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

细胞的定向迁移在早期发育、感染的炎症反应、伤口愈合以及肿瘤的侵袭和转移过程中都是重要的。由于这一过程的解除与各种病理事件有关，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与细胞极化和运动有关的保守信号。几类信号分子，包括参与肌醇磷脂转化和修饰的酶和控制Rho GTP酶的信号网络组件，在这些过程中发挥着关键作用。最近使用先进的荧光显微镜的研究表明，理解细胞内信号如何控制细胞的定向运动严重依赖于它们在时间和空间上的动态组织。荧光显微镜需要用荧光分子(‘荧光团’)‘标记’感兴趣的蛋白质，例如可以用来标记活细胞中特定蛋白质的基因表达的荧光蛋白。荧光团在它们吸收的波长上被光激发，由此产生的发射(荧光)用成像探测器记录下来。这项名为Förster Resonant Energy Transfer(FRET)的技术的工作原理是用不同的荧光团标记两种蛋白质，或者将不同的荧光团结合到单个蛋白质中，该蛋白质会根据特定的信号改变形状。两个荧光团的性质是这样选择的，即其中一个(受主)的激发光谱与另一个(施主)的发射光谱重叠，只有当它们很接近时，才会导致能量从受主转移到受主。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成像FRET，但由于背景噪声的存在，这种基于强度的FRET往往是不可靠的。更可靠的技术包括绘制受体与供体的比率、荧光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到基态时需要多长时间衰减来测量的。使用超快相机技术，可以对样品上的荧光衰变进行成像，并绘制荧光寿命图。由于FRET为被激发的供体荧光团提供了另一条失去能量的途径，FLIM可以通过观察由此导致的供体荧光寿命的缩短来绘制FRET发生的位置。在BBSRC最近的一个项目中，我们开发了一种新型的高速薄膜显微镜，能够对两个探针(用于蛋白质-蛋白质相互作用或生物传感器)的FRET成像进行多路传输。这允许我们同时映射活细胞中两个不同信令事件的时空属性。在这里，我们打算扩展该方法以多路传输更多的同时信号事件，并使用FRET来专注于控制细胞定向运动的一些关键组件，特别是那些与来自膜磷脂酰肌醇PIP2的细胞内信号相关的组件。我们的目标是将这些信号与同一极化移动细胞中的其他细胞内信号相关联，并分析它们的时序和定位的动态方面(例如，极化细胞的前面和后面)。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治疗因细胞定向运动放松管制而导致的各种病理事件非常重要。本文提出的FRET方法学的技术创新将在生物学中得到广泛的应用。

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.