細胞極性確立とmRNA局在のin vivo イメージング

• Project/Area Number: 11242202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University (2001-2003); Nagoya University (1999-2000)
• Principal Investigator: 入江 賢児 大阪大学, 医学系研究科, 助教授 (90232628)
• Project Period (FY): 1999 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥55,900,000 (Direct Cost: ¥55,900,000); Fiscal Year 2003: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 2002: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 2001: ¥12,700,000 (Direct Cost: ¥12,700,000); Fiscal Year 2000: ¥12,700,000 (Direct Cost: ¥12,700,000); Fiscal Year 1999: ¥13,500,000 (Direct Cost: ¥13,500,000)
• Keywords: 非対称分裂 / mRNA / RNA結合蛋白質 / 酵母 / 細胞骨格 / アクチン / 翻訳 / ミオシン / RNA結合タンパク質 / ASHI / シオシン / GFP

Research Abstract

本研究では、細胞極性の分子レベルでの研究が最も進んでいる出芽酵母を用いて、mRNAや細胞極性決定因子の局在のin vivoイメージングの系を確立し、その系を用いてmRNAの細胞内局在の分子機構機構および細胞極性の確立や維持に関与する因子のmRNA局在における機能を明らかにする。また、動物細胞から酵母相同遺伝子を機能的に分離することにより、動物細胞におけるmRNAの局在機構およびその細胞極性との関連を明らかにする。さらに、動物細胞において細胞極性の形成に重要な役割を果たす細胞間接着の形成機構について解析する。本年度は、酵母および培養細胞を用いた細胞極性、非対称分裂、mRNAの局在、細胞間接着形成の機構の解析から、以下の結果を得た。
(1)NucleaseドメインをもつMkt1がPbp1と複合体を形成して、HO mRNAの3'非翻訳領域を介して、HO遺伝子の発現を正に制御することを明らかにした。
(2)ASH1 mRNAの娘細胞の先端への局在に必要なShe4が、ASH1 mRNAを輸送するMyo4モータータンパク質のシャペロンであることを明らかにした。
(3)上皮細胞における細胞間接着形成時において、接着分子ネクチン-3が、運動している細胞の先端部に局在するネクチン様分子Nec1-5とヘテロな接着を形成し、この接着がアドヘレンスジャンクション形成の初期に機能することを見出した。
(4)上皮細胞のアドヘレンスジャンクションにおいて、アドヘレンスジャンクション形成における2つの細胞間接着機構、ネクチン-アファディン系とカドヘリン-カテニン系を連結する因子ADIP,LMO7を見出した。
このように、本年度の研究は予想以上に進展し、当初の目的を達成することができた。

