Imaging of Synamics of Cellular Functions using GFP-based probes

我们的主要目标是更好地了解生命分子在空间和时间中的行为。信号转导级联涉及多个酶，并由特定的蛋白质-蛋白质相互作用协调。这种动力学通过诸如荧光读出的光学手段来揭示。水母Arequioreare维多利亚的绿色荧光蛋白（GFP）是一种自发荧光蛋白，可以通过基因融合整合到其他蛋白质中。我们的方法之一是使用两种不同颜色的GFP，以允许荧光共振能量转移（FRET），这是高度敏感的相对方向和两个荧光团之间的距离，并改变其发射强度的比例，和理想的快速成像和共聚焦显微镜读出。基于使用GFP的FRET，Cameleon是遗传编码的Ca^<2+>-荧光指示剂。另一种方法是使用环状排列的GFP（cpGFP），其中氨基和羧基部分已经互换，并通过原始末端之间的短间隔子重新连接。我们已经创建了Ca^<2+>敏感的cpGFP，“pericam”。由于可以从遗传学角度对钙离子和钙包膜进行定位，并通过单光子或双光子激发显微镜成像，因此它们可以监测整个生物体、组织、细胞器和亚微观环境中的Ca^2+，而这些在以前是无法测量的。细胞内Ca^2+浓度的动态变化控制着许多重要的细胞事件.我们已经扩展了光学方法，开发了一些荧光指示剂，用于可视化Ca^2+相关事件，目前通过研磨数百万个细胞进行分析。此外，我们还开发了用于多色荧光成像的光学硬件：在装有两种或更多种荧光指示剂的细胞中，同时观察Ca ^2+相关事件和Ca^2+动力学。

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.