MAGI1 Recruits Dll1 to Cadherin-based Adherens Junctions and Stabilizes It on the Cell Surface
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m500375200
ISSN1083-351X
AutoresEri Mizuhara, Tomoya Nakatani, Yasuko Minaki, Yoshimasa Sakamoto, Yūichi Ono, Yoshimi Takai,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoDelta-Notch signaling plays an essential role in cell fate determination in many tissue types, including the central nervous system. Although the signaling mechanism of Notch has been extensively studied, the behaviors of its ligands are not well understood. In the present study, we found that, in the developing neural tube, Dll1(Delta-like 1) was mainly localized on the processes extending from nascent neurons toward both the pia and the ventricle and accumulated at apical termini, where adherens junctions (AJs) were formed. To understand the mechanism of Dll1 localization, we searched for binding proteins for Dll1 and identified a scaffolding molecule, MAGI1. In the developing spinal cord, MAGI1 mRNA was highly expressed in the ventricular zone, where Dll1 mRNA was expressed. MAGI1 protein accumulated at the AJs formed around the termini of apically extending processes and was partially colocalized with Dll1. MAGI1 bound not only to Dll1 but also to N-cadherin-β-catenin complexes. In cultured AJ-forming fibroblasts, MAGI1 was localized at AJs, and Dll1 was recruited to these AJs through binding to MAGI1. In addition, Dll1 was stabilized on the cell surface by MAGI1. Taken together, these results suggest that Dll1 is presented on the surface of AJs formed at the apical termini of processes through interaction with MAGI1 to activate Notch on neighboring cells in the developing central nervous system. Delta-Notch signaling plays an essential role in cell fate determination in many tissue types, including the central nervous system. Although the signaling mechanism of Notch has been extensively studied, the behaviors of its ligands are not well understood. In the present study, we found that, in the developing neural tube, Dll1(Delta-like 1) was mainly localized on the processes extending from nascent neurons toward both the pia and the ventricle and accumulated at apical termini, where adherens junctions (AJs) were formed. To understand the mechanism of Dll1 localization, we searched for binding proteins for Dll1 and identified a scaffolding molecule, MAGI1. In the developing spinal cord, MAGI1 mRNA was highly expressed in the ventricular zone, where Dll1 mRNA was expressed. MAGI1 protein accumulated at the AJs formed around the termini of apically extending processes and was partially colocalized with Dll1. MAGI1 bound not only to Dll1 but also to N-cadherin-β-catenin complexes. In cultured AJ-forming fibroblasts, MAGI1 was localized at AJs, and Dll1 was recruited to these AJs through binding to MAGI1. In addition, Dll1 was stabilized on the cell surface by MAGI1. Taken together, these results suggest that Dll1 is presented on the surface of AJs formed at the apical termini of processes through interaction with MAGI1 to activate Notch on neighboring cells in the developing central nervous system. Delta-Notch signaling is an evolutionarily conserved signaling pathway that controls cell fate determination, cellular differentiation, and pattern formation in many tissue types (1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4896) Google Scholar, 2Lewis J. Semin. Cell Dev. Biol. 1998; 9: 583-589Crossref PubMed Scopus (350) Google Scholar). During vertebrate central nervous system development, Delta-Notch signaling is involved in maintaining the undifferentiated neural progenitor pool to regulate