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• W2010901230 abstract "In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion. In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion. Axon guidance cues can act as attractants or repellents and lay out a specific grid for axons and neurons to navigate in the developing nervous system (1Tessier-Lavigne M. Goodman C.S. The molecular biology of axon guidance.Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2688) Google Scholar, 2Dodd J. Jessell T.M. 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The mammalian receptors of netrins are deleted in colorectal cancer (DCC) 2The abbreviations used are: DCCdeleted in colorectal cancerDSCAMDown syndrome cell adhesion moleculeUNC5uncoordinated-5EGLexternal granule layerPCLPurkinje cell layerIGLinternal granule layerFAKfocal adhesion kinasePAK1p21-activated kinase 1CASCrk-associated substrateGEFguanine nucleotide exchange factor. (13Keino-Masu K. Masu M. Hinck L. Leonardo E.D. Chan S.S. Culotti J.G. Tessier-Lavigne M. Deleted in Colorectal Cancer (DCC) encodes a netrin receptor.Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 14Fearon E.R. Cho K.R. Nigro J.M. Kern S.E. Simons J.W. Ruppert J.M. Hamilton S.R. Preisinger A.C. Thomas G. Kinzler K.W. Identification of a chromosome 18q gene that is altered in colorectal cancers.Science. 1990; 247: 49-56Crossref PubMed Scopus (1535) Google Scholar, 15Cooper H.M. Armes P. Britto J. Gad J. Wilks A.F. 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Mouse Neogenin, a DCC-like molecule, has four splice variants and is expressed widely in the adult mouse and during embryogenesis.Oncogene. 1997; 15: 691-700Crossref PubMed Scopus (78) Google Scholar, 18Meyerhardt J.A. Look A.T. Bigner S.H. Fearon E.R. Identification and characterization of neogenin, a DCC-related gene.Oncogene. 1997; 14: 1129-1136Crossref PubMed Scopus (76) Google Scholar), DSCAM (19Andrews G.L. Tanglao S. Farmer W.T. Morin S. Brotman S. Berberoglu M.A. Price H. Fernandez G.C. Mastick G.S. Charron F. Kidd T. Dscam guides embryonic axons by Netrin-dependent and -independent functions.Development. 2008; 135: 3839-3848Crossref PubMed Scopus (90) Google Scholar, 20Ly A. Nikolaev A. Suresh G. Zheng Y. Tessier-Lavigne M. Stein E. DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.Cell. 2008; 133: 1241-1254Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar), and UNC5 (22Ackerman S.L. Kozak L.P. Przyborski S.A. Rund L.A. Boyer B.B. Knowles B.B. The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein.Nature. 1997; 386: 838-842Crossref PubMed Scopus (317) Google Scholar, 23Leonardo E.D. Hinck L. Masu M. Keino-Masu K. Ackerman S.L. Tessier-Lavigne M. Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors.Nature. 1997; 386: 833-838Crossref PubMed Scopus (426) Google Scholar, 24Przyborski S.A. Knowles B.B. Ackerman S.L. Embryonic phenotype of Unc5h3 mutant mice suggests chemorepulsion during the formation of the rostral cerebellar boundary.Development. 1998; 125: 41-50Crossref PubMed Google Scholar, 25Engelkamp D. Cloning of three mouse Unc5 genes and their expression patterns at mid-gestation.Mech. Dev. 2002; 118: 191-197Crossref PubMed Scopus (80) Google Scholar). While DCC can mediate netrin-1 induced axon attraction, the binding of DCC and UNC5 cytoplasmic domains is sufficient to convert netrin-1 induced DCC attraction into repulsion (26Hong K. Hinck L. Nishiyama M. Poo M.M. Tessier-Lavigne M. Stein E. A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion.Cell. 1999; 97: 927-941Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 27Killeen M. Tong J. Krizus A. Steven R. Scott I. Pawson T. Culotti J. UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but its UNC-40-independent functions also require a region between the ZU-5 and death domains.Dev. Biol. 2002; 251: 348-366Crossref PubMed Scopus (49) Google Scholar, 28Keleman K. Dickson B.J. Short- and long-range repulsion by the Drosophila Unc5 netrin receptor.Neuron. 