我々は生体分子の動きや生体分子間に起こる動的な相互作用を生きている細胞内でイメージングすることは、動的な生体機能を可視化することに直結すると考えた。そこで細胞核内の構造を蛍光相関分光法(FCS; Fluorescence Correlation Spectroscopy)を利用して，分子運動の特性に注目して解析し，これまでのイメージング手法では明らかにできなかった核の機能と密接関連したダイナミックな機構が存在する事を示した（特定領域公募研究H17-H18）。さらに拡散測定に基礎をおくFCSだけでは分子量比が極端に大きくないと分子間の相互作用が検出できない、また内在性タンパク質の影響が無視できないなどの限界が判明した。その限界を打ち破るために時間と空間の相互相関測定（FCCS）を利用してこれまでの研究をさらに発展させていくことを計画した。そのためにまず走査型の多点FCS装置の試作と細胞内相互作用解析を確立した（図１）。

　次に具体的なターゲットとしてGlucocorticoid Receptor（GR）に注目した。GRはステロイドホルモンレセプターの一種であり、グルココルチコイドホルモン（リガンド）と結合することで細胞質から核内への移行し、ホモ二量体としてターゲット遺伝子のエンハンサー部位（Glucocorticoid Response Element：GRE）へ結合することで転写を調節している。我々のこれまでFCS測定の結果、最終的に二量体化することで転写調節が開始されるというステップを示唆しているが、細胞のどこで（空間）、何時（時間）に２量化するかは不明のままである。その解明のため、FCCS時間相関解析における、蛍光寿命測定による補正方法を完成させることを目指した。これまでさまざまなリンカー長を有するGFP２量体を作成し，その長さとFCSにおける一分子蛍光強度との関係を明らかにした。FCCSの弱点であった内在性たんぱく質見積もりを可能として、内在性のタンパク質の影響が定量的に推定できるようになることが期待できる。つまり、GFP単量体とGFP２量体、RFP単量体とRFP２量体量を補正し、寿命とFCCSからGFP-RFP複合体量の補正を行い、定量的な結合量を推定することが可能となることが期待できる。

