-binding peptide and heterozygous synj animals (synj1/+). Error bars indicate SEM; **P < 0.01; ***P < 0.001; n is indicated in the bars. (Scale bar in M for MR: 50 μm.) ns, not significant.

Contrary to NMJ development, a role for PI(4,5)P2 in synaptic vesicle cycling has been established (20), and we therefore explored the effectiveness of the PLCδ1-PH-GFP probe as a dominant negative by measuring synaptic function. Although voltage clamp experiments indicate similar neurotransmitter release characteristics (excitatory junctional current [EJC] and miniature EJC [mEJC]) in animals that express PLCδ1-PH-GFP and in controls (Fig. 1 DI), synaptic vesicle cycling during intense stimulation is reduced (Fig. 1 JL) in animals that express PLCδ1-PH-GFP. These data suggest that expression of PLCδ1-PH-GFP results in synaptic vesicle cycling deficits as well as a concomitant overgrowth of the NMJ.

We further substantiate the hypothesis that PI(4,5)P2 limits NMJ growth by expressing RNAi to phosphatidylinositol-4-kinase IIIα (PI4KIIIα), which mediates the formation of phosphatidylinositol-4-monophosphate [PI(4)P], a precursor of PI(4,5)P2, or by knock down of phosphatidylinositol-4-phosphate-5-kinase (PI4P5K), an enzyme that produces PI(4,5)P2. Both tools result in reduced α-adaptin labeling (Fig. S1), and quantification of NMJ morphology in these genotypes indicates excessive NMJ growth (Fig. 1 MO and S and Table S1). Similarly, we also observe larger NMJs in tweek mutants that harbor reduced synaptic PI(4,5)P2 (15), and this effect is rescued by a wild-type copy of the tweek gene (Fig. 1 P, Q, and S and Table S1). Finally, we also counteracted the reduced PI(4,5)P2 availability in animals that express PLCδ1-PH-GFP by removing a copy of synaptojanin (synj). synj encodes a synaptic phosphoinositide phosphatase (2123), and mutants harbor increased PI(4,5)P2 levels (15, 21). Although heterozygous synj animals do not show NMJ morphology defects, removal of one copy of synj restores the excessive NMJ growth observed upon expression of PLCδ1-PH-GFP (Fig. 1 R and S), suggesting that reduced PI(4,5)P2 dephosphorylation in heterozygous synj animals that express PLCδ1-PH-GFP restores their NMJ overgrowth. Without excluding an effect on other cellular processes as well, our different genetic manipulations indicate that lower PI(4,5)P2 levels result in larger NMJs.

We surmise the increase in NMJ size is specific to reduced PI(4,5)P2 availability, because the interaction between this lipid and PLCδ1-PH-GFP is specific (24). Furthermore, presynaptic expression of general receptor for phosphoinositides-1 (GRP1)-PH-GFP, which interacts with phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3], and FYVE-domain-GFP (2XFYVE-GFP), which binds PI(3)P, does not cause NMJ overgrowth (Fig. 1S). In line with our observations, overexpression of phosphatidylinositol-3-kinase (PI3K), an enzyme that phosphorylates PI(4,5)P2, also results in NMJ overgrowth (25, 26).

Wiskott-Aldrich Syndrome Protein Is Recruited by PI(4,5)P2 to Mediate Synaptic Growth.

PI(4,5)P2 together with CDC42 binds and activates WSP, which is suggested to regulate actin branching (27), and wsp null mutants also show enlarged NMJs (13); Fig. 2 A and B and Table S2). To determine if PI(4,5)P2 restricts NMJ growth in a WSP-dependent manner, we neuronally expressed PLCδ1-PH-GFP in wsp null mutants and quantified NMJ morphology. As shown in Fig. 2 AC and G, the total NMJ length in wsp mutants expressing presynaptic PLCδ1-PH is not additive but is very similar to that in wsp mutants alone or in wsp mutants expressing the mutant PLCδ1-PHS39R (Table S2). These observations indicate that PI(4,5)P2-mediated control of NMJ morphology is WSP-dependent.

Fig. 2.

Fig. 2.

