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Multidimensional fluorescence imaging of PIP2-derived intracellular signals in directional cell movement

定向细胞运动中 PIP2 衍生的细胞内信号的多维荧光成像

基本信息

批准号：
BB/H00713X/1
负责人：
Paul Michael William French
金额：
$ 48.29万
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2010
资助国家：
英国
起止时间：
2010 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FH00713X%2F1
关键词：
Multidimensional fluorescence imaging PIP2 derived

项目摘要

Directional cell migration is important during early development, inflammatory responses to infection, wound healing, and also during tumor invasion and metastasis. Since deregulation of this process has been linked to various pathological events, it has become an active area of research for therapies. Different lines of experimental evidence suggest that various types of migratory cells share a conserved set of signals involved in cell polarization and motility. Several classes of signalling molecules, including enzymes involved in turnover and modification of phosphoinositides and components of signalling networks controlling Rho GTP-ases, play key roles in these processes. Recent studies using advanced fluorescence microscopy suggest that understanding how intracellular signals control directional cell movement is critically dependent on their dynamic organization in time and space. Fluorescence microscopy entails 'labelling' proteins of interest with fluorescent molecules ('fluorophores') such as genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Fluorophores are 'excited' by illumination at a wavelength that they absorb and the resulting emission (fluorescence) is recorded using an imaging detector. The technique called Förster Resonant Energy transfer (FRET) works by labelling two proteins with different fluorophores or incorporating different fluorophors into single protein that changes shape in response to specific signals. The property of the two fluorophores is chosen so that the excitation spectrum on one (the 'acceptor') overlaps with the emission spectrum of the other (the 'donor') and results in the transfer of energy from the excited donor to the acceptor only if they are in close proximity. This provides a basis to measure protein/protein interactions (not just co-localisation) and to use single protein probes as biosensors. One can image FRET by observing the donor or acceptor fluorescence intensity distributions but such intensity-based FRET is often unreliable because of background noise. More reliable techniques include mapping the ratio of acceptor to donor fluorescence and fluorescence lifetime imaging (FLIM) of the donor signal. 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 ultrafast camera technology, it is possible to image fluorescence decays across a sample and map the fluorescence lifetime. Because FRET provides an additional route for excited donor fluorophores to lose their energy, FLIM can map where FRET is occurring by observing the resulting reduction in donor fluorescence lifetime. In a recent BBSRC project we developed a novel high-speed FLIM microscope able to 'multiplex' FRET imaging of two probes (either for protein-protein interaction or biosensors). This permits us to simultaneously map the spatiotemporal properties of two different signalling events in live cells. Here we intend to extend the approach to multiplex more simultaneous signalling events and to use FRET to focus on some of the key components controlling the directional movement of cells, in particular those associated with intracellular signals derived from a membrane phosphoinositide, PIP2. Our goal is to correlate these with other intracellular signals in the same polarized, moving cell and to analyse dynamic aspects of their timing and localisation (e.g. front and back of polarized cell). This would provide new insights into the sequence of cell signalling interactions and some underlying molecular mechanisms important for the development of therapies for various pathological events resulting from deregulation of directional cell movement. The technical innovations of FRET methodology proposed here would find wide application in biology.

定向细胞迁移在早期发育、对感染的炎症反应、伤口愈合以及在肿瘤侵袭和转移期间是重要的。由于这一过程的失调与各种病理事件有关，因此它已成为治疗研究的一个活跃领域。不同的实验证据表明，不同类型的迁移细胞共享一组保守的信号参与细胞极化和运动。几类信号分子，包括参与磷酸肌醇的周转和修饰的酶和控制Rho GTP酶的信号网络的组分，在这些过程中发挥关键作用。最近的研究使用先进的荧光显微镜表明，了解细胞内信号如何控制定向细胞运动是至关重要的依赖于它们在时间和空间的动态组织。荧光显微镜需要用荧光分子（“荧光团”）“标记”感兴趣的蛋白质，例如可用于标记活细胞中特定蛋白质的遗传表达的荧光蛋白。荧光团被它们吸收的波长的照明“激发”，并且使用成像检测器记录所产生的发射（荧光）。这种被称为Förster共振能量转移（FRET）的技术通过用不同的荧光团标记两种蛋白质或将不同的荧光团掺入单个蛋白质中来工作，该蛋白质会响应特定信号而改变形状。选择两个荧光团的性质，使得一个（“受体”）上的激发光谱与另一个（“供体”）的发射光谱重叠，并且仅当它们非常接近时才导致能量从激发的供体转移到受体。这为测量蛋白质/蛋白质相互作用（不仅仅是共定位）和使用单一蛋白质探针作为生物传感器提供了基础。人们可以通过观察供体或受体的荧光强度分布来对FRET成像，但由于背景噪声，这种基于强度的FRET通常是不可靠的。更可靠的技术包括绘制受体与供体荧光的比率和供体信号的荧光寿命成像（FLIM）。一般来说，荧光寿命是通过用短脉冲光激发荧光团并观察荧光信号在松弛回到基态时衰减多久来测量的。使用超快相机技术，可以对样品中的荧光衰减进行成像，并绘制荧光寿命。由于FRET提供了一个额外的途径激发供体荧光团失去其能量，FLIM可以通过观察供体荧光寿命的减少来映射FRET发生的位置。在最近的BBSRC项目中，我们开发了一种新型的高速FLIM显微镜，能够“多重”FRET成像的两个探针（无论是蛋白质-蛋白质相互作用或生物传感器）。这使我们能够同时映射活细胞中两个不同信号事件的时空特性。在这里，我们打算扩展的方法，多路复用更多的同时信号事件，并使用FRET集中在一些控制细胞的定向运动的关键组件，特别是那些与来自膜磷酸肌醇，PIP 2的细胞内信号。我们的目标是将这些信号与同一极化运动细胞中的其他细胞内信号相关联，并分析其时序和定位的动态方面（例如极化细胞的前后）。这将为细胞信号相互作用的序列和一些潜在的分子机制提供新的见解，这些分子机制对于开发由定向细胞运动失调引起的各种病理事件的疗法很重要。本文提出的FRET方法的技术创新将在生物学中得到广泛的应用。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fluorescence lifetime imaging: FLIM for cell biology, drug discovery and label-free diagnosis

荧光寿命成像：用于细胞生物学、药物发现和无标记诊断的 FLIM

DOI：
发表时间：
2011
期刊：
Optics InfoBase Conference Papers
影响因子：
0
作者：
French P.M.W.
通讯作者：
French P.M.W.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

利用自动荧光寿命成像显微镜进行开源高内涵分析

DOI：
10.3791/55119-v
发表时间：
2017
期刊：
Journal of Visualized Experiments
影响因子：
0
作者：
French P
通讯作者：
French P

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

DOI：
10.1002/jbio.201500258
发表时间：
2016-04
期刊：
Journal of biophotonics
影响因子：
2.8
作者：
Andrews N;Ramel MC;Kumar S;Alexandrov Y;Kelly DJ;Warren SC;Kerry L;Lockwood N;Frolov A;Frankel P;Bugeon L;McGinty J;Dallman MJ;French PM
通讯作者：
French PM

Fluorescence Lifetime Imaging for Biomedicine

生物医学荧光寿命成像