DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth
© Wu et al.; licensee BioMed Central Ltd. 2007
Received: 23 March 2007
Accepted: 15 August 2007
Published: 15 August 2007
The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance.
We identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants – there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth.
The F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.
The connectivity and functionality of a neural circuit depends on the structure of its constituent neurons' presynaptic fields. Different neurons make very different synaptic trees – a serotonergic neuron will synapse with thousands of neurons throughout the brain, while climbing fibers from the medulla may synapse with only a single Purkinje cell in the cerebellum. Individual neurons with the same identity can also have different sized synaptic arbors and this has functional consequences: during synaptic competition at the vertebrate neuromuscular junction (NMJ), motoneurons with larger arbors are at a disadvantage when competing against those with smaller arbors . Molecular mechanisms that control the size and structure of a neuron's presynaptic field are key regulators of the development, function, and plasticity of neural circuits.
In Drosophila, genetic studies have identified a number of signaling pathways that regulate the morphology of the presynaptic arbor made by motoneurons onto muscles (reviewed in ). One such pathway requires highwire – in highwire mutants there is a dramatic increase in the complexity of the synaptic terminal, with a large increase in the number of synaptic boutons and branches . This function for highwire is evolutionarily conserved: studies in worms, fish, and mammals all suggest that highwire homologs are required for normal synaptic development. In Caenorhabditis elegans, the homolog rpm-1 regulates the number, spacing, and morphology of presynaptic active zones [4, 5], mutations in the zebrafish homolog esrom disrupt retinotectal projections , while mice carrying large deletions that remove the murine homolog phr1 and adjacent genes disrupt NMJ morphology .
Highwire and its homologs are huge proteins on the order of 5,000 amino acids that share a series of domains that likely perform distinct functions. These include an amino-terminal Ran GTPase exchange factor domain that can inhibit adenylate cyclase , two Phr repeats of unknown function, a Myc-binding domain , and a carboxy-terminal RING domain that can function as an E3 ubiquitin ligase. Numerous studies demonstrate a central role for this ubiquitin ligase activity in the molecular mechanism of Highwire action. In both C. elegans and zebrafish, the isolated RING domain can promote ubiquitination in vitro [6, 10], and mutations that disrupt the RING finger abolish function [6, 11]. In addition, overexpression of ubiquitin hydrolases enhances the highwire phenotype and promotes synaptic terminal overgrowth at the Drosophila NMJ .
What are the key targets of Highwire regulation? As befits a large, multi-domain protein, Highwire and its homologs interact with many candidate targets, including the co-SMAD Medea, adenylate cyclases, members of the tuberous sclerosis (TSC) complex, and the oncogenic protein Myc [8, 9, 13, 14]. One particularly compelling candidate is the mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) Wallenda in Drosophila and its C. elegans homologue dual leucine zipper kinase (DLK) [10, 15]. In both worms and flies, levels of the Wallenda/DLK kinase are increased in the absence of highwire/rpm-1, the synaptic defects of highwire/rpm-1 mutants are suppressed by mutations in either Wallenda/DLK or their downstream MAP kinases, and increasing the levels of Wallenda/DLK is sufficient to phenocopy the synaptic defects of highwire/rpm-1 mutants. In C. elegans, DLK is required for some but not all rpm-1 phenotypes, indicating that rpm-1 may have other targets. In Drosophila, wallenda is necessary for all of the synaptic morphology defects in a highwire mutant, but it is not required for highwire-dependent regulation of synaptic function . Since the regulation of synaptic transmission by highwire requires a functional ubiquitin ligase domain , Highwire very likely targets at least one protein in addition to Wallenda.
RING finger E3 ubiquitin ligases can function to promote ubiquitination of target proteins on their own or as members of large E3 protein complexes . In C. elegans, RPM-1 binds to Cullin-1, Skp-1, and the F-box protein FSN-1, leading to the hypothesis that RPM-1 functions as part of an atypical SCF (Skp1-cullin-F box protein) E3 ubiquitin ligase complex . F-box proteins such as FSN-1 determine the substrate specificity of the E3 ligase complex . Mutations in C. elegans fsn-1 lead to many of the same phenotypes as rpm-1 mutants, consistent with the hypothesis that they function together to regulate synapse development. However, genetic and biochemical data indicate that the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) is the functionally relevant target of FSN-1, since levels of ALK increase in an fsn-1 mutant and the fsn-1 phenotype is suppressed by mutations in ALK . However, rpm-1 mutants are suppressed much more strongly by DLK than by ALK , so rpm-1 and fsn-1 may target different substrates. In fact, it is not known if FSN-1 biochemically or genetically interacts with DLK. This presents a paradox – two components of the same E3 ligase work together to regulate synaptic development, but apparently via different molecular pathways.