2001; 32: 605-617Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), suggesting that the coordination of intracellular domains of these receptors plays a crucial role in netrin-1 attractive or repulsive signaling. deleted in colorectal cancer Down syndrome cell adhesion molecule uncoordinated-5 external granule layer Purkinje cell layer internal granule layer focal adhesion kinase p21-activated kinase 1 Crk-associated substrate guanine nucleotide exchange factor. Drosophila Dscam is a contact-dependent homophilic cell repulsive molecule with thousands of alternatively spliced isoforms involved in axon guidance and targeting, segregation of axon branches and dendritic patterning (29Chen B.E. Kondo M. Garnier A. Watson F.L. Püettmann-Holgado R. Lamar D.R. Schmucker D. The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila.Cell. 2006; 125: 607-620Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 30Hummel T. Vasconcelos M.L. Clemens J.C. Fishilevich Y. Vosshall L.B. Zipursky S.L. Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam.Neuron. 2003; 37: 221-231Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 31Schmucker D. Clemens J.C. Shu H. Worby C.A. Xiao J. Muda M. Dixon J.E. Zipursky S.L. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.Cell. 2000; 101: 671-684Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 32Wang J. Zugates C.T. Liang I.H. Lee C.H. Lee T. Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons.Neuron. 2002; 33: 559-571Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 33Zhu H. Hummel T. Clemens J.C. Berdnik D. Zipursky S.L. Luo L. Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe.Nat. Neurosci. 2006; 9: 349-355Crossref PubMed Scopus (136) Google Scholar, 34Zhan X.L. Clemens J.C. Neves G. Hattori D. Flanagan J.J. Hummel T. Vasconcelos M.L. Chess A. Zipursky S.L. Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies.Neuron. 2004; 43: 673-686Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). While the vertebrate DSCAM gene does not encode multiple isoforms, it shares functional similarities to fly Dscam. In DSCAM deficient mice, retinal ganglion cells have defects in neuronal spacing and dendritic arborization patterns exhibiting neuronal self-avoidance phenotypes (35Fuerst P.G. Bruce F. Tian M. Wei W. Elstrott J. Feller M.B. Erskine L. Singer J.H. Burgess R.W. DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina.Neuron. 2009; 64: 484-497Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 36Fuerst P.G. Koizumi A. Masland R.H. Burgess R.W. Neurite arborization and mosaic spacing in the mouse retina require DSCAM.Nature. 2008; 451: 470-474Crossref PubMed Scopus (229) Google Scholar). In contrast, studies in the chicken retina have shown that DSCAM plays a role in synapse formation by promoting the targeting of retinal ganglion cell dendrites and bipolar cell axons to the same layer (37Yamagata M. Sanes J.R. Dscam and Sidekick proteins direct lamina-specific synaptic connections in vertebrate retina.Nature. 2008; 451: 465-469Crossref PubMed Scopus (316) Google Scholar). As a conserved netrin receptor in flies and vertebrates, DSCAM collaborates with DCC involved in netrin-1 attraction (19Andrews G.L. Tanglao S. Farmer W.T. Morin S. Brotman S. Berberoglu M.A. Price H. Fernandez G.C. Mastick G.S. Charron F. Kidd T. Dscam guides embryonic axons by Netrin-dependent and -independent functions.Development. 2008; 135: 3839-3848Crossref PubMed Scopus (90) Google Scholar, 20Ly A. Nikolaev A. Suresh G. Zheng Y. Tessier-Lavigne M. Stein E. DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.Cell. 2008; 133: 1241-1254Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar). These results suggest that DSCAM may function as a bifunctional guidance receptor involved in either attractive or repulsive signaling pathways. We report here that DSCAM functions as a repulsive receptor associating with UNC5C to mediate netrin-1-induced axon growth cone collapse, and FAK, Fyn, and PAK1 are involved in coordinating netrin-1/DSCAM and netrin-1/UNC5C repulsive signaling. These studies not only reveal the role of DSCAM in netrin-mediated repulsion, but help us better understand the complexity of netrin signaling in the developing nervous system. Plasmids encoding the full-length human DSCAM-Flag, DSCAMΔN, DSCAMΔC, the full-length human UNC5C-HA and UNC5C truncation mutants (ΔIg1, ΔIg2, ΔIgs, ΔTSP) have been described previously (21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar, 38Kruger R.P. Lee J. Li W. Guan K.L. Mapping netrin receptor binding reveals domains of Unc5 regulating its tyrosine phosphorylation.J. Neurosci. 