New approaches for imaging and controlling the molecules of life.

成像和控制生命分子的新方法。

• 批准号: 17017039
• 负责人: MIYAWAKI Atsushi
• 金额: $ 35.14万
• 依托单位: The Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2009
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17017039/
• 关键词: 蛍光蛋白質; 細胞周期; レシオイメージング; 膜電位; 蛍光イメージング; 神経前駆細胞; 蛍光タンパク質; バイオイメージング

We harnessed the regulation of cell-cycle-dependent ubiquitination to develop a genetically encoded indicator for cell-cycle progression, Fucci. This technology permits us to visualize the cell-cycle behavior of individual cells within complex tissues of experimental animals, such as mice and fish. We used two new coral fluorescent proteins as FRET donor and acceptor to develop a voltage sensor, Mermaid. This technology allows for direct visualization of electrical activities in excitable cells. We developed a far-red fluorescent protein endowed with a large Stokes shift, Keima. We show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy and two-photon excitation microscopy.The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. We have also developed numerous mutants of Dronpa, which change between bright and dark states with different speeds. These mutants were used to improve performance of super-resolution microscopy. KikGR is a fluorescent protein engineered to display green-to-red photoconvertibility that is induced by irradiation with violet light. Through crystallographic studies on both green and red states, we obtained evidence that the β-elimination reaction governing the green-to-red photoconversion follows an E1 (unimolecular) mechanism. Using KikGR as an optical highlighter, we demonstrate that diffusion barrier across the nuclear envelope is less restrictive during nuclear reassembly.

我们利用细胞周期依赖性泛素化的调节来开发一种遗传编码的细胞周期进展指标Fucci。这项技术使我们能够可视化实验动物（如小鼠和鱼类）复杂组织中单个细胞的细胞周期行为。我们使用两种新的珊瑚荧光蛋白作为FRET供体和受体，开发了一种电压传感器，美人鱼。该技术允许直接可视化可兴奋细胞中的电活动。我们开发了一种具有大斯托克斯位移的远红荧光蛋白，Keima。我们展示了Keima在双色荧光成像技术（例如荧光互相关光谱和双光子激发显微镜）中的有用性。荧光蛋白Dronpa中光致变色的结构基础知之甚少。我们在环境温度下对溶液中的Dronpa进行了NMR分析，以发现蛋白质在黑暗状态下的结构灵活性。我们还开发了许多Dronpa突变体，它们以不同的速度在明亮和黑暗状态之间变化。这些突变体被用于提高超分辨率显微镜的性能。KikGR是一种荧光蛋白，其被工程化以显示由紫光照射诱导的绿色至红色光转换性。通过对绿色态和红色态的晶体学研究，我们得到了控制绿色到红色光转换的β-消除反应遵循E1（单分子）机制的证据。使用KikGR作为光学荧光笔，我们证明了在核重组过程中，穿过核被膜的扩散屏障的限制性较小。

项目成果

Medical Bio(細胞周期イメージング技術, 6(11))

医学生物（细胞周期成像技术，6（11））

• 发表时间: 2009
• 作者: 阪上-沢野朝子;正井久雄;宮脇敦史
• 通讯作者: 宮脇敦史

蛍光蛋白質

荧光蛋白

• 发表时间: 2005

Development of microscopic systems for high-speed dual-excitation ratiometric Ca(2+) imaging.

开发用于高速双激发比率 Ca(2 ) 成像的显微系统。

• 发表时间: 2008
• 期刊: Brain Cell Biology 36
• 作者: Fukano T;Shimozono S;Miyawaki A.
• 通讯作者: Miyawaki A.

Innovations in fluorescence imaging of brain functions using fluorescent proteins

使用荧光蛋白进行脑功能荧光成像的创新

• 发表时间: 2008
• 作者: Miyawaki;A.;Miyawaki A.;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史;宮脇敦史
• 通讯作者: 宮脇敦史

液晶(高輝度発光ダイオードと強誘電性液晶シャッターを用いたビデオレート細胞内カルシウムイオンイメージング, 11)

液晶（使用高亮度发光二极管和铁电液晶快门的视频速率细胞内钙离子成像，11）

• 发表时间: 2007
• 作者: 深野天;宮脇敦史
• 通讯作者: 宮脇敦史