発表論文リスト：

1. Yumi Noda, Saburo Horikawa, Eiichiro Kanda, Maho Yamashita, Hu Meng, Kayoko Eto, Yuhua Li, Michio Kuwahara, Keiji Hirai, Changi Pack, Masataka Kinjo, Shigeo Okabe, and Sei Sasaki.Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking.J. Cell Biol., Aug 2008; 182: 587-601.
2. Nagao I, Aoki Y, Tanaka M, Kinjo M.Analysis of the molecular dynamics of medaka nuage proteins by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. FEBS J. 275(2), 341-349 (2008)
3. Nagaya H, Tamura T, Higa-Nishiyama A, Ohashi K, Takeuchi M, Hashimoto H,Hatsuzawa K, Kinjo M, Okada T, Wada I.Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum. J Cell Biol. 180(1), 129-43 (2008)
4. Fumihiko Fujii and Masataka, Kinjo.Detection of antigen protein using fluorescence cross-correlation spectroscopy and quantum-dots labeled antibody , ChemBioChem 8, 2199-2203, (2007)
5. Fujii F, Horiuchi M, Ueno M, Sakata H, Nagao I, Tamura M, Kinjo M.Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy.. Anal Biochem, 370, 131-141(2007)
6. Takahashi Y, Okamoto Y, Popiel HA, Fujikake N, Toda T, Kinjo M, Nagai Y.Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy.. J Biol Chem,282, 24039-24048 (2007)
7. Kabayama K, Sato T, Saito K, Loberto N, Prinetti A, Sonnino S, Kinjo M, Igarashi Y, Inokuchi J.Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance. Proc Natl Acad Sci U S A, 104(34), 13678-13683. (2007)
8. Masafumi Shimizu, Satoshi Sasaki and Masataka Kinjo.Triplet Fraction Buildup Effect of the DNA-YOYO Complex Studied with Fluorescence Correlation Spectroscopy.Analytical Biochem., 336(1), 87-92, (2007)
9. Yoichi Araki, Takanori Kawano, Hidenori Taru, Yuhki Saito, Sachiyo Wada, Kanako Miyamoto, Hisako Kobayashi, Hiroyuki O. Ishikawa, Yu Ohsugi, Tohru Yamamoto, Kenji Matsuno, Masataka Kinjo, and Toshiharu Suzuki.The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport.EMBO J 26, 1475-1486 (2007)
10. Shintaro Mikuni, Changi Pack, Mamoru Tamura and Masataka Kinjo.Diffusion analysis of glucocorticoid receptor and antagonist effect in living cell nucleus. Experimental and Molecular Pathology, 82(2), 163-168, (2007)
11. Shintaro Mikuni, Mamoru Tamura and Masataka Kinjo.Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy. FEBS Letters 581, 389-393, (2007)
12. Hiroaki Kii, Takuya Takagi, Akira Sasaki, Takaharu Okajima, and Masataka Kinjo.DNA Microstructure Based on Self-Assembly of 4-Sticky-End Holiday Junctions in Aqueous Solution.Journal of Nanoscience and Nanotechnology 7, 726-729 (2007)
13. Rieko Oyama, Hideaki Takashima, Masato Yonezawa, Nobuhide Doi, Etsuko Miyamoto-Sato, Masataka Kinjo and Hiroshi Yanagawa.Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy.Nucleic Acids Research, Vol. 34, No. 14 e102 (2006)
14. Changi Pack, Kenta Saito, Mamoru Tamura, and Masataka Kinjo.Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs.Biophys J. vol 91 3921-3936 (2006)
15. Yu Ohsugi, Kenta Saito, Mamoru Tamura and Masataka Kinjo.Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy.Biophys J. 91, 3456-3464 (2006)
16. Y. Ohsugi and Masataka Kinjo.Analysis of membrane-binding protein mobility in living cells using total internal reflection fluorescence correlation spectroscopy.Biophysical Reviews and Letters, Vol. 1, No. 3, 293-299 (2006)
17. Hiroki Konno, Tomoe Murakami, Fumihiko Fujii, Fumie Koyama, Hanayo Ueoka-Nakanishi, Chan-Gi Pack, Masataka Kinjo, and Toru Hisabori.The regulator of the F1 motor: Inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.EMBO J. 25, 4596-4604 (2006)
18. Akira Kitamura, Hiroshi Kubota, Chan-Gi Pack, Gen Matsumoto, Shoshiro Hirayama, Yasuo Takahashi, Hiroshi Kimura, Masataka Kinjo, Richard I. Morimoto and Kazuhiro Nagata.Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state.Nature Cell Biology 8(10):1163-1169 (2006)
19. Shigeko Kawai-Noma Satoru Ayano, Chan-Gi Pack, Masataka Kinjo, Masasuke Yoshida, Kenji Yasuda and Hideki Taguchi.Dynamics of yeast prion aggregates in single living cells.Genes to Cells 11, 1085-1096, (2006)
20. Yasutomo Nomura, Hirobumi Fuchigami, Hiroaki Kii, Zhonggang Feng, Takao Nakamura and Masataka Kinjo.Direct quantification of gene expression using fluorescence correlation.spectroscopy" Current Pharmaceutical Biotechnology. (2006)
21. Hideki Muto, Issei Nagao, Taku Demura, Hiroo Fukuda, Masataka Kinjo, and Kotaro T. Yamamoto.Fluorescence cross-correlation analyses of molecular interaction between an Aux/IAA protein, MSG2/IAA19, and protein-protein interaction domains of auxin response factors of Arabidopsis expressed in HeLa cells.Plant Cell Physiol., 47, 1095-1101 (2006)
22. Takako Kogure, Satoshi Karasawa, Toshio Araki, Kenta Saito, Masataka Kinjo, and Atsushi Miyawaki A. fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.Naturt Biotechnol 30, 577- 581 (2006).
23. Yasutomo Nomura, Hirobumi Fuchigami, Hiroaki Kii, Zhonggang Feng, Takao Nakamura and Masataka Kinjo.Quantification of size distribution of restriction fragments in mitochondrial genome using fluorescence correlation spectroscopy.Experimental and Molecular Pathology, 80, 275-278 (2006)
24. Yasutomo Nomura, Hirobumi Fuchigami, Hiroaki Kii, Zhonggang Feng, Takao Nakamura and Masataka Kinjo.Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy.Analytical Biochemistry, 350, 196-201 (2006)
25. Maya Hirose, Hideki Tohda, Yuko Giga-Hama, Reiko Tsushima, Tamotsu Zako, Ryo Iizuka, Changi Pack, Masataka Kinjo, Noriyuki Ishii, and Masafumi Yohda.Interaction of a small heat shock protein of the fission yeast, Schizosaccharomyces pombe, with a denatured protein at elevated temperature.J. Biol. Chem; 280, 32586-32593 (2005) .
26. Yasuo Takahashi, Ryuji Sawada, Kiyochika Ishibashi, Sintarou Mikuni and Masataka Kinjo.Analysis of Cellular Functions by Multipoint Fluorescence Correlation Spectroscopy Curr. Pharm. Biotech. 6, 159-156 (2005)