2004; 24: 10826-10834Crossref PubMed Scopus (58) Google Scholar). The targeted sequences of DSCAM shRNA, control DSCAM shRNA, UNC5C shRNA, and control UNC5C shRNA are: 5′-AAAGAGTTTAGCTGAAATGCT-3′ (DSCAM shRNA), 5′-AATGCATCTCTGCAAGAGGTA-3′ (control DSCAM shRNA), 5′-AAGAACCCAAGGCTCTTCATT-3′ (UNC5C shRNA) and 5′-AACTGTACTGTGTCAGAGGAA-3′ (control UNC5C shRNA). The designed hairpin siRNA templates were inserted into the mU6pro vector between SalI and XbaI sites and verified by sequencing. The following antibodies were used: rabbit anti-Flag (Abcam, Cambridge, MA), mouse anti-myc (9E10, Upstate Biotechnology, Lake Placid, NY), rabbit anti-HA (Santa Cruz Biotechnology, Lexington, NY), rabbit anti-FAK (BD Biosciences, Franklin Lakes, NJ), rabbit anti-PAK (Cell Signaling Technology, Beverly, MA), mouse anti-Fyn (Santa Cruz Biotechnology, Lexington, NY), goat anti-UNC5C (R&D Systems, Minneapolis, MN), and the HRP-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies (Santa Cruz Biotechnology, Lexington, NY). The purified rabbit anti-DSCAM was described previously (39Li W. Guan K.L. The Down syndrome cell adhesion molecule (DSCAM) interacts with and activates Pak.J. Biol. Chem. 2004; 279: 32824-32831Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). B27, Alexa Fluor 555 phalloidin and DAPI were purchased from Invitrogen (Carlsbad, CA). Netrin-1 protein and the sham-purified control were made from the conditioned media of HEK293 cells as previously described (21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar, 40Liu G. Beggs H. Jürgensen C. Park H.T. Tang H. Gorski J. Jones K.R. Reichardt L.F. Wu J. Rao Y. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction.Nat. Neurosci. 2004; 7: 1222-1232Crossref PubMed Scopus (218) Google Scholar, 41Liu G. Li W. Gao X. Li X. Jürgensen C. Park H.T. Shin N.Y. Yu J. He M.L. Hanks S.K. Wu J.Y. Guan K.L. Rao Y. p130CAS is required for netrin signaling and commissural axon guidance.J. Neurosci. 2007; 27: 957-968Crossref PubMed Scopus (44) Google Scholar). HEK293 cells were transfected using polyethylenimine (PEI) at 2:1 ratio with plasmids. Dissociated E15 cortical and P4 cerebellar neurons were plated on PLL-coated tissue culture dishes. Cells were lysed in mild lysis buffer (MLB: 20 mm Tris-HCl pH 7.4, 100 mm NaCl, 1% Nonidet P-40, and 0.1 mm phenylmethylsulfonyl fluoride) containing protease inhibitor mixture followed by incubation with specific antibodies for 2 h as described previously (21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar, 40Liu G. Beggs H. Jürgensen C. Park H.T. Tang H. Gorski J. Jones K.R. Reichardt L.F. Wu J. Rao Y. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction.Nat. Neurosci. 2004; 7: 1222-1232Crossref PubMed Scopus (218) Google Scholar). Protein extracts were boiled and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After transferring, membranes were blocked with 5% milk and incubated in primary antibody overnight at 4 °C. Membranes were washed with PBST (1× PBS + 0.1% Tween-20) and placed in secondary antibody for 1 h. Western blots were visualized on autoradiography film (Denville Scientific, Metuchen, NJ) with the enhanced chemiluminescence kit (Thermo Fisher Scientific Inc., Waltham, MA). The dissociated culture procedure of the E15 mouse cortex and cerebellum, and the P2–4 mouse cerebellum was conducted as described previously (21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar, 40Liu G. Beggs H. Jürgensen C. Park H.T. Tang H. Gorski J. Jones K.R. Reichardt L.F. Wu J. Rao Y. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction.Nat. Neurosci. 2004; 7: 1222-1232Crossref PubMed Scopus (218) Google Scholar, 41Liu G. Li W. Gao X. Li X. Jürgensen C. Park H.T. Shin N.Y. Yu J. He M.L. Hanks S.K. Wu J.Y. Guan K.L. Rao Y. p130CAS is required for netrin signaling and commissural axon guidance.J. Neurosci. 