High-speed imaging of FRET in live cells applied to investigate the role of PLCepsilon in intracellular signal pathways

活细胞中 FRET 的高速成像用于研究 PLCepsilon 在细胞内信号通路中的作用

• 批准号: BB/E002250/1
• 负责人: Matilda Katan
• 金额: $ 30.29万
• 依托单位: Institute of Cancer Research
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE002250%2F1
• 关键词: speed; imaging; FRET; live; cells

This multidisciplinary joint proposal between Imperial College London and the Institute of Cancer Research is to develop new technology for imaging interactions between protein molecules in live cells, to be applied to study intracellular signal pathways that are important for cancer. These protein interactions will be imaged using the fluorescence-based technique of Forster Resonant Energy transfer (FRET). Fluorescence imaging entails 'labelling' proteins of interest with fluorescent molecules (called 'fluorophores') that absorb and emit light in a characteristic manner. A recent breakthrough has been the development of genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Conventionally the proteins of interest would be 'excited' by irradiating them with photons that can absorbed by the fluorophore labels. These would then emit light (fluorescence) and relax back to their original state. By imaging the intensity of this fluorescence, one can visualise the distribution of the fluorophores / and therefore the proteins to which they are attached. To study interactions between different proteins, one can label each kind of protein with a different fluorophore emitting at a different wavelength (i.e. different colour light). By recording images of different colours / corresponding to the distributions of each kind of protein / and superimposing them, one can see where different proteins occur in the same place, i.e. co-localisation. The problem with this technique is that the spatial resolution of the optical microscopes that are used to image living cells is limited to ~ the wavelength of the light in question (about 400-700 nm) but the proteins themselves are much smaller (~ 1-10 nm). Therefore, even if two proteins appear to occur in the same place in the fluorescence image, they can be completely independent, on a molecular scale. FRET provides a way to determine when the fluorophores are within ~ 10 nm of each other / a distance at which the proteins would be interacting. It works by observing the transfer of 'excitation energy' from one fluorophore (called the 'donor' to another (called the 'acceptor') than only occurs over this very short distance. The most straightforward way to observe FRET is to see where the donor fluorescence intensity decreases or the acceptor fluorescence intensity increases. Unfortunately this kind of intensity-based imaging is often unreliable because of background noise. The most reliable way to image FRET is by fluorescence lifetime imaging (FLIM). In general fluorescence lifetime is measured by exciting fluorophores with a short pulse of light and observing how long it takes the fluorescence signal to decay away as they relax back to their ground state. Using very fast camera technology, it is possible to image fluorescence decays across a sample and obtain a value of fluorescence lifetime for each pixel in the image. Because FRET provides an additional way for excited fluorophores to lose their energy, one can determine where FRET is occurring by observing a reduction in the donor fluorescence lifetime. Unfortunately most FLIM technology is rather slow, taking several minutes to acquire a FLIM image (map of fluorescence lifetime values) and this makes it difficult to use FLIM-FRET to follow dynamics in live cells. The goal of this project is to combine novel high-speed FLIM and microscopy expertise at Imperial with the biological expertise at the ICR to develop new FLIM-FRET imaging systems. This will involve designing new molecules with appropriate donor and acceptor fluorophore labels and combining high-speed FLIM technology with novel microscope configurations. A particularly ambitious part of this proposal will be to design experiments in which we can 'multiplex FRET imaging, i.e. image two protein-protein interactions in parallel to study how different events in cells are associated in a signalling pathway.

伦敦帝国理工学院和癌症研究所之间的这项多学科联合提案旨在开发对活细胞中蛋白质分子之间的相互作用进行成像的新技术，并将其应用于研究对癌症很重要的细胞内信号通路。这些蛋白质相互作用将使用基于荧光的福斯特共振能量转移 (FRET) 技术进行成像。荧光成像需要用荧光分子（称为“荧光团”）“标记”感兴趣的蛋白质，这些荧光分子以特有的方式吸收和发射光。最近的一项突破是基因表达荧光蛋白的开发，可用于标记活细胞中的特定蛋白质。传统上，通过用荧光团标记吸收的光子照射感兴趣的蛋白质，可以“激发”它们。然后它们会发出光（荧光）并恢复到原始状态。通过对这种荧光的强度进行成像，人们可以可视化荧光团/以及它们所附着的蛋白质的分布。为了研究不同蛋白质之间的相互作用，可以用以不同波长（即不同颜色的光）发射的不同荧光团来标记每种蛋白质。通过记录不同颜色的图像/对应于每种蛋白质的分布/并将它们叠加，人们可以看到不同蛋白质出现在同一位置的位置，即共定位。这项技术的问题在于，用于对活细胞成像的光学显微镜的空间分辨率仅限于~所讨论的光的波长（约400-700 nm），但蛋白质本身要小得多（~ 1- 10 纳米）。因此，即使两种蛋白质出现在荧光图像中的同一位置，它们在分子尺度上也可以是完全独立的。 FRET 提供了一种方法来确定荧光团何时彼此相距约 10 nm/蛋白质相互作用的距离以内。它的工作原理是观察“激发能量”从一个荧光团（称为“供体”）到另一个荧光团（称为“受体”）的转移，而这种转移仅发生在很短的距离内。观察 FRET 最直接的方法是查看荧光团在何处不幸的是，由于背景噪声，这种基于强度的成像通常不可靠，最可靠的 FRET 成像方法是通过荧光寿命成像 (FLIM) 来测量。用短光脉冲激发荧光团，并观察荧光信号在放松回到基态时衰减需要多长时间，使用非常快的相机技术，可以对样品中的荧光衰减进行成像并获得值。由于 FRET 为激发的荧光团失去能量提供了另一种方式，因此可以通过观察供体荧光寿命的缩短来确定发生 FRET 的位置。不幸的是，大多数 FLIM 技术速度相当慢，需要几分钟才能获取 FLIM 图像（荧光寿命值图），这使得很难使用 FLIM-FRET 来跟踪活细胞的动态。该项目的目标是将帝国理工学院的新型高速 FLIM 和显微镜专业知识与 ICR 的生物专业知识相结合，开发新的 FLIM-FRET 成像系统。这将涉及设计具有适当供体和受体荧光团标记的新分子，并将高速 FLIM 技术与新颖的显微镜配置相结合。该提案的一个特别雄心勃勃的部分是设计实验，在这些实验中我们可以“多重 FRET 成像”，即并行成像两种蛋白质-蛋白质相互作用，以研究细胞中的不同事件如何在信号传导途径中相关联。