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细胞周期进展的基因编码指标 Fucci，这项技术使我们能够可视化实验动物（例如小鼠和小鼠）复杂组织内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 供体和受体来开发电压传感器 Mermaid，该技术可以直接可视化可兴奋细胞中的电活动。 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。我们对荧光蛋白 Dronpa 的光致变色的结构基础知之甚少。 Dronpa 在环境温度下的溶液中寻找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。 KikGR 是一种荧光蛋白，可通过紫光照射诱导显示绿光到红光的光电转换，通过对绿色和红色状态的晶体学研究，我们获得了 β-消除反应控制的证据。绿色到红色的光转换遵循 E1（单分子）机制，使用 KikGR 作为光学荧光笔，我们证明核膜上的扩散势垒在核过程中受到的限制较小。重新组装。

项目成果

Spatially restricted generation of fluorescence signals using simultaneous 488- and 405-nm irradiation

使用同时 488 和 405 nm 照射产生空间受限的荧光信号

• 发表时间: 2007
• 作者: Miyawaki A.;Sakaue-Sawano A.;Ando R.;Mizuno H.;Kogure T.;Kurokawa H.;Sugiyama M.;Sugimura K.;宮脇敦史;Miyawaki A.;宮脇敦史;Miyawaki A.;Miyawaki A.;Miyawaki A.;Miyawaki A.
• 通讯作者: Miyawaki A.

Fast dual-excitation ratiometry with light-emitting diodes and high-speed liquid crystal shutters.

使用发光二极管和高速液晶快门进行快速双激发比率测定。

• 发表时间: 2005
• 期刊: Biochem Biophys Res Commun. 330
• 作者: Fukano T;Shimozono S;Miyawaki A.
• 通讯作者: Miyawaki A.

Spatio-temporal Patterns of Intracellular Signaling

细胞内信号传导的时空模式

• 发表时间: 2006
• 作者: Miyawaki A.;Sakaue-Sawano A.;Ando R.;Mizuno H.;Kogure T.;Kurokawa H.;Sugiyama M.;Sugimura K.;宮脇敦史;Miyawaki A.;宮脇敦史;Miyawaki A.;Miyawaki A.;Miyawaki A.;Miyawaki A.;Miyawaki A.;Miyawaki A.
• 通讯作者: Miyawaki A.

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 Biol. 36
• 作者: Fukano T;Shimozono S;Miyawaki A.
• 通讯作者: Miyawaki A.

GFP-like proteins stably accumulation in lysosomes.

GFP 样蛋白在溶酶体中稳定积累。

• 发表时间: 2008
• 期刊: Cell Struct. Funct. In Press
• 作者: katayama H.;et. al.
• 通讯作者: et. al.