the correct number and timing of generation of neurons and glial cells (2Lewis J. Semin. Cell Dev. Biol. 1998; 9: 583-589Crossref PubMed Scopus (350) Google Scholar, 3Kageyama R. Ohtsuka T. Cell Res. 1999; 9: 179-188Crossref PubMed Scopus (282) Google Scholar). Developing neural tubes consist of two distinct layers, the ventricular zone (VZ), 1The abbreviations used are: VZ, ventricular zone; ML, mantle layer; AJ, adherens junction; ICD, intracellular domain; aa, amino acid(s); WT, wild-type; TX-100, Triton X-100; PIPES, 1,4-piperazinediethanesulfonic acid; HA, hemagglutinin; E3, ubiquitin-protein isopeptide ligase. 1The abbreviations used are: VZ, ventricular zone; ML, mantle layer; AJ, adherens junction; ICD, intracellular domain; aa, amino acid(s); WT, wild-type; TX-100, Triton X-100; PIPES, 1,4-piperazinediethanesulfonic acid; HA, hemagglutinin; E3, ubiquitin-protein isopeptide ligase. which is composed of undifferentiated neural progenitors, and the mantle layer (ML), where differentiating neurons accumulate. After progenitors in the VZ exit the cell cycle, these neuronally fated cells immediately migrate toward the ML and differentiate into mature neurons (4Nadarajah B. Parnavelas J.G. Nat. Rev. Neurosci. 2002; 3: 423-432Crossref PubMed Scopus (511) Google Scholar). These migrating precursor cells in the VZ transiently express Notch ligand molecules, such as Dll1 (Delta-like 1) and Jagged1, to activate Notch receptors expressed on the neighboring progenitors (5Bettenhausen B. Hrabe de Angelis M. Simon D. Guenet J.L. Gossler A. Development (Camb.). 1995; 121: 2407-2418Crossref PubMed Google Scholar, 6Chitnis A. Henrique D. Lewis J. Ish-Horowicz D. Kintner C. Nature. 1995; 375: 761-766Crossref PubMed Scopus (613) Google Scholar, 7Henrique D. Adam J. Myat A. Chitnis A. Lewis J. Ish-Horowicz D. Nature. 1995; 375: 787-790Crossref PubMed Scopus (919) Google Scholar, 8Henrique D. Hirsinger E. Adam J. Le Roux I. Pourquie O. Ish-Horowicz D. Lewis J. Curr. Biol. 1997; 7: 661-670Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 9Myat A. Henrique D. Ish-Horowicz D. Lewis J. Dev. Biol. 1996; 174: 233-247Crossref PubMed Scopus (276) Google Scholar). Activation of Notch receptors mediated by the downstream target genes, such as the Hes family basic helix-loop-helix transcriptional repressors, prevents the cells from undergoing neuronal differentiation by repressing proneural basic helix-loop-helix factors, such as Mash1 (1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4896) Google Scholar, 3Kageyama R. Ohtsuka T. Cell Res. 1999; 9: 179-188Crossref PubMed Scopus (282) Google Scholar). In addition to these "lateral inhibition" functions, constructive roles of Notch signaling in gliogenesis have also been reported (10Gaiano N. Nye J.S. Fishell G. Neuron. 2000; 26: 395-404Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 11Wang S. Barres B.A. Neuron. 2000; 27: 197-200Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The mechanism of signal transduction by the Notch pathway has been extensively studied. Ligand binding on the cell surface leads to proteolytic cleavage of the Notch receptor at an extracellular region by tumor necrosis factor α-converting enzyme metalloprotease (12Lai E.C. Development (Camb.). 2004; 131: 965-973Crossref PubMed Scopus (861) Google Scholar). This triggers a second cleavage at a transmembrane domain by a presenilin-dependent protease to release the Notch intracellular domain from the plasma membrane. The Notch intracellular domain is then translocated into the nucleus, where it acts as a transcriptional coactivator for a DNA-binding factor, RBP-J/CBF1, to induce expression of downstream target genes (12Lai E.C. Development (Camb.). 