PI(4,5)P2 activates presynaptic WSP. (AC) NMJs labeled with anti-HRP in controls (w1118) (A), wsp mutants (wsp1/Df) (B), and wsp mutants that neuronally express PLCδ1-PH-GFP (C). (DF) Anti-HRP–labeled NMJs of wsp mutants that express full-length wild-type WSP (WSPfl) in neurons (D), of wsp mutants neuronally expressing the CDC42-binding mutant WSPH242D (GBD*) (E) and of wsp mutants neuronally expressing the PI(4,5)P2-binding mutant WSPΔBR (ΔBR) (F). (Scale bar in A for AF: 50 μm.) (G) Quantification of NMJ branch length in genotypes indicated (WSPH242D-ΔBR harbors both the CDC42- and the PI(4,5)P2-binding domain mutations). Error bars indicate SEM; **P < 0.01; n is indicated in the bars; ns, not significant.

We further substantiated this finding using a WSP mutant that lacks its PI(4,5)P2-binding domain (WSPΔBR), one that lacks the CDC42-binding domain (WSPH242D), and one that lacks both its PI(4,5)P2- and CDC42-binding domains (WSPΔBR,H242D) (28). Neuronal expression of wild-type WSP in a wsp-null mutant shows normal NMJ size (Fig. 2 D and G) (13), indicating an important presynaptic role for WSP. However, neuronal expression of WSPΔBR,H242D or WSPΔBR in wsp mutants does not rescue NMJ overgrowth. Conversely, expression of WSPH242D in wsp null mutants shows normal NMJ morphology (Fig. 2 DG). Hence, our data suggest that WSP-mediated restriction of NMJ growth is dependent on PI(4,5)P2 binding but not on CDC42 binding (Fig. 2 DG and Table S2). Similarly, bristle development on the fly notum is dependent on WSP, but CDC42 binding of WSP is dispensable in this context as well (28). Conceivably, alternative WSP-binding proteins may activate WSP. Indeed, several proteins containing Src homology 3 domains and small GTPases related to CDC42 have been shown to bind WSP, and these interactions may not be hampered by the WSP H242D mutation, allowing for WSP activation (29).

To determine if wsp mutants also show synaptic function defects, we measured vesicle cycling using FM 1–43 and neurotransmitter release using voltage clamp experiments. We find that wsp mutants take up less FM 1–43 upon stimulation (Fig. S3), and EJC amplitudes are not statistically different from controls. However, wsp mutants display smaller mini-amplitudes and an increased mini-frequency compared with controls. Hence, WSP may affect aspects of pre- and postsynaptic function (Fig. S3). Although our genetic analysis suggests a single overlapping pathway affects NMJ morphology in wsp mutants and in animals expressing PLCδ1-PH (Fig. 2 AC), the differences in synaptic function we observe between these genotypes (Fig. 1 DL and Fig. S3) suggests expression of PLCδ1-PH or wsp mutations affect aspects of NMJ function that are not entirely overlapping.

WSP is present at the pre- as well as postsynaptic sides of Drosophila NMJ (30, 31) (Fig. S4A). The presence of abundant postsynaptic WSP may hamper analyzing presynaptic protein localization. Therefore, to determine if PI(4,5)P2 is involved in localizing or stabilizing presynaptic WSP, we expressed WSP in the neurons of a wsp null mutant using nSyb-Gal4 and in addition neuronally expressed either the mutant PLCδ1-PHS39R-GFP or PLCδ1-PH-GFP. We then labeled these animals with anti-WSP antibodies. Although we observed ample presynaptic WSP upon expression of the mutant PLCδ1-PHS39R-GFP (Fig. S4C), we observed a marked reduction in presynaptic WSP when we shielded PI(4,5)P2 by expressing PLCδ1-PH-GFP (Fig. S4 AD and H). Concomitantly, in tweek mutants or in animals expressing PI4KIIIα RNAi or PLCδ1-PH-GFP, we found a marked reduction in presynaptic WSP, but postsynaptic WSP was not affected (Fig. S4 A and EH). Thus, presynaptic WSP is localized or stabilized by interacting with PI(4,5)P2.

Moesin-GFP Patches Localize to Sites of Bouton Growth and Are Increased When Wsp Signaling Is Reduced.