These findings in C. elegans raise a number of questions. First, is the binding between FSN-1 and RPM-1/Highwire conserved in Drosophila? Since the C. elegans genome encodes more than 300 F-box proteins and Drosophila encodes only 33, this is not a given [18, 19]. Second, does Fsn regulate synaptic terminal growth and synaptic function in Drosophila? Third, does Fsn collaborate with Highwire to regulate the levels of the Wallenda/DLK kinase and is this necessary for normal synaptic development? Fourth, does the regulation of other Highwire targets require collaboration with Fsn, or does Highwire function alone or with other F-box proteins? We identified the Drosophila homolog of FSN-1, DFsn, in a proteomic screen as a Highwire-binding protein. We have addressed the questions above by undertaking a genetic analysis of DFsn function in Drosophila. Our results demonstrate that DFsn collaborates with Highwire to restrain synaptic terminal growth by down-regulating the same target, the Wallenda/DLK kinase.
Drosophila Fsn is a Highwire-interacting protein
CG4643 is predicted to encode a 27 kDa protein containing an F-box domain, a WD-40 repeat, and a spla and ryanodine receptor (SPRY) domain. Its closest homolog in C. elegans is FSN-1, which is composed of the same functional domains and which binds to the RPM-1, the C. elegans homolog of Highwire. The two proteins share 41.9% homology, and define a small sub-family of F-box proteins with SPRY domains of which there is only a single member in worms, flies, mice (F-box protein 45 (GenBank:AAH26799)), and humans (hCG1734196 (GenBank:EAW53643)) with high homology across species (Figure 1b). Based on the sequence similarity of CG4643 and FSN-1, we have renamed CG4643 DFsn, for Drosophila Fsn.
The fact that TAP-HiwΔRING can pull down DFsn indicates that an intact RING domain is not required for the Highwire-DFsn interaction. To test whether wild-type Highwire also interacts with DFsn, we performed the following co-immunoprecipitation experiments. Since we have been unable to generate an antibody that recognizes endogenous DFsn, we generated transgenic flies that express His-Myc-tagged DFsn. As described below, the transgene encoding this is functional since it can rescue a DFsn mutant. We expressed His-Myc-tagged DFsn throughout the nervous system and performed immunoprecipitations from third instar larvae brains. Immunoprecipitations (IPs) and western blot analysis was performed with either affinity-purified polyclonal antibodies raised against Highwire  or a monoclonal antibody to Myc. As shown in Figure 1c, Myc-DFsn is present when endogenous Highwire is immunoprecipitated. Conversely, endogenous Highwire is detected in the IP fraction when Myc-DFsn is immunoprecipitated by the anti-Myc antibody. In addition, neither endogenous Highwire nor Myc-DFsn is detected when a control protein, Drosophila vesicular Glutamate transporter (DVGLUT), is immunoprecipitated from Myc-DFsn expressing larval brains (data not shown). These data demonstrate that endogenous Highwire physically interacts with DFsn. While eukaryotic genomes can express hundreds of F-box proteins and a similar number of RING domain ubiquitin ligases [18, 21], these data and the previous work of Liao et al.  demonstrate that the physical interaction between Highwire/RPM-1 and Fsn is evolutionarily conserved.
Since our efforts to generate an antibody against DFsn were unsuccessful, we generated a functional green fluorescent protein (GFP)-tagged DFsn transgene. When the transgene is expressed by a pan-neuronal Gal4 driver, GFP-DFsn localizes to the NMJ (Figure 1e,h). Double staining with markers for synaptic vesicles (Figure 1e–g) and periactive zones (Figure 1h–j) demonstrates that GFP-DFsn is not restricted to any of these compartments. GFP-DFsn is also detectable in the axon and neuronal cell body (data not shown), although the staining of GFP-DFsn is much stronger in synaptic boutons than in the preterminal axon (Figure 1e, arrow), suggesting a synaptic enrichment. GFP-Highwire encoded by a pan-neuronally expressed transgene has a very similar localization pattern . These data are consistent with the presence of a Highwire-DFsn complex at the NMJ.