2004; 131: 965-973Crossref PubMed Scopus (861) Google Scholar). The Delta gene is well conserved from flies to mammals and encodes a transmembrane ligand for the Notch receptor (5Bettenhausen B. Hrabe de Angelis M. Simon D. Guenet J.L. Gossler A. Development (Camb.). 1995; 121: 2407-2418Crossref PubMed Google Scholar, 6Chitnis A. Henrique D. Lewis J. Ish-Horowicz D. Kintner C. Nature. 1995; 375: 761-766Crossref PubMed Scopus (613) Google Scholar, 7Henrique D. Adam J. Myat A. Chitnis A. Lewis J. Ish-Horowicz D. Nature. 1995; 375: 787-790Crossref PubMed Scopus (919) Google Scholar). Thus, Delta-Notch signaling mediates local cell-cell communication. Regulation of the ligand as well as the receptor seems to play important roles in signal transduction by the Delta-Notch pathway. The soluble extracellular domain of Notch ligands can bind to the Notch receptor; however, it cannot activate Notch in vitro (13Varnum-Finney B. Wu L. Yu M. Brashem-Stein C. Staats S. Flowers D. Griffin J.D. Bernstein I.D. J. Cell Sci. 2000; 113: 4313-4318Crossref PubMed Google Scholar) but rather acts as a dominant-negative mutant that inhibits the native Delta-Notch pathway in vivo (14Hukriede N.A. Gu Y. Fleming R.J. Development (Camb.). 1997; 124: 3427-3437Crossref PubMed Google Scholar, 15Sun X. Artavanis-Tsakonas S. Development (Camb.). 1997; 124: 3439-3448Crossref PubMed Google Scholar). Furthermore, deletion mutants of Notch ligands lacking the intracellular domain also act as dominant-negative mutants (6Chitnis A. Henrique D. Lewis J. Ish-Horowicz D. Kintner C. Nature. 1995; 375: 761-766Crossref PubMed Scopus (613) Google Scholar, 8Henrique D. Hirsinger E. Adam J. Le Roux I. Pourquie O. Ish-Horowicz D. Lewis J. Curr. Biol. 1997; 7: 661-670Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 16Sun X. Artavanis-Tsakonas S. Development (Camb.). 1996; 122: 2465-2474Crossref PubMed Google Scholar). These findings suggest the possible importance of the intracellular domain of Delta in Notch activation. In support of this, one of the factors involved in the Delta-Notch signaling cascade in Drosophila, Neuralyzed (Neur), has been shown to bind the cytoplasmic domain of Delta and act as an E3 ubiquitin ligase for Delta protein (17Yeh E. Dermer M. Commisso C. Zhou L. McGlade C.J. Boulianne G.L. Curr. Biol. 2001; 11: 1675-1679Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 18Lai E.C. Deblandre G.A. Kintner C. Rubin G.M. Dev. Cell. 2001; 1: 783-794Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 19Pavlopoulos E. Pitsouli C. Klueg K.M. Muskavitch M.A. Moschonas N.K. Delidakis C. Dev. Cell. 2001; 1: 807-816Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Neur-dependent internalization, possibly triggered by its ubiquitination, facilitates Notch activation (18Lai E.C. Deblandre G.A. Kintner C. Rubin G.M. Dev. Cell. 2001; 1: 783-794Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 19Pavlopoulos E. Pitsouli C. Klueg K.M. Muskavitch M.A. Moschonas N.K. Delidakis C. Dev. Cell. 2001; 1: 807-816Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Consistent with this, dynamin-dependent endocytosis in Delta-expressing signaling cells is required for Notch activation (20Seugnet L. Simpson P. Haenlin M. Dev. Biol. 1997; 192: 585-598Crossref PubMed Scopus (225) Google Scholar). Furthermore, trans-endocytosis of the Notch extracellular domain by its ligand has been observed (21Klueg K.M. Muskavitch M.A. J. Cell Sci. 1999; 112: 3289-3297Crossref PubMed Google Scholar, 22Parks A.L. Klueg K.M. Stout J.R. Muskavitch M.A. Development (Camb.). 