Intracellular fluorescent probes for chemical processes in single living cells

用于单个活细胞化学过程的细胞内荧光探针

• 批准号: 13554030
• 负责人: UMEZAWA Yoshio
• 金额: $ 9.15万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13554030/
• 关键词: fluorescent probes; protein; protein interaction; non-destructive analysis; second messengers; protein phosphorylation; ミトコンドリア; エストロゲン; GFP; 蛋白質間相互作用; ルシフェラーゼ; 疎水場感受性蛍光試薬; サイクリックGMP

For nondestructive analysis of chemical processes in living cells, we have developed some general methods and new intracellular fluorescent probes for detecting 1) second messengers, cGMP, diacylglycerol and phosphatidylinositol-3,4,5-triphosphate, 2) protein phosphorylation, 3) protein confbrmational changes, 4) protein-protein interactions, and 5) protein localizations in live cells under a confocal laser microscope.The approaches for the present probe developments are use of fluorescence resonance energy transfer for reporting binding of substrates (analytes) to molecular recognition domains in dual-fluorophore conjugated probe molecules, and use of protein splicing chemistry for detecting protein-protein interactions.Key molecules and steps of cellular signaling pathways were visualized in relevant live cells using developed fluorescent probe molecules. These probes were found of general importance not only for fundamental biology studies, but also for assay and screening methods for chemicals that inhibit or facilitate cellular signaling pathways.

为了对活细胞内的化学过程进行无损分析，我们开发了一些通用的方法和新的细胞内荧光探针，用于检测1）第二信使，cGMP，二酰基甘油和磷脂酰肌醇-3，4，5-三磷酸，2）蛋白质磷酸化，3）蛋白质构象变化，4）蛋白质-蛋白质相互作用，5）激光共聚焦显微镜下活细胞中蛋白质的定位。目前探针开发的方法是利用荧光共振能量转移来报告底物的结合（分析物）到双荧光团缀合探针分子中的分子识别结构域，以及使用蛋白质剪接化学检测蛋白质-蛋白质相互作用。使用开发的荧光探针分子在相关活细胞中可视化细胞信号传导途径的关键分子和步骤。发现这些探针不仅对于基础生物学研究具有普遍重要性，而且对于抑制或促进细胞信号传导途径的化学物质的测定和筛选方法也具有普遍重要性。

项目成果

Y. Umezawa: "Seeing What Was Unseen. New Analytical Methods for Molecular Imaging"The Chemical Record. 3. 22-28 (2003)

Y. Umezawa：“看到看不见的东西。分子成像的新分析方法”化学记录。

T. Ozawa, Y. Sako, M. Sato, T. Kitamura, and Y Umezawa: "A Genetic Approach to Identifying Mitochondrial Proteins"Nature Biotech.. 21. 287-293 (2003)

T. Ozawa、Y. Sako、M. Sato、T. Kitamura 和 Y Umezawa：“识别线粒体蛋白的遗传方法”Nature Biotech.. 21. 287-293 (2003)

M.Sata, et al.: "Fluorescent Indicators for Imaging Protein Phosphorylation in Single Living Cells"Nature Biotech. (in press). (2002)

M.Sata 等人：“单个活细胞中蛋白质磷酸化成像的荧光指示剂”《自然生物技术》。

T.Ozawa et al.: "A Genetic Approach to Identifying Mitochondrial Proteins"Nature Biotechnology. 21. 287-293 (2003)

T.Ozawa 等人：“识别线粒体蛋白的遗传方法”《自然生物技术》。

T. Ozawa, and Y. Umezawa: "Peptide Assemblies in Living Cells : Methods for Detecting Protein-Protein Interactions"Supramolecular Chemistry. 14. 271-280 (2002)

T. Ozawa 和 Y. Umezawa：“活细胞中的肽组装：检测蛋白质-蛋白质相互作用的方法”超分子化学。