Development of high-throughput system of visible immunoprecipitation assay

• Project/Area Number: 15K14456
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Kyoto University
• Principal Investigator: Nakayama Kazuhisa 京都大学, 薬学研究科, 教授 (40192679)
• Co-Investigator(Renkei-kenkyūsha): KATOH Yohei 京都大学, 大学院薬学研究科, 助教 (90568172)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: タンパク質間相互作用 / GFP / Nanobody / 繊毛内タンパク質輸送複合体 / マイクロプレート / ポリスチレン親和性タグ

Outline of Final Research Achievements

The visible immunoprecipitation (VIP) assay is a convenient, flexible method to detect protein-protein interactions. In this study, I attempted to improve the VIP assay to detect protein-protein interactions on a microplate and enable high-throughput screening.
By fusing polystyrene (PS)-affinity peptide sequence to anti-GFP-Nanoboy, I successfully immobilized it to PS microplate. The sensitivity was further enhanced by ELISA, through optimizing the concentrations of the secondary and tertiary antibodies.

Research Products

• [Journal Article] Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E. (2017)
  • Author(s): Nozaki S, Katoh Y, Terada M, Michisaka S, Funabashi T, Takahashi S, Kontani K, Nakayama K.
  • Journal Title: J. Cell. Sci. Volume: 130 Issue: 3 Pages: 563-576
  • DOI: 10.1242/jcs.197004
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein-coupled receptors (2017)
  • Author(s): Hirano T, Katoh Y, Nakayama K
  • Journal Title: Mol. Biol. Cell Volume: 28 Issue: 3 Pages: 429-439
  • DOI: 10.1091/mbc.e16-11-0813
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Ciliary entry of KIF17 is dependent on its binding to the IFT-B complex via IFT46-IFT56 as well as on its nuclear localization signal (2017)
  • Author(s): Funabashi T, Katoh Y, Michisaka S, Terada M, Sugawa M, Nakayama K
  • Journal Title: Mol. Biol. Cell Volume: 28 Issue: 5 Pages: 624-633
  • DOI: 10.1091/mbc.e16-09-0648
  • NAID: 120005997941
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system (2017)
  • Author(s): Katoh Y, Michisaka S, Nozaki S, Funabashi T, Hirano T, Takei R, Nakayama K.
  • Journal Title: Mol. Biol. Cell Volume: 28 Issue: 7 Pages: 898-906
  • DOI: 10.1091/mbc.e17-01-0051
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Overall architecture of the intraflagellar transport (IFT)-B complex containing Cluap1/IFT38 as an essential component of the IFT-B peripheral subcomplex. (2016)
  • Author(s): Katoh Y, Terada M, Nishijima Y, Takei R, Nozaki S, Hamada H, Nakayama K
  • Journal Title: The Journal of Biological Chemistry Volume: 291 Issue: 21 Pages: 10962-10975
  • DOI: 10.1074/jbc.m116.713883
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Alteration of transbilayer phospholipid compositions is involved in cell adhesion, cell spreading, and focal adhesion formation (2016)
  • Author(s): Miyano R, Matsumoto T, Takatsu H, Nakayama K, Shin HW
  • Journal Title: FEBS Lett. Volume: 590 Issue: 14 Pages: 2138-2145
  • DOI: 10.1002/1873-3468.12247
  • NAID: 120007159976
  • Peer Reviewed
• [Journal Article] The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane (2016)
  • Author(s): Tanaka Y, Ono N, Shima T, Tanaka G, Katoh Y, Nakayama K, Takatsu H, Shin HW
  • Journal Title: Mol. Biol. Cell Volume: 27 Issue: 24 Pages: 3883-3893
  • DOI: 10.1091/mbc.e16-08-0586
  • NAID: 120005997942
  • Peer Reviewed / Open Access
• [Journal Article] Modulation of primary cilia length by melanin-concentrating hormone receptor 1. Cell Signaling (2016)
  • Author(s): Hamamoto A, Yamato S, Katoh Y, Nakayama K, Yoshimura K, Takeda S, Kobayashi Y, *Saito Y
  • Journal Title: Cellular Signalling Volume: 28 Issue: 6 Pages: 272-584
  • DOI: 10.1016/j.cellsig.2016.02.018
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Class I Arfs (Arf1 and Arf3) and Arf6 are localized to the Flemming body and play important roles in cytokinesis. (2016)
  • Author(s): Hanai, A., Ohgi, M., Yagi, C., Ueda, T., Shin, H.-W., and Nakayama, K.