2000; 127: 1373-1385Crossref PubMed Google Scholar). In vertebrates, similar E3 ligases, such as Neur and Mind bomb, have been identified as regulators of Delta protein and Notch signaling (23Deblandre G.A. Lai E.C. Kintner C. Dev. Cell. 2001; 1: 795-806Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 24Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). Thus, endocytosis of the ligand regulated by its intracellular domain seems to be an important mechanism of Notch activation, and this mechanism is conserved from flies to vertebrates. The expression pattern and function of the Delta gene have been extensively elucidated. However, the high internalization efficiency that is required for its function and the resulting instability of the Delta protein on the cell surface make it difficult to understand the molecular behavior of Delta protein. In particular, where and how Delta protein is presented to Notch-expressing signal-receiving cells remain largely unknown. Recently, Delta-induced filopodia formation was reported to mediate long-range lateral inhibition of neural differentiation of sensory organ precursors in Drosophila (25De Joussineau C. Soule J. Martin M. Anguille C. Montcourrier P. Alexandre D. Nature. 2003; 426: 555-559Crossref PubMed Scopus (147) Google Scholar), but it is currently unknown whether a similar cellular structure is involved in the presentation of Delta protein to neighboring cells in vertebrate systems. The adherens junction (AJ) is a specialized structure for the cell-cell adhesion machinery in epithelial cells and consists of cadherin and nectin family cell adhesion molecules, which are linked to the actin cytoskeleton through their binding proteins catenins and afadin, respectively (26Barth A.I. Nathke I.S. Nelson W.J. Curr. Opin. Cell. Biol. 1997; 9: 683-690Crossref PubMed Scopus (485) Google Scholar, 27Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (483) Google Scholar). Neuroepithelial cells in the developing neural tube extend processes toward both the ventricle and the pia to form the bipolar morphology and then form AJs at the termini of their apically extending processes with the termini of neighboring progenitors (28Aaku-Saraste E. Hellwig A. Huttner W.B. Dev. Biol. 1996; 180: 664-679Crossref PubMed Scopus (206) Google Scholar). AJs are thought to be involved in maintenance of the undifferentiated progenitor pool and neural production by asymmetric cell division at the neurogenesis stage (29Chenn A. Zhang Y.A. Chang B.T. McConnell S.K. Mol. Cell. Neurosci. 1998; 11: 183-193Crossref PubMed Scopus (172) Google Scholar, 30Zechner D. Fujita Y. Hulsken J. Muller T. Walther I. Taketo M.M. Crenshaw III, E.B. Birchmeier W. Birchmeier C. Dev. Biol. 2003; 258: 406-418Crossref PubMed Scopus (413) Google Scholar, 31Chae T.H. Kim S. Marz K.E. Hanson P.I. Walsh C.A. Nat. Genet. 2004; 36: 264-270Crossref PubMed Scopus (133) Google Scholar). Recently, we observed that migrating nascent neural precursors expressing Dll1 mRNA in the developing spinal cord extended processes toward the ventricle and formed AJs at the apical termini of the extending processes (32Minaki Y. Mizuhara E. Morimoto K. Nakatani T. Sakamoto Y. Inoue Y. Satoh K. Imai T. Takai Y. Ono Y. Neurosci. Res. 2005; 52: 250-262Crossref PubMed Scopus (26) Google Scholar). AJs are known to both regulate cell-cell adhesion and mediate the cell-cell signaling involved in cell growth and differentiation (26Barth A.I. Nathke I.S. Nelson W.J. Curr. Opin. Cell. Biol. 1997; 9: 683-690Crossref PubMed Scopus (485) Google Scholar). Thus, it is possible that Dll1-expressing nascent precursors in the VZ communicate with neighboring progenitors through the AJs to regulate the differentiation of these