WSP has been implicated in regulating the structure of the actin cytoskeleton, proposed to play a role in NMJ development (32). To start understanding the involvement of WSP in the control of NMJ growth, we assessed the distribution of the actin-binding protein Moesin-GFP at NMJs in living animals. Moesin-GFP expressed at NMJs of controls forms patches and concentrates in sub-boutonic accumulations (Fig. 3A and Table S3). These patches probably are constituted of bundled filamentous actin, because feeding larvae jasplakinolide (15 μM), which promotes actin polymerization and stabilization (33, 34), results in an increase in the number of Moesin-GFP patches (Fig. 3 B, L, and M), whereas feeding larvae the actin-depolymerizing drug latrunculin (10 μM) (33, 34) leads to a significant reduction in the number of Moesin patches (Fig. 3 C, L, and M) (Materials and Methods). Interestingly, in animals that express PLCδ1-PH-Cherry or in wsp mutants, the number of Moesin-GFP patches per NMJ area is markedly increased (Fig. 3 D and G), suggesting these conditions result in increased actin polymerization or stabilization at NMJ boutons. Indeed, unlike controls, growing wsp mutants or larvae expressing PLCδ1-PH-Cherry in the presence of jasplakinolide does not further increase the number of Moesin-GFP patches (Fig. 3 E and H), whereas growing wsp mutants or animals expressing PLCδ1-PH-Cherry in the presence of latrunculin significantly reduces the number of boutonic Moesin-GFP patches (Fig. 3 F and I). Taken together, our data suggest that loss of WSP function or expression of PLCδ1-PH results in an accumulation of Moesin-GFP patches associated with polymerized or stabilized actin.

Fig. 3.

Fig. 3.

Moesin-GFP patches correlate with bouton growth. (AC) GFP expression in NMJ boutons of controls neuronally expressing Moesin-GFP (moe-GFP) (nSyb > moe-GFP) (A), in larvae grown on 15 μM jasplakinolide (JAS) (B), or in larvae grown on 10 μM latrunculin (LAT) (C). (DF) GFP expression at NMJ boutons of wsp mutants neuronally expressing Moesin-GFP (nSyb > moe-GFP, wsp1/Df) (D), in wsp-mutant larvae grown on 15 μM jasplakinolide (E), or wsp-mutant larvae grown on 10 μM latrunculin (F). (GI) GFP expression at NMJ boutons of larvae neuronally expressing both PLCδ1-PH-Cherry and Moesin-GFP (nSyb > moe-GFP, PLCδ1-PH-Cherry) (G) and in the same animals grown on 15 μM jasplakinolide (H) or on 10 μM latrunculin (I). (J and K) Higher magnification of GFP and RFP expression in NMJ boutons of nSyb > moe-GFP, PLCδ1-PH-Cherry larvae indicating accumulation of Moesin-GFP (but not PLCδ1-PH-Cherry) in satellite boutons (arrows). (L and M) NMJ branch length (L) and satellite bouton number (M) plotted against the number of Moesin-GFP patches per NMJ area in controls (nSyb > moe-GFP) (black), in larvae expressing PLCδ1-PH-Cherry (nSyb > moe-GFP, PLCδ1-PH-Cherry) (red), and in wsp mutants (nSyb > moe-GFP, wsp1/Df) (blue) not fed actin-modifying drugs (open squares), fed the actin-depolymerizing drug latrunculin (crosses), or fed the actin-polymerizing and actin-stabilizing drug jasplakinolide (solid squares).

Moesin has been suggested to bridge actin and membranes, and Moesin concentrates in membrane protrusions (35, 36). In boutons of larvae expressing PLCδ1-PH-Cherry or in boutons of wsp mutants, Moesin-GFP patches accrue in satellite boutons that are small protrusions from larger synaptic boutons (63.7 ± 4.9% of the satellite boutons in larvae expressing PLCδ1-PH-Cherry and 74.8 ± 2.5% of the satellite boutons in wsp mutants harbor Moesin-GFP patches) (Fig. 3J). Finally, we also induced the formation of new boutons using a previously established stimulation protocol (Fig. S5) (37). In this case, too, our data indicate an enrichment of Moesin-GFP in newly formed bouton protrusions and in the axonal segments that connect them (Fig. S5). Hence, Moesin-GFP appears to enrich at sites of NMJ growth.