  • Journal Title: Journal of Biochemistry Volume: 159 Issue: 2 Pages: 201-208
  • DOI: 10.1093/jb/mvv088
  • Peer Reviewed
• [Journal Article] Modulation of ciliary length by melanin-concentrating hormone receptor 1. (2016)
  • Author(s): Hamamoto, A., Yamato, S., Katoh, Y., Nakayama, K., Yoshimura, K., Takeda, S.,
  • Journal Title: Cell. Signal. Volume: 28 Pages: 572-584
  • Peer Reviewed
• [Journal Article] GM130 is a parallel tetramer with a flexible rod-like structure and N-terminally open (Y-shaped) and closed (I-shaped) conformations. (2015)
  • Author(s): Ishida R, Yamamoto A, Nakayama K, Sohda M, Misumi Y, Yasunaga T, Nakamura N.
  • Journal Title: FEBS J. Volume: 印刷中 Issue: 11 Pages: 2232-2244
  • DOI: 10.1111/febs.13271
  • Peer Reviewed
• [Journal Article] Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. (2015)
  • Author(s): Katoh Y, Nozaki S, Hartanto D, Miyano R, Nakayama K
  • Journal Title: Journal of Cell Science Volume: 128 Issue: 12 Pages: 2351-2362
  • DOI: 10.1242/jcs.168740
  • Peer Reviewed / Open Access
• [Journal Article] Phospholipid flippase ATP10A translocates phosphatidylcholine and is involved in plasma membrane dynamics. (2015)
  • Author(s): Naito, T., Takatsu, H., Miyano, R., Takada, N., Nakayama, K., and Shin, H.-W.
  • Journal Title: Journal of Biological Chemistry Volume: 290 Issue: 24 Pages: 15004-15017
  • DOI: 10.1074/jbc.m115.655191
  • NAID: 120005690225
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles. (2015)
  • Author(s): Kubo K, Kobayashi M, Nozaki S, Yagi C, Hatsuzawa K, Katoh Y, Shin HW, Takahashi S, Nakayama K
  • Journal Title: Biology Open Volume: 4 Issue: 7 Pages: 910-920
  • DOI: 10.1242/bio.012146
  • NAID: 120005997718
  • Peer Reviewed / Open Access
• [Journal Article] COPI-mediated retrieval of SCAP is critical for regulating lipogenesis under basal and sterol-deficient conditions. (2015)
  • Author(s): Takashima, K., Saitoh, A., Funabashi, T., Hirose, S., Yagi, C., Nozaki, S., Sato, R., Shin, H.-W., and Nakayama, K.
  • Journal Title: Journal of Cell Science Volume: 128 Pages: 2805-2815
  • DOI: 10.1242/jcs.164137
  • Peer Reviewed
• [Journal Article] ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells. (2015)
  • Author(s): Takada, N., Takatsu, H., Miyano, R., Nakayama, K., and Shin, H.-W.
  • Journal Title: Jounal of Lipid Research Volume: 56 Issue: 11 Pages: 2151-2157
  • DOI: 10.1194/jlr.m062547
  • NAID: 120005707794
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Phospholipid flippase ATP10A, a me mber of type IV P-type ATPases, translocates phosphatidylcholine and is involved in plasma membrane dynamics. (2016)
  • Author(s): Naito, T., Takatsu, H., Miyano, R., Takada, N., Nakayama, K. & Shin, H.-W.
  • Organizer: 2016 ASCB Annu al Meeting.
  • Place of Presentation: San Francisco, California, USA,
  • Year and Date: 2016-12-03
  • Int'l Joint Research
• [Presentation] Regulation of ciliary retrograde protein trafficking by Joubert syndrome proteins Arl13b and INPP5E. (2016)
  • Author(s): Nozaki, S., Katoh, Y. & Nakayama, K.
  • Organizer: 2016 ASCB Annu al Meeting.
  • Place of Presentation: San Francisco, California, USA,
  • Year and Date: 2016-12-03
  • Int'l Joint Research
• [Presentation] Arl13b-dependant localization of INPP5E within cilia maintains ciliary r etrograde protein trafficking. (2016)
  • Author(s): Nozaki, S., Katoh, Y. & Nakayama, K.
  • Organizer: The 28th CDB Meeting, “Cilia and Centrosomes: Current Advances and Future Directions”
  • Place of Presentation: 神戸
  • Year and Date: 2016-11-27
  • Int'l Joint Research
• [Presentation] Overall architecture of the intraflagellar transport (IFT)-B complex rev ealed by a visible immunoprecipitation assay. (2016)
  • Author(s): Katoh, Y., Terada, M. & Nakayama, K.