2007; 27: 957-968Crossref PubMed Scopus (44) Google Scholar) with some modifications. Neurons were grown on PLL-coated glass coverslips for growth cone collapse or immunostaining assays. For immunocytochemistry of DSCAM and UNC5C in dissociated primary neurons, cells on coverslips were fixed for 10 min in 4% pre-warmed paraformaldehyde (PFA) solution (127 mm NaCl, 5 mm KCl, 1.1 mm NaH2PO4, 0.4 mm KH2PO4, 2 mm MgCl2, 5.5 mm glucose, 1 mm EGTA, 10 mm Pipes) and permeabilized with 0.1% Triton X-100 for 15 min. After being washed three times with PBS, neurons were blocked in 10% normal goat serum for 1 h and incubated with the primary antibody solution for 2 h at 37 °C (rabbit anti-DSCAM, goat anti-UNC5C). Cells were washed in PBS three times followed by incubating with the secondary antibodies (anti-rabbit-488 and anti-goat-633) at 37 °C for 1 h. Neurons on coverslips were counterstained with DAPI and mounted onto slides using Fluorogel (EMS, PA). Images were taken under a confocal microscope (Olympus IX71 Fluoview). For studying the expression pattern of DSCAM and UNC5C in the developing nervous system, mouse embryos, and postnatal pups were collected. P2-P4 pups were perfused with 4% PFA intracardially and brain tissues transferred into cold 4% PFA overnight for further fixation. E15 mouse embryos were directly placed in cold 4% PFA overnight. Tissues were washed in PBS, transferred into sucrose-infiltration solution at 4 °C until the brain sank into the sucrose solution, and embedded in OCT. 14 μm coronal slices were cryosectioned and fixed with cold acetone on superfrost plus slides. After post-fixation with 4% PFA at room temperature for 30 min, brain slices were subjected to heat-mediated antigen retrieval using citric acid (pH 6) and blocked with 3% BSA for 1 h in 1× PBST (1× PBS with 0.1% Tween-20), and incubated with the primary antibodies overnight (rabbit anti-DSCAM and goat anti-UNC5C). After washing three times in permeabilization buffer, slices were incubated with the secondary antibody (anti-rabbit-488 and anti-goat-633) for 2 h at 37 °C. Images were taken under a confocal microscope (Olympus IX71 Fluoview). The P2-4 mouse cerebellum was dissected and sliced into 200 μm sections using a tissue chopper (Vibratome, Bannockburn, IL). Only the external granule layer (EGL) was finely dissected using a sharpened tungsten needle and trypsinized at 37 °C for 15 min (42Alcántara S. Ruiz M. De Castro F. Soriano E. Sotelo C. Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system.Development. 2000; 127: 1359-1372Crossref PubMed Google Scholar). After dissociation, neurons (1 × 104) were nucleofected (Amaxa, Walkersville, MD) with Venus yellow fluorescent protein (Venus-YFP) only or Venus-YFP plus DSCAM shRNAs, UNC5C shRNAs or different truncation mutants of DSCAM and UNC5C (DSCAM wild type, DSCAMΔC, DSCAMΔN, UNC5C wild type, UNC5CΔC, and UNC5CΔN) using program G-013. After electroporation, cells were plated onto PLL-coated coverslips and cultured in warm media (DMEM + B27 + 20 units/ml of penicillin/streptomycin) at 37 °C with 5% CO2. After 4 h, the media was replaced with fresh media to remove toxic nucleofection solution and neurons were cultured for 14 h. Neurons were starved in DMEM + 0.1% BSA + B27 for 4 h and stimulated with HEK293 control or netrin-1 conditioned media for 30 min. Cells were fixed with 4% PFA for 15 min and stained with phalloidin and DAPI. Images of only YFP-labeled cells were taken using an epifluorescent microscope. For quantification, a single EGL cell with a clearly polarized axon was considered collapsed if it had no lamellipodia and two or fewer filopodia (43Bartoe J.L. McKenna W.L. Quan T.K. Stafford B.K. Moore J.A. Xia J. Takamiya K. Huganir R.L. Hinck L. Protein interacting with C-kinase 1/protein kinase Calpha-mediated endocytosis converts netrin-1-mediated repulsion to attraction.J. Neurosci. 2006; 26: 3192-3205Crossref PubMed Scopus (63) Google Scholar). At least 150 random growth cones were measured per group and experiments were done in triplicate. A one-way ANOVA followed by post hoc testing with Fisher's Least Significant Difference (LSD) was performed to detect statistical significance among different groups. The protein structure of DSCAM is similar to DCC and both receptors are required for netrin signaling (19Andrews G.L. Tanglao S. Farmer W.T. Morin S. Brotman S. Berberoglu M.A. Price H. Fernandez G.C. Mastick G.S. Charron F. Kidd T. Dscam guides embryonic axons by Netrin-dependent and -independent functions.Development. 