To test this notion further, we investigated whether the presence of Moesin-GFP patches is associated with NMJ growth. Therefore we quantified NMJ length and satellite bouton number in control animals, in wsp mutants, and in animals that express PLCδ1-PH-Cherry and were fed jasplakinolide or latrunculin. As indicated in Fig. 3 L and M and Table S3, NMJ size and satellite bouton number correlate very well with the concentration of Moesin-GFP patches at NMJ boutons (R2 > 0.96), providing further evidence that accumulation of Moesin patches is connected with NMJ growth.

WSP activation of the ARP2/3 complex is involved in the formation of branched actin that accumulates in lamellipodia (38). Although branched actin meshes localize at the leading edge of cells (39), unbranched actin concentrates in filopodia that are thought to induce membrane protrusions, may induce spine growth in vitro, and conceivably also may induce NMJ growth. Consistent with this notion, in primary hippocampal neurons, expression of Cordon Blue, a protein that stimulates the formation of unbranched actin filaments, promotes new spine sprouting (40). Our data thus suggest that when WSP-mediated actin-branching activity is reduced or absent, actin in boutons may polymerize in unbranched filaments that support new bouton sprouting. This model is consistent with studies of loss of formin function in Drosophila that are diaphanous mutants. Formins promote linear, unbranched elongation of actin filament (41, 42), and NMJs are much smaller in diaphanous mutants than in wsp mutants (3). Thus, the data suggest that PI(4,5)P2-mediated activation of WSP in motor neuron boutons may control the balance between actin branching and bundling and constitutes an important regulator of NMJ growth.

Nervous Wreck and Wishful Thinking Act Genetically in a Pathway Parallel to PI(4,5)P2.

NWK is a presynaptic scaffold that binds WSP in vitro and can stimulate ARP2/3-mediated actin polymerization in a WSP-dependent manner, albeit inefficiently. Hence, it is thought that NWK cooperates with WSP to restrict NMJ growth (13, 14). To determine if PI(4,5)P2-mediated control of NMJ morphology is NWK-dependent, we measured NMJ size in nwk mutants that neuronally express PLCδ1-PH-GFP. Interestingly, unlike wsp mutants, nwk mutants that express PLCδ1-PH show significantly larger NMJs than nwk mutants or nwk mutants that express the mutant PLCδ1-PHS39R (Fig. 4 A–C and G and Table S4). These results suggest that PI(4,5)P2 regulates NMJ morphology independently from NWK, and this notion is supported further by the observation that NMJs are larger in nwk;wsp double-null mutants than in either of the single mutants (Fig. S6) (13). Hence, both reduced PI(4,5)P2 availability and the wsp mutation exacerbate the overgrowth phenotype in nwk mutants, consistent with a model in which WSP harbors NWK-independent functions to restrict NMJ growth.

Fig. 4.

Fig. 4.

WSP harbors NWK- and WIT-independent functions. (AC) NMJs labeled with anti-HRP in controls (w1118) (A), nwk mutants (B), and nwk mutants that neuronally express the PI(4,5)P2-binding peptide PLCδ1-PH-GFP (C). (DF) Anti-HRP–labeled NMJs of wit mutants (D), wsp mutants (E), and wit;wsp double mutants (F). (Scale bar in A for AF: 50 μm.) (G) Quantification of total NMJ branch length (muscle 6/7, segment A2) of genotypes shown in AF and in animals neuronally expressing a dominant active TKVact and wsp mutants expressing TKVact. Error bars indicate SEM; **P < 0.01; ***P < 0.001; n is indicated in the bars. (H) Quantification of anti-pMAD labeling intensity in the indicated genotypes. magenta: quantification of pMAD intensity in boutons; white: in the ventral nerve cord. Error bars indicate SEM; ***P < 0.001; n is indicated in the bars; ns, not significant. (IP) Maximum intensity projections of NMJs (IL) labeled with anti-pMAD (magenta) and anti-Dlg (green) and ventral nerve cords labeled with anti-pMAD (MP) in controls (I and M), nwk mutants (J and N), wsp mutants (K and O), and larva expressing PLCδ1-PH-GFP in neurons (L and P). (Scale bars in I for IL and in M for MP: 5 μm.)