  • Organizer: he 28th CDB Meeting, “Cilia and Centrosomes: Current Advances and Future Dir ections”
  • Place of Presentation: 神戸
  • Year and Date: 2016-11-27
  • Int'l Joint Research
• [Presentation] Differential roles of KIF17 and heterotrimeric kinesin-II interacting with the IFT-B complex in biogenesis of primary cilia. (2016)
  • Author(s): Funabashi, T., Katoh, Y. & Nakayama, K.
  • Organizer: The 28th CDB Meeting, “Cilia and Centrosomes: Current Advances and Future Directions”.
  • Place of Presentation: 神戸
  • Year and Date: 2016-11-27
  • Int'l Joint Research
• [Presentation] IFT-A複合体と相互作用するC11orf74の繊毛内タンパク質輸送における役割． (2016)
  • Author(s): 高原万梨子、平野友章、加藤洋平、中山和久
  • Organizer: 第89回日 本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
• [Presentation] キネシン2とIFT複合体の相互作用の様式の解明と繊毛形成における役割． (2016)
  • Author(s): 船橋輝記、加藤洋平、中山和久
  • Organizer: 第89回日 本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
• [Presentation] P4-ATPaseによるリン脂質フリップが膜変形に与える影響． (2016)
  • Author(s): 高田直人、中山和久、申 惠媛
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
• [Presentation] リン脂質フリッパーゼATP11CのPKCによる制御機構． (2016)
  • Author(s): 高津宏之、高山真裕、中山和久、申 惠媛
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
• [Presentation] ジュベール症候群原因遺伝子産物Arl13bによって制御される繊毛内タンパク質輸送の分子機構． (2016)
  • Author(s): 加藤洋平、野崎梢平、中山和久
  • Organizer: 第 89回日本生化学会大会シンポジウム「Arfファミリー低分子量Gタンパク質群が介在する多彩な生理機能と関連疾患」
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
  • Invited
• [Presentation] 繊毛内のイノシトールリン脂質代謝とタンパク質の局在におけるArl13bとIFT-Bの役割． (2016)
  • Author(s): 野崎梢平、加藤洋平、中山和久
  • Organizer: 第15回 次 世代を担う若手ファーマ・バイオフォーラム2016
  • Place of Presentation: 大阪
  • Year and Date: 2016-09-10
• [Presentation] 繊毛内タンパク質の輸送を担うIFT-A複合体の構築様式と機能の解明． (2016)
  • Author(s): 平野友章、加藤洋平、中山和久
  • Organizer: 第15回 次 世代を担う若手ファーマ・バイオフォーラム2016
  • Place of Presentation: 大阪
  • Year and Date: 2016-09-10
• [Presentation] 中心体/基底小体で複合体を形成するRabL2-Cep19-FOPの機能の解析． (2016)
  • Author(s): 西島侑哉、萩谷遥平、加藤洋平、中山和久
  • Organizer: 第15回 次 世代を担う若手ファーマ・バイオフォーラム2016
  • Place of Presentation: 大阪
  • Year and Date: 2016-09-10
• [Presentation] 繊毛内のイノシトールリン脂質代謝とタンパク質の局在におけるArl13bとIFT-Bの役割． (2016)
  • Author(s): 野崎梢平、加藤洋平、中山和久
  • Organizer: 第68回日本細胞生物学会大会
  • Place of Presentation: 京都
  • Year and Date: 2016-06-16
• [Presentation] 繊毛内タンパク質輸送複合体IFT-Aの構築様式と機能の解明． (2016)
  • Author(s): 平野友章、加藤洋平、中山和久
  • Organizer: 第68回日本細胞生物学会大会
  • Place of Presentation: 京都
  • Year and Date: 2016-06-15
• [Presentation] KIF17とIFT複合体の相互作用様式の解明と繊毛形成における役割． (2016)
  • Author(s): 船橋輝記、加藤洋平、中山和久
  • Organizer: 第68回日本細胞生物学会大会
  • Place of Presentation: 京都
  • Year and Date: 2016-06-15
• [Presentation] 観るだけでわかるタンパク質間相互作用解析法」を活用した繊毛内タンパク質輸送複合体 IFT-Bの構築様式の解明． (2016)
  • Author(s): 加藤洋平、寺田将也、野崎梢平
  • Organizer: 第68回日本細胞生物学会大会
  • Place of Presentation: 京都
  • Year and Date: 2016-06-15
• [Presentation] Trafficking Machineries within Cilia: Arc hitectures and Functions of the IFT-A and IFT-B complexes. (2016)
  • Author(s): Nakayama, K., Katoh, Y., Terada, T., Nozaki, S., Hirano, T. & Funabashi, T.