cells. In addition, Dll1 mRNA was selectively transported to the apically extending processes (32Minaki Y. Mizuhara E. Morimoto K. Nakatani T. Sakamoto Y. Inoue Y. Satoh K. Imai T. Takai Y. Ono Y. Neurosci. Res. 2005; 52: 250-262Crossref PubMed Scopus (26) Google Scholar), raising the possibility that Dll1 functions on these processes to activate Notch on neighboring progenitors. To address these issues, we first examined the localization of Dll1 in the developing mouse spinal cord and observed that it was mainly localized at the processes. To understand the mechanism of Dll1 localization and regulation of its activity in the Notch signaling pathway, we screened for proteins that interacted with the intracellular domain of Dll1 and identified the multiple PDZ domain-containing scaffolding molecule MAGI1 as a binding partner for the C terminus of Dll1. During the course of this study, Wright et al. (33Wright G.J. Leslie J.D. Ariza-McNaughton L. Lewis J. Development (Camb.). 2004; 131: 5659-5669Crossref PubMed Scopus (47) Google Scholar) reported that zebrafish MAGI proteins bind to DeltaC and DeltaD proteins and that the MAGI1-binding domain of DeltaD is required for proper development of Rohon-Beard neurons. However, the mode of action of MAGI1 in Delta-Notch signaling has not yet been clarified. Thus, in the current study, we tried to address this issue by focusing on the role of MAGI1 in regulating Dll1 localization. In developing neural tubes, MAGI1 was localized at AJs formed at the termini of apical processes extending from both proliferating progenitors and neuronally fated precursors expressing Dll1 in the VZ and partially colocalized with Dll1. By using an in vitro culture system involving AJ-forming fibroblast cells, we found that Dll1 was recruited to AJs through its interaction with MAGI1. Finally, we showed that Dll1 was stabilized on the cell surface by MAGI1. Taken together, these results suggest a role for MAGI1 in Dll1 localization and the possible involvement of cadherin-based AJs in Delta-Notch signaling. Plasmid Construction—pcDNA-SS-FLAG was constructed by ligating the annealed oligonucleotides 5′-GAT-CGG-CCA-CCA-TGT-CTG-CAC-TTC-TGA-TCC-TAG-CTC-TTG-TTG-GAG-CTG-CAG-TTG-CTG-3′, 5′-GGA-TCA-GAA-GTG-CAG-ACA-TGG-TGG-CC-3′,5′-GAT-CGG-ATT-ACA-AGG-ATG-ACG-ACG-ATA-AGG-TCG-ACC-TCG-AGG-GAT-CCG-AAT-TCG-CGG-CCG-CG-3′, 5′-CTT-ATC-GTC-GTC-ATC-CTT-GTA-ATC-CGA-TCC-AGC-AAC-TGC-AGC-TCC-AAC-AAG-AGC-TA-3′, and 5′-TCG-ACG-CGG-CCG-CGA-ATT-CGG-ATC-CCT-CGA-GGT-CGA-C-3′ into the BamHI/XhoI site of pcDNA3.1+ (Invitrogen). pMX-SS-HA and pcDNA-SS-HA were constructed by ligating the annealed oligonucleotides 5′-GAT-CGG-CCA-CCA-TGT-CTG-CAC-TTC-TGA-TCC-TAG-CTC-TTG-TTG-GAG-CTG-CAG-TTG-CTG-3′, 5′-GGA-TCA-GAA-GTG-CAG-ACA-TGG-TGG-CC-3′, 5′-GAT-CGG-ACT-ACC-CAT-ACG-ACG-TCC-CAG-ACT-ACG-CTG-TCG-ACC-TCG-AGG-GAT-CCG-AAT-TCG-CGG-CCG-CG-3′, 5′-AGC-GTA-GTC-TGG-GAC-GTC-GTA-TGG-GTA-GTC-CGA-TCC-AGC-AAC-TGC-AGC-TCC-AAC-AAG-AGC-TA-3′, and 5′-TCG-ACG-CGG-CCG-CGA-ATT-CGG-ATC-CCT-CGA-GGT-CGA-C-3′ into the BamHI/XhoI site of pMXII (35Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Crossref PubMed Scopus (55) Google Scholar) and pcDNA3.1+, respectively. cDNAs of Dll1, MAGI1a, Jagged1, β-catenin, and N-cadherin were amplified by PCR using the following primer sets: Dll1 WT, 5′-GAG-CTC-GAG-TCC-GGC-GTA-TTT-GAG-CTG-AAG-CTG-CA-3′ and 5′-GAG-GAA-TTC-TTA-CAC-CTC-AGT-CGC-TAT-AAC-ACA-CTC-3′; Dll1 ΔC, 5′-GAG-CTC-GAG-TCC-GGC-GTA-TTT-GAG-CTG-AAG-CTG-CA-3′ and 5′-GAG-GAA-TTC-TCA-TAT-AAC-ACA-CTC-ATC-CTT-TTC-TGC-AG-3′; Dll1 ICD, 5′-GAG-GTC-GAC-GTC-CGG-CTG-AAG-CTA-CAG-AAA-CAC-CAG-3′ and 5′-GAG-GCG-GCC-GCT-TAC-ACC-TCA-GTC-GCT-ATA-ACA-CAC-TC-3′; MAGI1a WT, 