NWK limits NMJ size by inhibiting WIT, a type II BMP receptor that facilitates NMJ growth, and wit;nwk double mutants show the same small NMJs observed in wit mutants, indicating that WIT acts downstream of NWK (6, 7, 12, 43). In contrast to wit;nwk double mutants, NMJs are significantly larger in wit;wsp double mutants than in wit mutants, suggesting that WSP acts, at least in part, in a pathway parallel to WIT to restrict NMJ growth (Fig. 4 DG and Table S4).

To substantiate these data, we also expressed dominant active Thickveins (TKVact), a type I BMP receptor that cooperates with WIT to control NMJ growth. Neuronal expression of TKVact in wild-type controls does not alter NMJ size (Fig. 4G), but expression of the active receptor in nwk mutants leads to NMJ overgrowth when compared with NMJs in nwk mutants. This observation is consistent with a role for NWK in restricting WIT signaling (12). In contrast, expression of TKVact in wsp mutants shows NMJs very similar to those observed in wsp mutants (Fig. 4G and Table S4), further suggesting WSP harbors functions beyond limiting BMP receptor signaling to restrict NMJ growth.

Finally, we indirectly tested WIT activation by analyzing phosphorylation of the downstream target Mothers Against Decapentaplegic (MAD), which translocates to the nucleus and induces transcription of target genes (6, 7, 44). However, although p-MAD levels at boutons and in cell bodies are markedly increased in nwk mutants, we did not observe such a strong increase in phosphorylated MAD (pMAD) labeling in wsp null mutants or in cells expressing PLCδ1-PH-GFP (Fig. 4 HP and Tables S1S10). Although we do not exclude overlapping roles for WSP and NWK in inhibiting WIT, our data suggest that WSP can restrict NMJ growth by acting in parallel to NWK and WIT.

Tweek Cooperates with WSP and NWK to Regulate NMJ Growth.

Tweek is a 560-kDa protein present in neurons, and mutants show reduced boutonic PI(4,5)P2 availability and less vesicle cycling (15). Furthermore, as shown in Fig. 1, NMJ synapses in tweek mutants are larger than in controls or in nwk or wsp mutants (Fig. 1S). To determine if the NMJ overgrowth caused by lower PI(4,5)P2 availability and reduced WSP function is tweek-dependent, we expressed PLCδ1-PH-GFP in tweek-null mutants and also created tweek;wsp double-null mutants. As shown in Fig. 5 AC and G, NMJ length and bouton number in tweek mutants expressing PLCδ1-PH-GFP is not increased compared with tweek mutants or with tweek mutants expressing PLCδ1-PHS39R-GFP. Furthermore, we did not observe a difference in NMJ length or bouton number in tweek;wsp double mutants as compared with tweek mutants (Fig. 5 B, D, and G and Tables S1S10). These data suggest WSP and PI(4,5)P2 act in a Tweek-dependent pathway.

Fig. 5.

Fig. 5.

Tweek acts in WSP and NWK signaling. (AC) NMJs labeled with anti-HRP in controls (A), tweek mutants that neuronally express PLCδ1-PHS39R-GFP (B), and tweek mutants that express PLCδ1- PH-GFP (C). (DF) NMJ labeling with anti-HRP in tweek;wsp double mutants (D), in tweek;nwk double mutants (E), and in nwk wsp double mutants neuronally expressing PLCδ1-PH-GFP (F). (G) Quantification of data shown in AF. Error bars indicate SEM; ***P < 0.001; n is indicated in the bars; ns, not significant. (H) Model of the genetic pathway encompassing Tweek, NWK, and WSP (Wasp).

