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• W2046141144 abstract "Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. Here we identify such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require wallenda. Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. Here we identify such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require wallenda. The growth of new synaptic contacts shapes the initial development and subsequent refinement of neural circuits. Genetic studies have identified highwire as a highly conserved presynaptic regulator of synapse growth and morphology. In Drosophila, loss of highwire causes dramatic synaptic overgrowth of the neuromuscular junction (NMJ) (Wan et al., 2000Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Highwire regulates synaptic growth in Drosophila.Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). In C. elegans, mutations in the homolog rpm-1 cause defects in the morphology, spacing, and number of presynaptic active zones (Schaefer et al., 2000Schaefer A.M. Hadwiger G.D. Nonet M.L. rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans.Neuron. 2000; 26: 345-356Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Zhen et al., 2000Zhen M. Huang X. Bamber B. Jin Y. Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain.Neuron. 2000; 26: 331-343Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Mutations in the zebrafish homolog esrom disrupt retinotectal projections (D'Souza et al., 2005D'Souza J. Hendricks M. Le Guyader S. Subburaju S. Grunewald B. Scholich K. Jesuthasan S. Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc).Development. 2005; 132: 247-256Crossref PubMed Scopus (76) Google Scholar), and deficiencies that remove the mouse homolog phr1 and neighboring genes result in altered NMJ development (Burgess et al., 2004Burgess R.W. Peterson K.A. Johnson M.J. Roix J.J. Welsh I.C. O'Brien T.P. Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice.Mol. Cell. Biol. 2004; 24: 1096-1105Crossref PubMed Scopus (91) Google Scholar). Highwire and its homologs are enormous proteins, ranging from 418 kDa in worms to 564 kDa in flies, and all share a series of domains that likely perform distinct biochemical activities. These include an N-terminal Ran GTP exchange factor-like domain that can inhibit adenylate cyclase (Pierre et al., 2004Pierre S.C. Hausler J. Birod K. Geisslinger G. Scholich K. PAM mediates sustained inhibition of cAMP signaling by sphingosine-1-phosphate.EMBO J. 2004; 23: 3031-3040Crossref PubMed Scopus (45) Google Scholar), two PHR repeats of unknown function, a domain shown to bind the myc oncogene (Guo et al., 1998Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Identification of a large Myc-binding protein that contains RCC1-like repeats.Proc. Natl. Acad. Sci. USA. 1998; 95: 9172-9177Crossref PubMed Scopus (111) Google Scholar), and a C-terminal RING finger that can function as an E3 ubiquitin ligase. The molecular function for each domain during synaptic growth is thus far unknown; however, the prevailing model for Highwire function focuses on its E3 ubiquitin ligase activity. The C. elegans homolog rpm-1 interacts with an SCF ubiquitin ligase complex (Liao et al., 2004Liao E.H. Hung W. Abrams B. Zhen M. An SCF-like ubiquitin ligase complex that controls presynaptic differentiation.Nature. 2004; 430: 345-350Crossref PubMed Scopus (174) Google Scholar), and in both C. elegans and zebrafish the isolated RING domain can promote ubiquitination in vitro (D'Souza et al., 2005D'Souza J. Hendricks M. Le Guyader S. Subburaju S. Grunewald B. Scholich K. Jesuthasan S. Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc).Development. 2005; 132: 247-256Crossref PubMed Scopus (76) Google Scholar, Nakata et al., 2005Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In Drosophila, mutation of the C-terminal RING finger abolishes highwire function (Wu et al., 2005Wu C. Wairkar Y.P. Collins C.A. DiAntonio A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.J. Neurosci. 2005; 25: 9557-9566Crossref PubMed Scopus (86) Google Scholar). In addition, overexpression of ubiquitin hydrolases, which antagonize ubiquitination by removing ubiquitin from targeted proteins, promotes synaptic overgrowth and enhances the highwire phenotype (DiAntonio et al., 2001DiAntonio A. Haghighi A.P. Portman S.L. Lee J.D. Amaranto A.M. Goodman C.S. Ubiquitination-dependent mechanisms regulate synaptic growth and function.Nature. 