5′-GAG-GAA-TTC-ATG-TCG-AAA-GTG-ATC-CAG-AAG-AAG-AAC-CAC-3′ and 5′-GAG-GAA-TTC-TCA-TGG-AGT-CAT-GCC-AGG-GAA-GGA-AG-3′; Jagged1 ICD, 5′-GAG-GTC-GAC-TGT-GTA-CGG-AAG-CGG-CGG-AAG-CCC-AG-3′ and 5′-GAG-GCG-GCC-GCC-TAT-ACG-ATG-TAT-TCC-ATC-CGG-TTC-A-3′; β-catenin,5′-GAG-GTC-GAC-ATG-GCT-ACT-CAA-GCT-GAC-CTG-ATG-GAG-3′ and 5′-GAG-GCG-GCC-GCT-TAC-AGG-TCA-GTA-TCA-AAC-CAG-GCC-A-3′; and N-cadherin, 5′-GAG-GAA-TTC-GCC-ACC-ATG-TGC-CGG-ATA-GCG-GGA-GCG-CCG-CG-3′ and 5′-GAG-GCG-GCC-GCT-CAG-TCG-TCA-CCA-CCG-CCG-TAC-ATG-T-3′. The Dll1 WT and Dll1 ΔC fragments were digested with XhoI/EcoRI and cloned into the corresponding sites in pcDNA-SS-FLAG, pcDNA-SS-HA, and pMX-SS-HA. The MAGI1a fragment was digested with EcoRI and cloned into the corresponding sites in pcDNA-FLAG-NII, pcDNA-HA-NII, and pMX-Myc-NII (34Nakatani T. Mizuhara E. Minaki Y. Sakamoto Y. Ono Y. J. Biol. Chem. 2004; 279: 16356-16367Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The Dll1 ICD and Jagged1 ICD fragments were digested with SalI/NotI and cloned into the XhoI/NotI site of pcDNA-FLAG-NII. The β-catenin fragment was digested with SalI/NotI and cloned into the XhoI/NotI site of pcDNA-HA-NII. The N-cadherin fragment was digested with EcoRI/NotI and cloned into the corresponding site of pcDNA3.1+. Various mutant MAGI1a constructs containing the following amino acid (aa) residues were made (Fig. 1): MAGI1-WT, aa 1-1235 (full length); MAGI1ΔP0, aa 106-1235; MAGI1ΔP1, aa 1-459 + 537-1235; MAGI1ΔP2, aa 1-630 + 704-1235; MAGI1ΔP3, aa 1-828 + 906-1235; MAGI1ΔP4, aa 1-986 + 1074-1235; MAGI1ΔP5, aa 1-1138; and MAGI1P4: aa 974-1087. pMX-derived constructs were used for the subcellular localization experiments, and pcDNA-derived constructs were used for the coimmunoprecipitation assays. For in situ hybridization probes, Dll1 and MAGI1 cDNAs were amplified by PCR using the following primers: Dll1, 5′-GCG-AGA-AGG-ACG-TTT-CTG-TTA-GCA-TC-3′ and 5′-ATA-TAG-TCA-CAT-AGA-CCC-GAA-GTG-CC-3′; and MAGI1, 5′-TCA-TCG-ACA-GCT-GCA-AGG-AGG-CCG-TC-3′ and 5′-TTC-TGC-CCA-GAG-CTG-TCG-GTC-TGA-TC-3′. The amplified PCR fragments were cloned into pCRII (Invitrogen) and used as templates for the transcription of digoxigenin-labeled probes. In Situ Hybridization and Immunohistochemistry—In situ hybridization and immunohistochemistry were performed as described previously (34Nakatani T. Mizuhara E. Minaki Y. Sakamoto Y. Ono Y. J. Biol. Chem. 2004; 279: 16356-16367Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Polyclonal rabbit anti-MAGI1 and anti-Dll1 antibodies were raised against GST-MAGI1 (aa 379-454) and GST-Dll1 (aa 646-722), respectively, and affinity-purified. The primary antibodies used for double staining included anti-ZO1 (Zymed Laboratories Inc.), anti-N-cadherin (BD Biosciences), anti-β-catenin (Santa Cruz), and anti-Neph3 (32Minaki Y. Mizuhara E. Morimoto K. Nakatani T. Sakamoto Y. Inoue Y. Satoh K. Imai T. Takai Y. Ono Y. Neurosci. Res. 2005; 52: 250-262Crossref PubMed Scopus (26) Google Scholar). Immunofluorescence analysis of the transfected cells was performed as described previously (35Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Crossref PubMed Scopus (55) Google Scholar). Briefly, cells were transfected with the indicated combinations of plasmids using Lipofectamine (Invitrogen). After culture for 24 h, the transfected cells were replated onto glass coverslips, cultured for an additional 16 h, and subjected to immunofluorescence analyses using anti-Myc (Roche Applied Science), anti-E-cadherin (Takara), anti-Dll1, and anti-MAGI1 antibodies. Immunoprecipitation—Immunoprecipitation experiments using 293E cells and detection by Western blotting were performed as described previously (34Nakatani