Imaging of Synamics of Cellular Functions using GFP-based probes

使用基于 GFP 的探针对细胞功能进行成像

• 批准号: 11480186
• 负责人: MIYAWAKI Atsushi
• 金额: $ 10.05万
• 依托单位: The Institute of Physical and Chemical Research, RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480186/
• 关键词: GFP; RFP; circularly-permuted GFP; Ca^<2+> indicator; calmodulin; random mutagenesis; in vitro mutagenesis; pericam; カルシウムイメージング; cameleon; レーザー走査顕微鏡; プレートイメージアナライザー; 蛍光偏光度測定

Our primary goal is to gain a better understanding of how the molecules for life behave in space and time. Signal transduction cascades involve multiple enzymes and are orchestrated by specific protein-protein interactions. Such dynamics is revealed by optical means such as fluorescence readout. The green fluorescent protein (GFP)of the jellyfish Arequioreare victoria is a spontaneously fluorescent protein that can be ncorporated into other proteins by genetic fusion. One of our approaches is to use two GFPs of different colors to permit fluorescence resonance energy transfer(FRET), which is highly sensitive to the relative orientation and distance between the two fluorophores and alters the ratio of their emission intensities, and ideal readout for fast imaging and confocal microscopy. Cameleons are genetically-encoded fluorescent indicators for Ca^<2+>-based on FRET using GFPs. Another approach is to use circularly permuted GFP(cpGFP), in which the aminoand carboxy-portions have been interchanged and reconnected by a short spacer between the original termini. We have created Ca^<2+> -sensitive cpGFP, "pericam". Because cameleons and pericams can be targeted genetically and imaged by one- or two-photon excitation microscopy, they allowed monitoring Ca^<2+> in whole organisms, tissues, organelles, and submicroscopic environments where measurements were previously impossible. Dynamic changes in intracellular Ca^<2+> concentrations control many important celluar events. We have extended optical methods to develop some fluorescent indicators for visualization of the Ca^<2+> -related events, which are currently assayed by grinding millions of cells. Furthermore, we have developed optical hardwares for multi-color fluorescence imaging : the simultaneous observation of the Ca^<2+>-rerated events in conjunction with ca^<2+> dynamics in cells loaded with two or more fluorescent indicators.

我们的主要目标是更好地了解生命分子在时空中的行为方式。信号转导级联反应涉及多种酶，并由特定的蛋白质 - 蛋白质相互作用策划。这种动力学是通过诸如荧光读数之类的光学手段揭示的。水母的绿色荧光蛋白（GFP）是维多利亚的Quioreare Victoria是一种自发的荧光蛋白，可以通过遗传融合将其纳入其他蛋白质。我们的一种方法是使用两种不同颜色的GFP允许荧光共振能量转移（FRET），该荧光共振能量转移（FRET）对两个荧光团之间的相对取向和距离高度敏感，并改变了其发射强度的比率，并适合快速读取。成像和共聚焦显微镜。 Cameleons是基于GFP的FRET的遗传编码荧光指标。另一种方法是使用循环排列的GFP（CPGFP），其中氨基羧基段已经通过原始末端之间的短垫片互换和重新连接。我们创建了Ca^<2+> - 敏感的CPGFP，“ Pericam”。由于Cameleons和Cameleons和CapiCAM可以遗传靶向并通过一或两光子激发显微镜成像，因此它们允许在整个生物体，组织，细胞器和微观镜面环境中监测CA^<2+>以前是不可能进行测量的。细胞内Ca^<2+>浓度的动态变化控制着许多重要的均等事件。我们已经采用了扩展的光学方法来开发一些荧光指标，以可视化Ca^<2+>相关事件，这些事件目前通过研磨数百万个单元进行测定。此外，我们开发了用于多色荧光成像的光学硬件：同时观察Ca^<2+> - 延迟的事件以及带有两个或更多荧光指示剂的细胞中的CA^<2+>动力学。

项目成果

Mizuno H. , Sawano A. , Eli P. , Hama H. and Miyawaki A.: "Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer "Biochemistry. 40(8). 2502-2510 (2001)

Mizuno H.、Sawano A.、Eli P.、Hama H. 和 Miyawaki A.：“来自 Discosoma 的红色荧光蛋白作为融合标签和荧光共振能量转移的伙伴”生物化学。

Sawano A. and Miyawaki A.: "Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis"Nucleic Acids Research . 28, No. 16. e78 (2000)

Sawano A. 和 Miyawaki A.：“通过用于定点和半随机诱变的新型多功能 PCR 策略来定向进化绿色荧光蛋白”核酸研究。

Matsushita F.,Miyawaki A.,and Mikoshiba K.: "Vomeroglandin/CRP-ductin is strongly expressed in the glands associated with the mouse vomeronasal organ : Identification and characterization of mouse vomeroglandin"Biochemical and Biophysical Research Communi

Matsushita F.、Miyawaki A. 和 Mikoshiba K.：“犁骨素/CRP-ductin 在与小鼠犁鼻器官相关的腺体中强烈表达：小鼠犁骨素的鉴定和表征”生物化学和生物物理研究社区

Nagai T. , Sawano A. , Park E. , and Miyawaki A.: "Circularly permuted green fluorescent proteins engineered to sense to Ca^<2+>"Proceeding National Academy Science USA. 98, No. 6. 3197-3203 (2001)

Nagai T.、Sawano A.、Park E. 和 Miyawaki A.：“循环排列的绿色荧光蛋白被设计用于感知 Ca^<2>”，《美国国家科学院院刊》。

Miyawaki A. , Mizuno H. , Llopis J. , Tsien R. Y. and Jalink K.: "Cameleons as Cytosolic and intra-organellar calcium probes"Calcium Signalling . second Edition. 3-16 (2000)

Miyawaki A.、Mizuno H.、Llopis J.、Tsien R. Y. 和 Jalink K.：“Cameleons 作为细胞质和细胞器内钙探针”钙信号传导。