2001; 412: 449-452Crossref PubMed Scopus (321) Google Scholar). These results support a model in which Highwire restrains synaptic growth by downregulating levels of a signaling protein that promotes synaptic growth. What is the target of Highwire regulation? In Drosophila, Highwire has been proposed to regulate a TGF-β signaling pathway through an interaction with co-SMAD Medea (McCabe et al., 2004McCabe B.D. Hom S. Aberle H. Fetter R.D. Marques G. Haerry T.E. Wan H. O'Connor M.B. Goodman C.S. Haghighi A.P. Highwire regulates presynaptic BMP signaling essential for synaptic growth.Neuron. 2004; 41: 891-905Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). However, in C. elegans, the homolog rpm-1 regulates a p38 MAP kinase signaling pathway (Nakata et al., 2005Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) and may also regulate the tyrosine kinase ALK (Liao et al., 2004Liao E.H. Hung W. Abrams B. Zhen M. An SCF-like ubiquitin ligase complex that controls presynaptic differentiation.Nature. 2004; 430: 345-350Crossref PubMed Scopus (174) Google Scholar). Other candidate signaling pathways include cAMP metabolism in cultured neurons and rat spinal cord (Pierre et al., 2004Pierre S.C. Hausler J. Birod K. Geisslinger G. Scholich K. PAM mediates sustained inhibition of cAMP signaling by sphingosine-1-phosphate.EMBO J. 2004; 23: 3031-3040Crossref PubMed Scopus (45) Google Scholar), tuberous sclerosis complex (TSC) signaling in cultured neurons and the fly eye (D'Souza et al., 2005D'Souza J. Hendricks M. Le Guyader S. Subburaju S. Grunewald B. Scholich K. Jesuthasan S. Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc).Development. 2005; 132: 247-256Crossref PubMed Scopus (76) Google Scholar, Murthy et al., 2004Murthy V. Han S. Beauchamp R.L. Smith N. Haddad L.A. Ito N. Ramesh V. Pam and its ortholog highwire interact with and may negatively regulate the TSC1.TSC2 complex.J. Biol. Chem. 2004; 279: 1351-1358Crossref PubMed Scopus (67) Google Scholar), myc activity in cultured cells (Guo et al., 1998Guo Q. Xie J. Dang C.V. Liu E.T. Bishop J.M. Identification of a large Myc-binding protein that contains RCC1-like repeats.Proc. Natl. Acad. Sci. USA. 1998; 95: 9172-9177Crossref PubMed Scopus (111) Google Scholar), and pteridine biosynthesis in zebrafish (Le Guyader et al., 2005Le Guyader S. Maier J. Jesuthasan S. Esrom, an ortholog of PAM (protein associated with c-myc), regulates pteridine synthesis in the zebrafish.Dev. Biol. 2005; 277: 378-386Crossref PubMed Scopus (16) Google Scholar). While Highwire may interact with all of these varied pathways, it is not known which pathway or pathways are essential for controlling synaptic growth. Our unbiased approach to this question was to conduct a genetic screen in Drosophila for mutations that rescue the highwire synaptic overgrowth phenotype. The model for Highwire function predicts that the levels of a synaptogenic signaling protein would be increased in a highwire mutant. Therefore, genetically reducing the activity of such a protein should suppress the highwire synaptic overgrowth phenotype. In a genetic screen for such suppressors, we identified mutations in a single gene that we have named wallenda, which encodes a MAP kinase kinase kinase (MAPKKK) homologous to the vertebrate dual leucine zipper-bearing kinases DLK and LZK. Wallenda behaves as expected for a downstream target of Highwire. Both loss of highwire and overexpression of ubiquitin hydrolases cause dramatic increases in the level of Wallenda protein at synapses. wallenda is absolutely required for highwire-dependent synaptic overgrowth, and overexpression of wallenda is sufficient to cause synaptic overgrowth. Downstream of Wallenda, we find that the MAP kinase JNK and the transcription factor Fos are also essential for highwire synaptic overgrowth. Hence, Highwire controls synaptic growth by regulating a MAP kinase signal and its transcriptional output. The C. elegans Wallenda homolog DLK-1 is also required for the synaptic phenotypes of rpm-1 mutants (Nakata et al., 2005Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). This evolutionary conservation suggests that downregulation of a specific MAPKKK is a fundamental function for this large neuronal E3 ligase. At the Drosophila NMJ, mutations in highwire cause dramatic synaptic overgrowth (Wan et al., 2000Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Highwire regulates synaptic growth in Drosophila.Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Wu et al., 2005Wu C. Wairkar Y.P. Collins C.A. DiAntonio A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.J. Neurosci. 2005; 25: 9557-9566Crossref PubMed Scopus (86) Google Scholar). This implies that Highwire normally functions to downregulate a pathway that mediates synaptic growth; in its absence, this downstream pathway is overactive. A prediction from this model is that the synaptic overgrowth could be suppressed by decreasing the activity of this downstream pathway. To find potential components of this pathway, we conducted a genetic screen for mutations that can dominantly suppress the highwire phenotype. Such a screen should identify genes whose dosage is important for synaptic overgrowth. To conduct a large-scale screen, we took advantage of the observation that combining loss-of-function mutations in highwire with overexpression of the ubiquitin hydrolase fat facets (faf) is lethal (DiAntonio et al., 2001DiAntonio A. Haghighi A.P. Portman S.L. Lee J.D. Amaranto A.M. Goodman C.S. Ubiquitination-dependent mechanisms regulate synaptic growth and function.Nature. 2001; 412: 449-452Crossref PubMed Scopus (321) Google Scholar). This lethality could arise from the overactivity of a downstream target normally regulated by ubiquitination. If so, then mutating this hypothesized downstream target should rescue lethality. For the screen, flies carrying an upstream-activating sequence (UAS) promoter upstream of fat facets were chemically mutagenized and mated to highwire;elav-Gal4 virgins. Since highwire is on the X chromosome, all male offspring from this cross die because they are both mutant for highwire and overexpress fat facets in neurons. We screened over 20,000 mutagenized second and third chromosomes for suppression of lethality and found eighteen suppressors. Sixteen of these suppressors disrupt the fat facets gene, which was expected, since overexpression of nonfunctional fat facets is not lethal in combination with highwire (DiAntonio et al., 2001DiAntonio A. Haghighi A.P. Portman S.L. Lee J.D. Amaranto A.M. Goodman C.S. Ubiquitination-dependent mechanisms regulate synaptic growth and function.Nature. 2001; 412: 449-452Crossref PubMed Scopus (321) Google Scholar). The two suppressor mutations that did not map to fat facets were candidate suppressors of highwire. Genuine suppressors of highwire should not only suppress lethality, but should also suppress the cellular phenotype of synaptic overgrowth. Indeed, both mutations dominantly suppress the highwire synaptic morphology phenotype and, as transheterozygotes, completely suppress this phenotype (described in more detail below and in Figure 1). Both mutations map to a single locus on chromosome 3, and are phenocopied by deficiencies that delete this region (76B4–76D5). The mutations are therefore loss-of-function alleles of a single gene whose function is essential for the highwire synaptic overgrowth phenotype. We name this suppressor wallenda after the Flying Wallendas, an acrobat troupe famous for their world record stunts on the circus highwire. The ability of wallenda mutations to suppress the highwire synaptic morphology phenotype was characterized in detail (Figures 1A–1I) at the NMJ of muscle 4, which is formed from a single motoneuron, but similar results are observed at all glutamatergic type I NMJs (Figures 1A–1C, lower panels). Mutations in highwire cause dramatic synaptic overgrowth: a 4-fold increase in the number of boutons (Figure 1E) and synaptic branches (Figure 1F) and a 2-fold increase in total synaptic area (Figure 1G). The highwire mutant phenotype also causes a 66% reduction in bouton size (Figure 1H) and a 70% reduction in the average intensity (Figure 1I) and a 50% reduction in total intensity (data not shown) of staining for synaptic vesicle proteins. For our analysis, we use DVGLUT as a representative synaptic vesicle marker, but similar effects are observed for synaptotagmin (data not shown). All of the above parameters of the highwire mutant phenotype are suppressed by mutations in wallenda (Figures 1E–1I). Removing one copy of wallenda suppresses the highwire phenotype for each of these parameters by approximately 50% (p < 0.0001 for all parameters). Removing both copies of wallenda confers complete suppression. The highwire; wallenda double mutant (hiwND8;wnd1/wnd2) is not significantly different from wild-type (p > 0.9 for all parameters). Therefore, the wallenda gene is essential for the highwire synaptic overgrowth phenotype. The allele of highwire used in Figure 1 (hiwND8) contains a nonsense mutation in the N-terminal portion the protein (Wan et al., 2000Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Highwire regulates synaptic growth in Drosophila.Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). However, some full-length Highwire protein can be detected in this mutant due to read-through of the premature stop codon (Wu et al., 2005Wu C. Wairkar Y.P. Collins C.A. DiAntonio A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.J. Neurosci. 2005; 25: 9557-9566Crossref PubMed Scopus (86) Google Scholar). To test whether suppression requires residual highwire activity, we generated deletions that remove either the N-terminal or C-terminal halves of the gene (Wu et al., 2005Wu C. Wairkar Y.P. Collins C.A. DiAntonio A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.J. Neurosci. 2005; 25: 9557-9566Crossref PubMed Scopus (86) Google Scholar). Both hiwΔN and hiwΔC can be suppressed by wallenda similarly to hiwND8 (see Figure S1 in the Supplemental Data). These results rule out the possibility that wallenda functions as an upstream regulator of highwire, and instead support the model that wallenda functions downstream as an effector of the highwire synaptic overgrowth phenotype. In the absence of highwire, this downstream pathway is overactive. This pathway requires wallenda and is sensitive to its dose. It has been previously observed that mutations in the BMP/TGF-β signaling pathway can suppress highwire synaptic overgrowth, so we wished to compare this suppression to suppression by wallenda. Figure 1D shows suppression by mutations in the type II receptor wishful thinking (witA12/witB11). Additional alleles of wit that are genetic nulls are shown in Figure S2. Several differences are of note. First, mutation of wit and other TGF-β pathway components cause a very strong reduction in synaptic growth (Aberle et al., 2002Aberle H. Haghighi A.P. Fetter R.D. McCabe B.D. Magalhaes T.R. Goodman C.S. wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila.Neuron. 2002; 33: 545-558Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, Marques et al., 2002Marques G. Bao H. Haerry T.E. Shimell M.J. Duchek P. Zhang B. O'Connor M.B. The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function.Neuron. 2002; 33: 529-543Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) in genetic backgrounds that are either wild-type or mutant for highwire (McCabe et al., 2004McCabe B.D. Hom S. Aberle H. Fetter R.D. Marques G. Haerry T.E. Wan H. O'Connor M.B. Goodman C.S. Haghighi A.P. Highwire regulates presynaptic BMP signaling essential for synaptic growth.Neuron. 2004; 41: 891-905Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). When highwire is wild-type, mutation of wit causes a dramatic reduction in bouton number (64%), branching (80%), and synaptic area (64%). In contrast, wallenda mutants show no reduction in synaptic growth in an otherwise wild-type background (p > 0.9 compared to wild-type for all parameters). Second, TGF-β mutants do not suppress the reduced bouton size or synaptic vesicle protein intensity phenotypes of highwire nearly as well as they suppress the bouton number phenotype of highwire (McCabe et al., 2004McCabe B.D. Hom S. Aberle H. Fetter R.D. Marques G. Haerry T.E. Wan H. O'Connor M.B. Goodman C.S. Haghighi A.P. Highwire regulates presynaptic BMP signaling essential for synaptic growth.Neuron. 2004; 41: 891-905Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar; Figures 1D, 1H, and 1I, and Figure S2). In contrast, wallenda mutations suppress all parameters of the highwire morphology phenotype completely. These differences suggest that wallenda and TGF-β mutants may suppress highwire through different mechanisms. Consistent with this, no dominant genetic interactions were observed between wallenda and mutations in wit or the co-SMAD medea. The genetic interactions between loss-of-function mutations in the ubiquitin ligase highwire and gain-of-function mutations in the ubiquitin hydrolase fat facets suggest that both mutations may influence the same signaling pathway (DiAntonio et al., 2001DiAntonio A. Haghighi A.P. Portman S.L. Lee J.D. Amaranto A.M. Goodman C.S. Ubiquitination-dependent mechanisms regulate synaptic growth and function.Nature. 