To determine if Tweek and NWK cooperate to control NMJ morphology, we measured NMJ length and bouton number in tweek;nwk double mutants. Although NMJs are longer in both nwk and tweek mutants than in controls, we found no significant difference in NMJ size in tweek;nwk double mutants compared with tweek mutants, indicating that NWK-dependent restriction of NMJ growth is Tweek-dependent (Fig. 5 E and G; and Tables S1S10). Hence, our data suggest that Tweek controls the limitation of NMJ growth by both WSP and NWK. This conclusion is corroborated by the observation that NMJ size in tweek null mutants is very similar to that observed in wsp;nwk double mutants (Fig. S6 and Tables S1S10) (13). Finally we quantified NMJ morphology in wsp;nwk double mutants that express PLCδ1-PH-GFP and found no significant difference in bouton number or NMJ length when compared with tweek mutants (Fig. 5 F and G). Our data suggest Tweek orchestrates a genetic pathway that restricts NMJ growth by facilitating NWK and WSP signaling (Fig. 5H).

Conclusions

We have identified a role for PI(4,5)P2 in the control of WSP-mediated restriction of NMJ growth that is independent of BMP signaling and that correlates with altered presynaptic actin organization. Although PI(4,5)P2 signaling is involved in numerous cellular processes (45, 46), our manipulations of boutonic PI(4,5)P2 indicate an effect on NMJ growth that is associated with regulation of activity and/or localization of presynaptic WSP. Hence, the data suggest that WSP is a specific target that is critically sensitive to neuronal PI(4,5)P2 levels, regulating NMJ size by controlling actin dynamics at synaptic boutons.

Although NWK binds WSP in vitro, our data indicate that WSP and PI(4,5)P2 harbor an NWK-independent function, probably by affecting the actin cytoskeleton that controls synaptic morphology. Additional analyses also suggest that the gene tweek has a role in regulating both WSP- and NWK-dependent restriction of NMJ growth. Tweek recently was implicated in membrane cycling at the synapse and also plays a role in maintaining proper boutonic PI(4,5)P2 levels (15). Thus a model emerges in which parallel, antagonistic, Tweek-dependent pathways control NMJ size; NWK operates via WIT signaling and translocation of pMAD to the nucleus, whereas PI(4,5)P2 controls WSP activation. The function of WSP in mediating actin branching may antagonize the formation of dense actin filaments that appear to be correlated with additional NMJ growth. Because biochemical evidence suggests direct and functional interactions between NWK and WSP (13, 14), both pathways may meet at this level, allowing further fine-tuning of bouton sprouting.

Materials and Methods

Molecular Biology.

UAS-GFP-PH-GRP1, UAS-4C-PLCδ1-PH-Cherry, and UAS-WSPΔBR were obtained with standard cloning procedures using genomic DNA from tub::GFP-PH-GRP1 flies, from UAS-PLCδ1-PH-GFP flies, and from UAS-WSP flies as outlined in SI Materials and Methods.

Fly Stocks.

Genotypes of animals used are listed in Tables S1S10. w1118, nwk1, witA12, and witB11 (6, 13) and UAS-Moesin-GFP (35) were from the Bloomington Stock Center. wsp1, Df(3R)3450, UAS-Wspfl, UAS-WspH242D-ΔBR, and UAS-WspH242D were provided by Eyal Schejter (Weizmann Institute of Science, Rehovot, Israel) (47). Transgenic RNAi flies [PI4KinaseIIIα: (15993); PI4P5K-RNAi: (47027)] were obtained from the Vienna Drosophila RNAi Center (48). Tweek+ (HB69), tweek1, tweek2, yw; UAS-PLCδ1-PH-GFP, and yw; UAS-PLCδ1-PHS39R-GFP were from Hugo Bellen (Baylor College of Medicine, Houston) (15). UAS-PLCδ1-PH-GFP, UAS-PLCδ1-PHS39R-GFP, and UAS-PLCδ1-PH-Cherry were recombined with nSyb-Gal4 and D42-Gal4. We also recombined UAS-Moesin-GFP, UAS-PLCδ1-PH-Cherry, and nSyb-Gal4 and UAS-Moesin-GFP, wsp1, and nSyb-Gal4 as well as UAS-Moesin-GFP, Df(3R)3450, and nSyb-Gal4. Drivers and UAS constructs were homozygous.

Immunohistochemistry, Confocal Microscopy, and Quantification.