  • Organizer: 第68回日本細胞生物学会大会シンポジウム「高次生命機能を司るメンブレ ントラフィック：分子基盤からその破綻による疾患発症の理解に向けて」
  • Place of Presentation: 京都
  • Year and Date: 2016-06-15
  • Invited
• [Presentation] 「観るだけでわかるタンパク質間相互作用解析法」の開発と繊毛内タンパク質輸送複合体 IFT-B の 構築様式の解明． (2016)
  • Author(s): 加藤洋平、寺田将也、中山和久
  • Organizer: 第63回日本生化学会近畿支部例会
  • Place of Presentation: 神戸
  • Year and Date: 2016-05-21
• [Presentation] KIF17 と IFT 複合体の相互作用様式の解明と繊毛内輸送における役割． (2016)
  • Author(s): 船橋輝記、加藤洋平、中山和久
  • Organizer: 第63回日本生化学会近畿支部例会
  • Place of Presentation: 神戸
  • Year and Date: 2016-05-21
• [Presentation] ホスファチジルセリン取り込み異常のCHO-K1変異株（UPS-1）におけるflippaseの発現解析． (2015)
  • Author(s): 高田直人、高津宏之、宮野吏永、中山和久、申惠媛
  • Organizer: 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
• [Presentation] 繊毛内タンパク質輸送複合体IFT-Bの構築様式とCLUAP1の機能の解明． (2015)
  • Author(s): 寺田将也、加藤洋平、野崎梢平、武井領汰、中山和久
  • Organizer: 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
• [Presentation] 中心体/基底小体で複合体を形成するRabL2とCep19の機能の解析． (2015)
  • Author(s): 西島侑哉、萩谷遥平、加藤洋平、中山和久
  • Organizer: 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
• [Presentation] COPI被覆小胞を介するSCAP-SREBP複合体のゴルジ体から小胞体への逆行輸送による (2015)
  • Author(s): 船橋輝記、高島皓平、齋藤明奈、廣瀬祥平、申惠媛、中山和久
  • Organizer: 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
• [Presentation] Visible immunoprecipitation（VIP）アッセイによるExocyst複合体の構築様式の解明． (2015)
  • Author(s): 野崎梢平、加藤洋平、中山和久
  • Organizer: 第14回 次世代を担う若手ファーマ・バイオフォーラム2015
  • Place of Presentation: 千葉
  • Year and Date: 2015-09-12
• [Presentation] COPI被覆小胞を介するSCAP-SREBP複合体のゴルジ体-小胞体間の逆行輸送による脂質代謝調節． (2015)
  • Author(s): 船橋輝記、高島皓平、齋藤明奈、廣瀬祥平、申 惠媛、中山和久
  • Organizer: 第14回 次世代を担う若手ファーマ・バイオフォーラム2015
  • Place of Presentation: 千葉
  • Year and Date: 2015-09-12
• [Presentation] リン脂質フリッパーゼATP10AのPCフリップ活性および細胞膜ダイナ ミクスへの関与 (2015)
  • Author(s): 内藤朋樹、高津宏之、宮野吏永、高田直人、中山和久、申 惠媛
  • Organizer: 第14回 次世代を担う若手ファーマ・バイオフォーラム2015
  • Place of Presentation: 千葉
  • Year and Date: 2015-09-12
• [Presentation] Architectures of the BBSome, IFT-B, and Exocyst Complexes Revealed by Visible Immunoprecipitation (VIP) Assay Using Fluorescent Fusion Proteins. (2015)
  • Author(s): Nakayama, K., Katoh, Y., Nozaki, S., & Terada, M.
  • Organizer: FASEB Science Research Conference, “Biology o f Cilia and Flagella”
  • Place of Presentation: Snowmass Village, Colorado,
  • Year and Date: 2015-07-19
  • Int'l Joint Research
• [Presentation] Visible immunoprecipitation (VIP) アッセイによるマルチサブユニット複合体の構築様式の解明 (2015)
  • Author(s): 加藤洋平、野崎梢平、寺田将也、中山和久
  • Organizer: 第67回日本細胞生物学会大会
  • Place of Presentation: 東京
  • Year and Date: 2015-06-30
• [Presentation] リン脂質フリッパーゼATP10AのPCフリップ活性及び細胞膜ダイナミクスへの関与． (2015)
  • Author(s): 内藤朋樹、高津宏之、宮野吏永、高田直人、中山和久、申惠媛
  • Organizer: 第62回日本生化学会近畿支部例会
  • Place of Presentation: 草津
  • Year and Date: 2015-05-16