2001; 412: 449-452Crossref PubMed Scopus (321) Google Scholar). To test this hypothesis we assessed whether wallenda can suppress fat facets-induced synaptic overgrowth. Neuronal overexpression of fat facets, like loss-of-function highwire, leads to an increased number of synaptic boutons and branches and an increased synaptic span (Figure 1J, left panels). wallenda mutants suppress this phenotype (Figure 1K, p < 0.0005). Therefore, wallenda is required for the overgrowth caused by both overexpression of a ubiquitin hydrolase and loss of a ubiquitin ligase. wallenda thus behaves genetically like a candidate substrate for ubiquitination that could mediate synaptic overgrowth. highwire mutants have defects in synaptic function as well as synaptic morphology. Since wallenda mutants can suppress the morphological defects of highwire, we tested whether wallenda mutants can also suppress the synaptic transmission defects of highwire. highwire mutants show both reduced quantal size (response to a single vesicle) and reduced quantal content (number of vesicles released by the nerve) (Figure 2, and see Wan et al., 2000Wan H.I. DiAntonio A. Fetter R.D. Bergstrom K. Strauss R. Goodman C.S. Highwire regulates synaptic growth in Drosophila.Neuron. 2000; 26: 313-329Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, Wu et al., 2005Wu C. Wairkar Y.P. Collins C.A. DiAntonio A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.J. Neurosci. 2005; 25: 9557-9566Crossref PubMed Scopus (86) Google Scholar). wallenda mutants alone have normal synaptic function, allowing us to ask whether they could confer suppression to the highwire mutant phenotype. wallenda mutations do suppress the highwire defect in quantal size: both the amplitude and frequency of spontaneous miniature events in the highwire; wallenda double mutant are similar to wild-type (p > 0.9). In contrast, the evoked potentials of the highwire; wallenda double mutant are only modestly increased, and this is due entirely to the increased quantal size. The quantal content, calculated as the excitatory junction potential (EJP) amplitude divided by the miniature EJP (mEJP) amplitude, remains the same between highwire and highwire; wallenda (p > 0.9). Therefore, wallenda does not suppress the primary defect in highwire synaptic function, the reduced number of vesicles released by the nerve. While the quantal size phenotype of highwire may be secondary to the synaptic morphology phenotype, the defect in quantal content cannot be secondary to morphology. Therefore, highwire regulates synaptic physiology through a distinct molecular pathway that does not require wallenda. The genetic results described above suggest that wallenda is a downstream target of highwire that functions to promote synaptic growth. Using meiotic recombination and deficiency mapping, wallenda was mapped to 76B4–76D5 on the third chromosome. This location was refined by male recombination mapping (Chen et al., 1998Chen B. Chu T. Harms E. Gergen J.P. Strickland S. Mapping of Drosophila mutations using site-specific male recombination.Genetics. 1998; 149: 157-163PubMed Google Scholar) to a 20 kb region at 76B9. Four annotated genes reside within this region, including the predicted kinase CG8789. Sequencing of both wallenda alleles revealed mutations in CG8789: wnd1 has a mutation in a conserved residue in the kinase domain, and wnd2 has a mutation in the stop codon that adds an extra 104 amino acid tail to the protein (Figure 3). We serendipitously found a third allele of wallenda, wnd3, in a highwire mutant line (hiwPF253) that showed heterogeneity in the synaptic overgrowth phenotype. We mapped the source of this heterogeneity to a suppressor mutation at the wallenda locus. This mutation of spontaneous origin behaves exactly like a new loss-of-function mutation in wallenda: wnd3 can fully suppress the highwire synaptic overgrowth phenotype (Figure S1). Sequencing revealed that wnd3 contains a roo transposon insertion in the middle of the CG8789. CG8789 belongs to the family of mixed lineage kinases that function as MAPKKKs for JNK and p38 signaling pathways. Its closest vertebrate homologs are the dual leucine zipper-bearing kinases DLK and LZK (Figure 3) (Holzman et al., 1994Holzman L.B. Merritt S.E. Fan G. Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases.J. Biol. Chem. 1994; 269: 30808-30817Abstract Full Text PDF PubMed Google Scholar, Sakuma et al., 1997Sakuma H. Ikeda A. Oka S. Kozutsumi Y. Zanetta J.P. Kawasaki T. Molecular cloning and functional expression of a cDNA encoding a new member of mixed lineage protein kinase from human brain.J. Biol. Chem. 1997; 272: 28622-28629Crossref PubMed Scopus (69) Google Scholar). Its similarity to these vertebrate kinases (63% identity in the kinase domain) is much greater than to its closest Drosophila relative, the mixed lineage kinase slipper (which shares only 29% identity in the kinase domain). Vertebrate DLK is expressed in neurons and has been observed to localize to synapses (Hirai et al., 2005Hirai S. Kawaguchi A. Suenaga J. Ono M. Cui de F. Ohno S. Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo.Gene Expr. Patterns. 2005; 5: 517-523Crossref PubMed Scopus (39) Google Scholar, Mata et al., 1996Mata M. Merritt S.E. Fan G. Yu G.G. Holzman L.B. Characterization of dual leucine zipper-bearing kinase, a mixed lineage kinase present in synaptic terminals whose phosphorylation state is regulated by membrane depolarization via calcineurin.J. Biol. Chem. 1996; 271: 16888-16896Crossref PubMed Scopus (59) Google Scholar). The C. elegans homolog DLK-1 was identified as a suppressor of the highwire homolog rpm-1 (Nakata et al., 2005Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Hence, the regulation of this kinase is conserved between C. elegans and Drosophila. If wallenda functions downstream of highwire, it should be expressed in neurons where highwire function is required. In situ hybridization experiments in embryos detect wallenda transcript in the CNS at stage 13 and later, stages when axon guidance and synapse development take place (data not shown). To determine where Wallenda protein localizes, we raised polyclonal antisera in rabbits (WndA1) to a peptide near the C terminus (denoted in Figure 3). In embryos stage 13 and older, the α-Wallenda antibodies stain the neurite- and synapse-rich neuropil in the CNS. wnd3 mutants show no neuropil staining, demonstrating the specificity of the antibodies (Figures 4A and 4C). If wallenda is directly downstream of highwire then Highwire should regulate Wallenda protein levels. In embryos there is no significant difference in α-Wallenda staining between wild-type and highwire mutants (Figures 4A and 4C). In contrast, loss of highwire dramatically alters Wallenda staining in ventral nerve cords of third-instar larvae (Figures 4B and 4C). In wild-type larvae Wallenda is barely detectable in the neuropil, although there is slightly more (16%) staining than in the wnd3 mutant (p < 0.01). In highwire mutant larvae, Wallenda staining in the neuropil is dramatically increased. In addition, immunoblots of total protein extracts from dissected larval nerve cords demonstrate that mutation of highwire causes an increase in the total levels of Wallenda protein (2.5-fold, p < 0.01, Figures 4D and 4E). The difference in Wallenda levels between embryos and larvae, and the differential effect of highwire mutations during these two stages, indicates that Highwire functions to control Wallenda primarily in larvae. This is consistent with the finding that Highwire functions throughout larval development to control synaptic growth (Wu et al., 2005Wu C. Wairkar Y.P. Collins C.A. DiAntonio A. Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.J. Neurosci. 2005; 25: 9557-9566Crossref PubMed Scopus (86) Google Scholar). Interestingly, this is the developmental period when the NMJ grows most dramatically (Schuster et al., 1996Schuster C.M. Davis G.W. Fetter R.D. Goodman C.S. Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth.Neuron. 1996; 17: 641-654Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). The increase in Wallenda" @default.
• W2046141144 created "2016-06-24" @default.
• W2046141144 creator A5027775268 @default.
• W2046141144 creator A5038450497 @default.
• W2046141144 creator A5049219339 @default.
• W2046141144 creator A5088178040 @default.
• W2046141144 date "2006-07-01" @default.
• W2046141144 modified "2023-10-16" @default.
• W2046141144 title "Highwire Restrains Synaptic Growth by Attenuating a MAP Kinase Signal" @default.
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