2008; 135: 3839-3848Crossref PubMed Scopus (90) Google Scholar, 20Ly A. Nikolaev A. Suresh G. Zheng Y. Tessier-Lavigne M. Stein E. DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.Cell. 2008; 133: 1241-1254Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar). UNC5 binds to DCC to switch DCC-mediated attraction into repulsion, raising the possibility that DSCAM may interact with UNC5 as well. To determine a potential interaction between DSCAM and UNC5, Flag-tagged full-length human DSCAM (DSCAM-Flag) and the full-length human UNC5C tagged with HA (UNC5C-HA) were expressed in HEK293 cells, cell lysates were immunoprecipitated with anti-Flag for DSCAM and immunoblots probed with either anti-HA for UNC5C or anti-Flag for DSCAM (Fig. 1A). UNC5C-HA coimmunoprecipitated with DSCAM (Fig. 1A). The reciprocal IP confirmed the interaction of DSCAM and UNC5C in HEK293 cells (data not shown). To examine whether endogenous DSCAM interacts with UNC5C, primary cortical neurons were used because these neurons express both DSCAM and UNC5C and cortex dissection, dissociation, and culturing is fairly straightforward (21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar, 23Leonardo E.D. Hinck L. Masu M. Keino-Masu K. Ackerman S.L. Tessier-Lavigne M. Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors.Nature. 1997; 386: 833-838Crossref PubMed Scopus (426) Google Scholar). After 24 h of primary culture, dissociated neurons were serum-starved in DMEM containing B27 for 4 h and stimulated with either purified netrin-1 (250 ng/ml) or sham-purified control. DSCAM was immunoprecipitated with anti-DSCAM and blotted for UNC5C and DSCAM (Fig. 1B). We found that UNC5C was co-immunoprecipitated with DSCAM and netrin-1 stimulation increased the DSCAM/UNC5C interaction (Fig. 1B). Netrin-1 enhanced the interaction of DSCAM and UNC5C within 5 min and the enhancement was sustained up to 20 min (Fig. 1B). To study whether DSCAM interacts with UNC5C in the postnatal mouse cerebellum, P2 mouse cerebellar neurons were dissociated and stimulated with or without netrin-1, as described previously (21Liu G. Li W. Wang L. Kar A. Guan K.L. Rao Y. Wu J.Y. DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar, 40Liu G. Beggs H. Jürgensen C. Park H.T. Tang H. Gorski J. Jones K.R. Reichardt L.F. Wu J. Rao Y. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction.Nat. Neurosci. 2004; 7: 1222-1232Crossref PubMed Scopus (218) Google Scholar, 41Liu G. Li W. Gao X. Li X. Jürgensen C. Park H.T. Shin N.Y. Yu J. He M.L. Hanks S.K. Wu J.Y. Guan K.L. Rao Y. p130CAS is required for netrin signaling and commissural axon guidance.J. Neurosci. 2007; 27: 957-968Crossref PubMed Scopus (44) Google Scholar). Cell lysates were immunoprecipitated with anti-DSCAM and blotted for DSCAM and UNC5C. Netrin-1 dramatically induced DSCAM/UNC5C interaction and continued to increase this interaction over 20 min (Fig. 1C). Netrin-1-induced interaction of endogenous DSCAM and UNC5C in E15 cortical and P2 cerebellar neurons was also observed in the reciprocal co-immunoprecipitation (data not shown). These above data demonstrate a physiological interaction between DSCAM and UNC5C, and this interaction is stimulated by netrin-1. DSCAM is a transmembrane protein belonging to the immunoglobulin (Ig) superfamily. Biochemical analysis of DSCAM truncation mutants (DSCAMΔC and DSCAMΔN) co-transfected with UNC5C in HEK293 cells revealed the DSCAM extracellular domain was essential for DSCAM interaction with UNC5C (Fig. 1, D and E and the reciprocal IP not shown). To investigate the domains of UNC5C that associate w" @default.
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• W2010901230 title "Down Syndrome Cell Adhesion Molecule (DSCAM) Associates with Uncoordinated-5C (UNC5C) in Netrin-1-mediated Growth Cone Collapse" @default.
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