T. Mizuhara E. Minaki Y. Sakamoto Y. Ono Y. J. Biol. Chem. 2004; 279: 16356-16367Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Briefly, transfected 293E cells were lysed with a lysis buffer containing 10 mm HEPES (pH 7.6), 250 mm NaCl, 5 mm EDTA, and 1% TX-100 for 1 h at 4 °C, and immunoprecipitation was performed with anti-FLAG M2 beads (Sigma). Western blotting was carried out with anti-FLAG M2 (Sigma), anti-MAGI1, anti-β-catenin (Sigma), and anti-N-cadherin (BD Biosciences) antibodies. Embryonic day (E) 12.5 mouse neural tubes were dissected and sonicated in a lysis buffer containing 10 mm HEPES (pH 7.6), 250 mm NaCl, 5 mm EDTA, and 1% TX-100. The cell extract was incubated with either an anti-MAGI1 antibody or control IgG at 4 °C for 16 h. The antibody-protein complexes were collected with protein G-Sepharose beads and subjected to Western blotting using anti-Dll1 and anti-MAGI1 antibodies. Reverse Transcription-PCR—Reverse transcription-PCR was performed as described previously (34Nakatani T. Mizuhara E. Minaki Y. Sakamoto Y. Ono Y. J. Biol. Chem. 2004; 279: 16356-16367Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). ExTaq polymerase (Takara) was used for amplification, which was carried out by denaturation at 94 °C for 30 s (2 min in the first cycle), annealing at 65 °C for 30 s, and extension at 72 °C for 1 min (3 min in the last cycle). There were 26 cycles for MAGI1 and 30 cycles for Dll1. The primer sequences were as follows: MAGI1, 5′-CTC-TGA-ACA-CTG-TGA-GCT-CTG-GCA-GC-3′ and 5′-TTG-GTT-TGG-TGG-TGG-TCC-TTG-TTT-CC-3′; and Dll1, 5′-GCG-AGA-AGG-ACG-TTT-CTG-TTA-GCA-TC-3′ and 5′-CTT-CTC-TTC-TCC-TGC-AGA-GCT-CTG-TG-3′. TX-100 Extraction—TX-100 extraction experiments were carried out as described previously (36Georgakopoulos A. Marambaud P. Efthimiopoulos S. Shioi J. Cui W. Li H.C. Schutte M. Gordon R. Holstein G.R. Martinelli G. Mehta P. Friedrich Jr., V.L. Robakis N.K. Mol. Cell. 1999; 4: 893-902Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Briefly, transfected EL cells on coverslips were extracted with CSK buffer (50 mm NaCl, 300 mm sucrose, 10 mm PIPES (pH 6.8), 3 mm MgCl2, and 0.5% TX-100) for 5 min at 4 °C, fixed with 1% formaldehyde, and subjected to immunofluorescence analyses using anti-E-cadherin (Takara), anti-Dll1, and anti-MAGI1 antibodies. Surface Protein Labeling—Transfected EL cells were surface-labeled with EZ-link Sulfo-NHS-SS-Biotin (Pierce) and sonicated in a lysis buffer containing 1% SDS, 10 mm Tris-HCl (pH 8.0), 100 mm NaCl, and 1 mm EDTA. Biotinylated proteins were collected with streptavidin beads, eluted by boiling in SDS sample buffer, and subjected to Western blotting with anti-HA (Roche Applied Science) and anti-E-cadherin (Takara) antibodies. Dll1 Protein Is Localized on the Processes Extending from Nascent Neuronally Fated Precursors in the Developing Spinal Cord VZ—We previously observed that Dll1 mRNA was selectively transported into the processes apically extending from nascent neural precursors in the developing spinal cord VZ (32Minaki Y. Mizuhara E. Morimoto K. Nakatani T. Sakamoto Y. Inoue Y. Satoh K. Imai T. Takai Y. Ono Y. Neurosci. Res. 2005; 52: 250-262Crossref PubMed Scopus (26) Google Scholar). This suggests that Dll1 functions on the processes. To examine this possibility, we first examined the localization of Dll1 protein in the developing mouse spinal cord. In the E11.5 spinal cord, Dll1 mRNA was expressed only in a subset of VZ cells (see Fig. 4B). Consistent with this, a high level of anti-Dll1 immunoreactivity was detected in the VZ region (Fig. 2A). Furthermore, the immunoreactivity was not detected in the ventral striped region where Dll1 