Research Products

• [Publications] Asada, M.: "ADIP, a novel afadin- and α-actinin-binding protein localized at cell-cell adherens junctions."J.Biol.Chem.. 278・6. 4103-4111 (2003)
• [Publications] Toi, H.: "She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae."Mol.Biol.Cell. 14・6. 2237-2249 (2003)
• [Publications] Katata, T.: "Involvement of nectin in the localization of IQGAP1 at the cell-cell adhesion sites through the actin cytoskeleton in Madin-Darby canine kidney cells."Oncogene. 22・14. 2097-2109 (2003)
• [Publications] Honda, T.: "Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions."Biochem.Biophys.Res.Commun.. 306・1. 104-109 (2003)
• [Publications] Honda, T.: "Antagonistic and agonistic effects of an extracellular fragment of nectin on formation of E-cadherin-based cell-cell adhesion."Genes Cells. 8・1. 51-63 (2003)
• [Publications] Honda, T.: "Cdc42 and Rac small G proteins activated by trans-interactions of nectins are involved in activation of c-Jun N-terminal kinase, but not in association of nectins and cadherin to form adherens junctions, in fibroblasts."Genes Cells. 8・5. 481-491 (2003)
• [Publications] Yamada, A.: "Nectin-dependent localization of synaptic scaffolding molecule (S-SCAM) at the puncta adherentia junctions formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the mouse hippocampus."Genes Cells. 8・12. 985-994 (2003)
• [Publications] Inagaki, M.: "Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus."J.Comp.Neurol.. 460・4. 514-524 (2003)
• [Publications] Takai, Y.: "Nectins and nectin-like molecules : Roles in cell adhesion, migration, and polarization."Cancer Science. 94・8. 655-667 (2003)
• [Publications] Yamamoto, Y.: "Vmac : A novel protein associated with vimentin-type intermediate filament in podocytes of rat kidney."Biochem.Biophys.Res.Commun.. 315・4. 1120-1125 (2004)
• [Publications] Yamamoto, Y.: "Direct binding of the human homologue of the drosophila disc large tumor suppressor gene to seven-pass transmembrane proteins, tumor endothelial marker 5 (TEM5) and a novel TEM5-like protein."Oncogene. in press. (2004)
• [Publications] Irie, K.: "Roles of nectins in cell adhesion, signaling, and polarization."Handbook of Experimental Pharmacology. in press. (2004)
• [Publications] Tadauchi, T.: "Post-transcriptional regulation of HO expression by the Mkt1-Pbp1 complex."Mol.Cell.Biol.. in press. (2004)
• [Publications] Irie, K.: "The Khd1 protein, which has three KH RNA-binding motifs, is required for proper localization of ASH1 mRNA in yeast"EMBO J.. 21・5. 58-67 (2002)
• [Publications] Ozaki-Kuroda, K.: "Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions"Curr. Biol.. 12・13. 1145-1150 (2002)
• [Publications] Fukuhara, A.: "Involvement of nectin in the localization of junctional adhesion molecule at tight junctions"Oncogene. 21・50. 7642-7655 (2002)
• [Publications] Momose, Y.: "Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion"Biochem. Biophys. Res. Commun.. 293・1. 45-49 (2002)
• [Publications] Tanaka, Y.: "Ectodomain shedding of nectin-1α by SF/HGF and TPA in MDCK cells"Biochem. Biophys. Res. Commun.. 299・3. 472-478 (2002)
• [Publications] Fukuhara, A.: "Role of nectin in organization of tight junctions in epithelial cells"Genes Cells. 7・10. 1059-1072 (2002)
• [Publications] Asada, M.: "ADIP, a novel afadin-and α-actinin-binding protein localized at cell-cell adherens junctions"J. Biol. Chem.. 278・6. 4103-4111 (2003)
• [Publications] Honda, T.: "Antagonistic and agonistic effects of an extracellular fragment of nectin on formation of E-cadherin-based cell-cell adhesion"Genes Cells. 8・1. 51-63 (2003)
• [Publications] Toi, H.: "She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae"Mol. Biol. Cell. (in press). (2003)
• [Publications] Katata, T.: "Involvement of nectin in the localization of IQGAP1 at the cell-cell adhesion sites through the actin cytoskeleton in Madin-Darby canine kidney cells"Oncogene. (in press). (2003)
• [Publications] Honda, T.: "Cdc42 and Rac small G proteins activated by trans-interactions of nectins are involved in activation of c-Jun N-terminal kinase, but not in association of nectins and E-cadherin to form adherens junctions, in fibroblasts"Genes Cells. (in press). (2003)
• [Publications] Inagaki, M.: "Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus"J. Comp. Neurol.. (in press). (2003)
• [Publications] Irie, K.: "The Khd1 protein, which has three KH RNA-binding motifs, is required for proper localization of ASH1 mRNA in yeast"EMBO J.. 21・5. 1158-1167 (2002)
• [Publications] Tadauchi, T.: "Post-transcriptional regulation through the HO 3´-UTR by Mpt5, a yeast homolog of Pumilio and FBF"EMBO J.. 20・3. 552-561 (2001)
• [Publications] Inoue, H.: "A Drosophila MAPKKK, D-MEKK1, mediates stress responses through activation of p38 MAPK"EMBO J.. 20・19. 5421-5430 (2001)
• [Publications] Ozaki-Kuroda, K.: "Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae."Mol Cell Biol.. 21・3. 827-839 (2001)
• [Publications] Yokoyama, S.: "α-catenin-independent recruitment of Zo-1 to nectin-based cell-cell adhesion sites through afadin"Mol.Biol.Cell. 12・6. 1595-1609 (2001)
• [Publications] Tadauchi et al.: "Post-transcriptional regulation through the HD 3'-UTR by Mpc5, a yeast homolog of Pumilio and FBF"EMBO Journal. 20. 552-561 (2001)
• [Publications] Ozaki-Kuroda et al.: "Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae"Molecular and Cellular Biology. 21. 827-839 (2001)
• [Publications] Takaesu et al.: "TAB2, a novel adaptor protein, mediates activation of TAKI MAPKKK by linking TAKI to TRAF6 in the IL-1 signal transduction pathway"Molecular Cell. 5. 649-658 (2000)
• [Publications] Takihara et al.: "14-3-3 protein-family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells"Experimental Cell Research. 260. 96-104 (2000)
• [Publications] Inagaki et al.: "PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast"Molecular and Cellular Biology. 19・12. 8344-8352 (1999)
• [Publications] Suzanne et al.: "The Drosophila p38 MAPK pathway is required during oogenesis for egg asymmetric development"Genes & Development. 13・11. 1464-1474 (1999)
• [Publications] Adachi-Yamada et al.: "p38 mitogen-activated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis"Molecular and Celluar Biology. 19・3. 2322-2329 (1999)
• [Publications] Yamaguchi et al.: "XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TABI-TAKI in the BMP singaling pathway"EMBO Journal. 18・1. 179-187 (1999)