DFsn is required to restrain synaptic terminal growth at the NMJ
Highwire regulates synaptic terminal growth presynaptically. If DFsn functions with Highwire to restrain this growth, then it should also be required in the presynaptic motoneuron. We generated transgenic flies capable of expressing DFsn in a tissue-specific manner (UAS-GFP-DFsn). We expressed GFP-DFsn in neurons with elav-Gal4, a pan-neural Gal4 driver, in the DFsn mutant background [P(f06595)/Df7872; UAS-GFP-DFsn/elav Gal4]. To assess rescue, we again quantified bouton number, synaptic branching, and synaptic span at the MN4-Ib NMJ onto muscle 4. All of these aspects of synaptic terminal overgrowth are rescued by the presynaptic expression of DFsn (Figure 3). The presence of the elav-Gal4 transgene without the UAS-GFP-DFsn transgene fails to rescue the DFsn mutant (p > 0.4 for number of boutons and branch points). Furthermore, synaptic terminal overgrowth of the DFsn mutant is also fully rescued when GFP-DFsn is expressed selectively in motoneurons (driven by D42; Additional file 1), indicating DFsn functions cell autonomously in the motoneuron to restrain synaptic terminal growth. Although we only quantified NMJs onto muscle 4, all type I NMJs, including those on muscle 6/7, show qualitatively similar rescue (Additional file 1). The transgene encoding the His-Myc-tagged DFsn also rescued the mutant phenotype (data not shown), so the fusion proteins used for immunoprecipitation and immunolocalization above are both functional. These data demonstrate that DFsn is responsible for the phenotype of the P(f06595) mutant, which we have renamed DFsn f06595 . These data demonstrate that DFsn is required in the presynaptic motoneuron to restrain synaptic terminal growth at the Drosophila NMJ.
Drosophila Fsn is required for normal synaptic function
DFsn genetically interacts with highwire to restrain synaptic terminal growth
DFsn down-regulates the levels of the Wallenda/DLK kinase to restrain synaptic terminal growth
How do Highwire and DFsn regulate synaptic terminal growth? In both fly and worm, Highwire/RPM-1 down-regulates the protein levels of the Wallenda/DLK kinase. Increased levels of Wallenda/DLK over-activate downstream MAP kinase cascades and lead to altered synapse development. Mutations in wallenda/DLK suppress the highwire/rpm-1 phenotypes. However, in worm, FSN-1 regulates the levels of the tyrosine kinase ALK and ALK mutants suppress the fsn-1 phenotype, leading to the suggestion that FSN-1 regulates synapse development by down-regulating ALK . If Highwire and DFsn collaborate as part of a ubiquitin ligase complex, then we predict they should both regulate the same target protein. We have found that ALK is postsynaptic at the Drosophila NMJ (AD, unpublished observation), so we have focused our attention on Wallenda.
Having demonstrated that DFsn regulates Wallenda levels, we tested whether Wallenda is required for synaptic terminal overgrowth in the DFsn mutant. As shown in Figure 6c–f, loss of wallenda suppresses the synaptic morphology defects of the DFsn mutant, restoring bouton number, branching number, and synaptic span to near wild-type levels. Therefore, Wallenda is necessary for synaptic terminal overgrowth in both the DFsn and highwire mutants. While wnd mutants suppress the morphological defects of the DFsn mutant, they do not suppress the electrophysiological phenotype. Quantal content is not significantly different between DFsn single mutants and DFsn, wnd double mutants, while both show impaired release compared to wild type (p < 0.001; Figure 4d). Therefore, DFsn, like Highwire, must regulate at least two pathways. In both mutants, the down-regulation of Wallenda is necessary to restrain synaptic terminal growth, while a genetically separable pathway is involved in promoting synaptic release.
Highwire and RPM-1 act as ubiquitin ligases to regulate synaptic development. Liao et al.  have proposed that, in C. elegans, RPM-1 participates in an atypical SCF ubiquitin ligase complex with the F-box protein FSN-1. Consistent with this hypothesis, RPM-1 binds to FSN-1 as well as to Skp-1 and Cullin-1, core components of SCF complexes. In addition, FSN-1 null mutants have very similar phenotypes to rpm-1 mutants at GABAergic synapses, but weaker phenotypes in DD motoneurons and sensory neurons. The difference in phenotypes suggests that RPM-1 interacts with other F-box proteins in addition to FSN-1, acts as a ubiquitin ligase without an F-box partner, or has ubiquitin-independent functions. The target of the RPM-1/FSN-1 complex in C. elegans is not clear. Biochemical and genetic data indicate that the receptor tyrosine kinase ALK is the functionally relevant target for FSN-1 , while the MAPKKK DLK is the functionally relevant target for RPM-1 . Our data in Drosophila support the model from worms that RPM-1 and FSN-1 form a functional ubiquitin ligase complex, but simplify the model by demonstrating that in Drosophila both components target the same substrate.