mRNA was absent (compare Figs. 2A and 4B). Together with the observation that this anti-Dll1 antibody did not cross-react with the most homologous protein (Dll4) and the Jagged family proteins in Western blotting (data not shown), these results confirm the specificity of the antibody. Importantly, Dll1 was detected not only at the cell body but also in the processes. In contrast to its mRNA, which was detected only in the apical processes extending toward the ventricle (32Minaki Y. Mizuhara E. Morimoto K. Nakatani T. Sakamoto Y. Inoue Y. Satoh K. Imai T. Takai Y. Ono Y. Neurosci. Res. 2005; 52: 250-262Crossref PubMed Scopus (26) Google Scholar), Dll1 protein was distributed in both the apical processes and the basal processes extending toward the pia. These results suggest that the localization of Dll1 is not simply regulated by local protein synthesis from localized mRNA.Fig. 2Localization of Dll1 in the developing spinal cord. Images of transverse sections of E11.5 spinal cord double-stained for Dll1 (green) and ZO1 (red) are shown. B-D show magnified images of A. Note that Dll1 is partially colocalized with ZO1 at AJs (arrowhead). Bars: A, 100 μm; B and C, 50 μm; D, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our previous observation that Dll1 mRNA accumulated at the termini of the apically extending processes (32Minaki Y. Mizuhara E. Morimoto K. Nakatani T. Sakamoto Y. Inoue Y. Satoh K. Imai T. Takai Y. Ono Y. Neurosci. Res. 2005; 52: 250-262Crossref PubMed Scopus (26) Google Scholar) suggests a possible involvement of the process termini, where AJs are formed, in Dll1 function. In support of this, Dll1 was detected in the processes contacting the ventricle, and its accumulation near the ventricle was observed (Fig. 2B). These signals were specific because such apical signals were not detected at Dll1 mRNA-negative regions (Fig. 2C). Furthermore, Dll1 was partially colocalized with an AJ marker, ZO1 (zonula occludens 1), at the termini (Fig. 2D). These results suggest that Dll1 is presented on the processes to activate Notch expressed on neighboring cells. MAGI1 Interacts with the C Terminus of Dll1—The selective localization of Dll1 in the processes suggests possible cell-intrinsic regulation through interacting proteins. To understand the mechanism of Dll1 localization, we searched for interacting partners for the ICD of Dll1 by yeast two-hybrid screening of an E12.5 central nervous system cDNA library. Sixteen of the 17 clones obtained encoded the multiple PDZ domain-containing scaffolding molecule, MAGI1 (38Laura R.P. Ross S. Koeppen H. Lasky L.A. Exp. Cell Res. 2002; 275: 155-170Crossref PubMed Scopus (77) Google Scholar). Most of the clones were derived from the cDNA portion encoding the fifth and sixth PDZ domains (PDZ4 and PDZ5) (data not shown). Although MAGI proteins have been reported to bind to the cytoplasmic domain of Delta proteins in vitro (33Wright G.J. Leslie J.D. Ariza-McNaughton L. Lewis J. Development (Camb.). 2004; 131: 5659-5669Crossref PubMed Scopus (47) Google Scholar, 37Pfister S. Przemeck G.K. Gerber J.K. Beckers J. Adamski J. Hrabe de Angelis M. J. Mol. Biol. 2003; 333: 229-235Crossref PubMed Scopus (33) Google Scholar), whether the full-length Delta interacts with MAGI1 in vivo has not yet been investigated. To examine this, we performed coimmunoprecipitation experiments using transfected 293E cells. It has been reported that many alternative splicing variants of MAGI1 are expressed in the brain (38Laura R.P. Ross S. Koeppen H. Lasky L.A. Exp. Cell Res. 2002; 275: 155-170Crossref PubMed Scopus (77) Google Scholar). We used t
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