Immunohistochemistry was performed as described except that we used TBS (0.02 M Tris, 0.15 M NaC1, pH 7.4) for pMAD labeling (49). Antibodies used were rabbit anti-HRP (1:1,000)(Jackson ImmunoResearch), mouse anti-CSP 1:50, mouse anti-Dlg (1:250) (Developmental Hybridoma Studies Bank), rabbit anti-WSP (1:500) (47), rabbit anti-pMAD (1:100) (Cell Signaling); and rabbit anti-α-adaptin (1:500) (M. Gonzales-Gaitan, University of Geneva, Geneva). Secondary antibodies were Alexa 488-, Alexa 555-, or Alexa 647-conjugated Abs (Invitrogen) used at 1:200. GFP and Cherry fluorescence was imaged without additional Ab amplification. Fluorescence was visualized with a Bio-Rad Radiance confocal on a Nikon microscope with Plan Fluor 40× NA 1.30 oil lens or a Zeiss 510 META confocal with a 63× NA 1.4 oil lens. Antibody labeling intensity was quantified as the mean gray value of boutonic fluorescence corrected for muscle background; synaptic length and bouton number on M6/7 in segment A2 were quantified as described (13).

Feeding Experiments.

Embryos of various genotypes used were reared on grape juice plates containing 10 μM latrunculin (1 mM stock in DMSO) or 15 μM jasplakinolide (1 mM stock in DMSO) and developed on these plates until the third-instar stage. We also reared embryos on plates containing 1 μM, 5 μM, 10 μM, 15 μM, and 30 μM of either drug, but >10 μM latrunculin and >15 μM jasplakinolide yielded poor survival.

Electrophysiology and Live Imaging

Two-electrode voltage clamp experiments were performed using HL-3 with 0.5 mM or 2 mM CaCl2 as described (15) and as outlined in SI Materials and Methods.

FM 1–43 uptake and unloading experiments were performed as described in (50) using 1 min stimulation with 90 mM KCl in 4 μM FM 1–43 (Invitrogen) in HL-3 with 1.5 mM CaCl2. Dye and KCl then were washed using HL-3, and uptake was imaged. Dye unloading was achieved by incubating labeled samples for 5 min in HL-3 with 90 mM KCl and 1.5 mM CaCl2. Images were captured with a Nikon Digital Sight DS2Mb-Wc camera using a 40× Nikon water immersion lens (NA 0.8), and quantification of labeling intensity (boutonic mean gray value minus muscle background) was performed in NIS elements AR 3.0 (Nikon).

Third-instar larvae expressing Moesin-GFP were dissected in HL-3, and GFP was visualized using a Zeiss 510 META confocal microscope equipped with a 63×, NA1.0 water immersion lens and a 505-nm LP filter. The number of Moesin-GFP patches per NMJ area was counted manually.

Activity-dependent bouton formation (ghost boutons) was induced as described in ref. 37 and as indicated in SI Materials and Methods.

Statistical Analysis.

Statistical analysis was performed using ANOVA followed by a post hoc Tukey's test for pairwise comparisons between groups.

Supplementary Material

Supporting Information

Acknowledgments

We thank the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, Eyal Schejter (Weizmann Institue of Science, Rehovot, Israel [wsp, UAS-wsp and anti-WSP]), Hugo Bellen (Baylor College of Medicine, Houston [tweek and UAS-PLCd-PH-GFP]); Graeme Davis (University of California San Francisco [UAS-MoeGFP]), Marcos Gonzales-Gaitan (Universite de Geneve, Geneva [anti alpha-adaptin], Avital Rodal (Massachusetts Institute of Technology, Cambridge, MA [anti-WSP]), Bassem Hassan, Carlos Dotti, Claudia Bagni, Jiekun Yan, Katarzyna Miskiewicz, Sven Vilain, Sabine Kuenen, and other members of the Verstreken laboratory for reagents and/or comments. Support was provided by Research Fund Katholieke Universiteit Leuven, a Methusalem grant from the Flemish Government, the Francqui Foundation, Flanders Interuniversity Institute for Biotechnology, Research Foundation–Flanders (FWO Vlaanderen) Grant G.0747.09, a Marie Curie Excellence Grant MEXT-CT-2006-042267 (to P.V.), and an Instituut Voor Wetenschap en Technologie (IWT) fellowship (to J.R.S).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1001794107/-/DCSupplemental.

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