We demonstrate that Highwire binds the Drosophila homolog of FSN-1, DFsn. Therefore, the physical association of Highwire/RPM-1 and DFsn/FSN-1 is evolutionarily conserved. While eukaryotic genomes can encode hundreds of F-box proteins like DFsn, in worms, flies, mice, and humans there is only a single F-box protein that also contains an SPRY domain, and each is more closely related by sequence to each other than to other F-box proteins. Since the binding of DFsn/FSN-1 to Highwire/RPM-1 is conserved, we speculate that the mouse and human homologs of DFsn, F-box protein 45 and hCG1734196, will bind to and function with Phr  and PAM , the mouse and human homologs of Highwire, respectively. Indeed, expression analysis demonstrates that both the F-box protein 45 and Phr are expressed in a very similar pattern in the mouse brain .
Our results suggest that the interaction of Highwire with DFsn is required for Highwire activity. Loss-of-function mutants for highwire and DFsn have qualitatively and quantitatively similar phenotypes – both are required to restrain synaptic terminal growth and promote synaptic release. Both Highwire and DFsn are necessary to down-regulate the levels of the MAPKKK Wallenda/DLK, and wallenda mutants suppress the morphological but not physiological phenotypes of both highwire and DFsn. Finally, genetic data support the model that Highwire and DFsn function together during synaptic development – DFsn mutants enhance the phenotype of a highwire hypomorph but not of a highwire null. All of these data are consistent with the model that Highwire and DFsn act together to form a functional ubiquitin ligase complex. In our model, this ligase complex targets Wallenda/DLK to restrain synaptic terminal growth, and an unknown substrate to promote synaptic function. We speculate that the targeting of the Wallenda/DLK MAPKKK by the Highwire/DFsn complex will be conserved from worms to mammals. While Highwire and DFsn collaborate for synaptic development, the male sterility of DFsn but not highwire mutants suggests that DFsn has Highwire-independent functions in other developmental processes.
Here we demonstrate that the F-box protein DFsn binds to the RING-domain ubiquitin ligase Highwire and can localize to the Drosophila NMJ. DFsn is required to restrain synaptic growth and promote synaptic transmission. Genetic interactions between DFsn and highwire mutants indicate that DFsn and highwire function in the same pathway. Finally, DFsn, like Highwire, regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for both DFsn- and Highwire-dependent synaptic terminal overgrowth. These results support the model that Highwire and DFsn form a functional ubiquitin ligase complex that down-regulates the MAPKKK Wallenda/DLK to restrain synaptic terminal growth.
Transgenic flies carrying the UAS-NTAP-HiwΔRING or UAS-TAP transgene driven by BG380 Gal4 driver were expanded. Fresh adult flies were deep frozen and then vortexed. Fly heads were then collected by passing the flies through sieves that separate the heads from other body parts. The purification procedure was performed at 4°C. Fly heads were homogenized with homogenization buffer (50 mM Tris-HCl pH 7.5, 125 mM NaCl, 5% Glycerol, 0.7% NP40, 1.5 mM MgCl2, 25 mM NaF, 1 mM EDTA, 0.2 mM DTT, 1 mM Na3VO4, 0.05 mM MG1115, 1 mM PMSF, Protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Two sequential centrifuge steps (17,000 rpm for 20 minutes and 45,000 rpm for 40 minutes) were used to clean the homogenate before loading the first column. The following purification steps were performed according to the Séraphin lab TAP protocol . Final elutions from the second column were divided into two portions. The first portion was loaded to a SDS-PAGE gel (Criterion XT 4–12%) followed by Sypro Ruby staining. Individual bands were then excised, digested with trypsin, and subjected to LC-MS/MS. The second portion was precipitated, digested with trypsin and then subjected to LC-MS/MS. The searching program MASCOT (Matrix Science Inc., Boston, MA, USA) was used to identify the protein species. As a control, the TAP-only complex was analyzed by SDS-PAGE and no bands higher than 15 kDa were detected by Sypro Ruby staining.
Flies were maintained at 25°C on normal food. The following strains were used in this study: Canton S (wild type), P-f06595 (Exelixis Collection at Harvard Medical School), Df(2R)7872 (Bloomington Stock Centre), elav Gal4 (neuron specific) , BG380 Gal4 (neuron specific) , D42 Gal4 (motor neuron specific) , ELAV-GeneSwitch , hiwND 51and hiwND 9[3, 12], two wallenda alleles: wnd1 and wnd3 .
Generation of TAP-tagged full-length highwire transgenes UAS-NTAP-Hiw and UAS-NTAP-HiwΔRING was described previously  using the TAP vector . A UAS-TAP transgene was also generated for control purposes. A full-length cDNA clone (Clone ID: LD47425) for DFsn (CG4643) was obtained from Research Genetics (Huntsville, AL, USA). The entire coding region of DFsn was subcloned into pUAST-EGFP or pUAST-HM vector  to generate UAS-GFP-DFsn and UAS-His-Myc-DFsn transgenes. Transformant lines were generated using standard techniques.
Immunoprecipitations and western blots
For immunoprecipitations, about 100 larval brains for each sample were collected by manual dissection. Larval brains were then homogenized in 0.5 ml lysis buffer (Tris-HCl 50 mM pH 7.4; NaCl 150 mM; EDTA 1 mM; NaF 2 mM; NP-40 0.2%; NaDOC (Na deoxycholate) 0.2% and protease inhibitors). Clean lysate was then incubated with the rabbit α-Highwire antibody  (HIW2b, 1:80), the mouse α-Myc antibody (9E10, 1:100), or the rabbit α-DVGLUT antibody  (1:80) for 2 hours. Protein A beads (25 ul; Roche) were added to the lysate. After overnight incubation, the beads were washed four times with lysis buffer before elution. HIW2b (1:250) and the mouse α-Myc antibody (1:1,000) were then used to probe the endogenous Highwire and the neuronally expressed Myc-DFsn (driven by BG380 Gal4), respectively, in the IP complex on western blots.
Endogenous Wallenda was detected using the WndA1 antisera as described . For all other antibodies, larvae were fixed in Bouin's solution (a 1:5:15 ratio of acetic acid/formalin/picric acid) for 8 minutes. The rabbit α-DVGLUT antibody was described previously , and used at 1:5,000 dilution. The α-fasciclin II (FasII; 1D4) antibody developed by Corey Goodman (Renovis, San Francisco, CA, USA) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA, USA, and used at 1:5. To examine the localization of GFP-DFsn, GFP-DFsn was expressed in neuronal tissues by driving the UAS-GFP-DFsn transgene with ELAV-GeneSwitch in the presence of RU486. The mouse monoclonal α-GFP antibody (3E6; Invitrogen, Carlsbad, CA, USA) was then used at 1: 800 dilution to detect the GFP-DFsn protein.
Imaging and analysis
Larvae were imaged on a Nikon (Tokyo, Japan) C1 confocal microscope. All images were acquired in third instar wild-type, mutant, and rescued larvae that had been simultaneously stained. The gain was chosen as the maximum gain that did not saturate the signal for each sample. A complete Z-stack was acquired for each NMJ or ventral nerve cord and rendered as a maximum projection. All quantifications were done at muscle 4 in segments A2–A4. The muscle sizes for all genotypes were similar. The number of type I boutons and the number of branch points of type I boutons were counted following α-DVGLUT and α-Fas II staining. A synaptic branch was defined as an arborization containing at least 2 type I boutons. Synaptic span was calculated by dividing the length of type I synapses by the length of muscle. The length of synapses and muscle were measured by an ocular micrometer (Nikon). Statistical analysis was performed and graphs were generated in Origin 7.0 (Origin Lab, Northampton, MA, USA). Each sample is compared to other samples in the group using ANOVA, with n for each condition described in the figure legends. All histograms are shown as mean ± standard error of the mean.
Intracellular electrophysiological recordings were performed as described previously . Both spontaneous mEJPs and evoked EJPs were recorded in HL-3 saline containing 0.47 mM Ca2+ . At least 70 consecutive events were measured per cell using MiniAnal (Synaptosoft, Decatur, GA, USA) and averaged to determine the mean mEJP. Events with a slow rise time were rejected as artifacts from neighboring electrically coupled muscle cells. Evoked EJPs were recorded by stimulating the cut end of the segmental nerve with a 1 ms depolarizing pulse as described by Broadie . Quantal content was estimated by dividing the mean EJP by the mean mEJP without correcting for non-linear summation, which would only have increased the observed differences.
This work is supported by grants from the Keck Foundation and NIH (DA020812) to AD. RWD is supported by NIH NRSA predoctoral grant # F31 NS054467. We would like to thank the Alvin J Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St Louis, Missouri, for the use of the Proteomics Core. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant #P30 CA91842. We would like to thank Bloomington Stock Centre and Exelixis Collection at Harvard Medical School for fly stocks, and Developmental Studies Hybridoma Bank for antibodies. We thank Shuo Luo and Xiaolu Sun for making the DFsn transgenic flies. We also thank Cathy Collins and Brad Miller for helpful comments.
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