Dynamics and function of the NF-kappaB signalling system

生物学的一个主要挑战是了解细胞如何识别外部信号并作出适当的反应。既然人类基因组的序列已经完成，那么为每个基因分配功能并识别控制关键细胞功能的相应蛋白质就变得很重要了。怀特和他的同事率先开发了基于显微镜的方法，用于单个活细胞中生物过程的可视化和时间间隔测量。我们使用了来自萤火虫、水母和荧光珊瑚的天然发光蛋白。这些蛋白质的合成（表达）使哺乳动物细胞变成发光（在黑暗中发光）或荧光（改变光的颜色）。通过将编码发光蛋白的基因放置在控制目标基因的启动子旁边，我们可以利用活细胞的发光作为一种测量目标基因正常开启和关闭时间的方法。荧光蛋白也被用于基因标记感兴趣的蛋白质，因此蛋白质的运动可以在活细胞中可视化。White和他的同事们以前使用延时荧光和发光显微镜结合计算机模拟来研究细胞的决策。我们发现一组重要的信号蛋白，称为NF-kappaB，反复进出细胞核，这表明细胞可能使用蛋白质作为计时器来编码复杂的信息（如摩尔斯电码）。这是一个惊喜，因为最初的NF-kappaB蛋白p65是在20年前发现的，它被认为是一个简单的开关，进入细胞核一次就能激活基因。只有在单个活细胞中进行时间间隔测量才能看到这一点。NF-kappaB系统被广泛认为对包括细胞分裂和细胞死亡在内的重要细胞过程的控制至关重要。它被认为与多种疾病有关，如癌症和炎症性疾病。我们现在将开发一个实质性的系统生物学项目来研究这个复杂系统的所有组成部分。虽然之前的工作提供了重要的见解，但我们现在需要更广泛的综合实验工具来研究它。此外，利用数学模型进行计算机预测对于帮助我们可视化这个系统是如何工作的至关重要。我们将对NF-kappaB信号中涉及的（更大的）一组蛋白质和受这些信号控制的基因进行精确测量。（非常有经验的）项目团队包括生物信息学家、细胞生物学家、计算机科学家、数学家、分子生物学家、显微镜学家和蛋白质化学家。项目将以结构化和有组织的方式进行管理，以便数学建模可以用于预测和设计生物实验。由实验官员组成的中心团队将负责协调实验、数据和模型的存储以及团队成员之间的信息交流。我们将研究细胞中每个NF-kappaB蛋白的分子数，它们的稳定性，化学状态以及彼此之间以及与其他蛋白质的相互作用。我们还将详细研究它们与哪些基因结合和控制。我们还将致力于了解单个蛋白质分子如何作用于单个基因来控制细胞生死的决定。为了理解这个复杂的系统，这种多学科的方法是必不可少的。该项目的另一个目标是为博士后和学生提供培训。在这方面，我们将受益于卡尔蔡司、滨松光电、相干和纳米成像设备等仪器公司赞助的培训课程和研讨会。该项目还将受益于与Genetix和阿斯利康的持续合作

A major challenge in biology is to understand how cells recognize external signals and give appropriate responses. Now that the sequence of the human genome is complete, it is important to assign functions to each gene and to identify the corresponding proteins that control key cellular functions. White and colleagues pioneered the development of microscopy-based methods for the visualization and timelapse measurement of biological processes in single living cells. We have used natural light-emitting proteins from fireflies, jelly fish and fluorescent corals. Synthesis (expression) of these proteins causes mammalian cells to become luminescent (light emitting in the dark) or fluorescent (change the colour of light). By placing the gene that codes for a luminescent protein next to a promoter that controls a gene of interest, we can use luminescence from living cells as a way of measuring when the gene of interest is normally switched on and off. Fluorescent proteins have also been used to genetically label proteins of interest, so that the movement of the protein can be visualized in a living cell. White and colleagues previously used timelapse fluorescence and luminescence microscopy coupled to computer simulations to investigate cell decision making. We discovered that a set of important signalling proteins, called NF-kappaB, move repeatedly into and out of the nucleus of the cell, suggesting that cells may use proteins as timers to encode complex messages (like Morse Code). This was a surprise since the original NF-kappaB protein, p65, was discovered 20 years ago and was thought to act as a simple switch that moves into the nucleus once to activate genes. Only timelapse measurements in single living cells were able to see this. The NF-kappaB system is widely recognised as crucial to the control of important cellular processes including both cell division and cell death. It is implicated as being involved in a variety of diseases, such as cancer and inflammatory disease. We will now develop a substantial systems biology project to study all of the components of this complex system. While the previous work has provided major insights, we now need a far broader range of integrated experimental tools to study it. Also the use of mathematical models to make computer predictions will be critical to help us to visualize how this system works. We will make accurate measurements of the (much larger) set of proteins that are involved in NF-kappaB signalling and the genes that are controlled by these signals. The (very experienced) project team includes bioinformaticians, cell biologists, computer scientists, mathematicians, molecular biologists, microscopists and protein chemists. The project will be managed in a structured and organized way, so that the mathematical modelling can be used to predict and design the biological experiments. A central team of experimental officers will be responsible for coordinating the experiments, data and model storage and communication of information between team members. We will study the numbers of molecules of each of the NF-kappaB proteins in the cell, their stability, chemical states and interactions with each other and with other proteins. We will also study in detail which genes that they bind to and control. We will also aim to understand how single protein molecules acting at single genes can act to control decisions of cell life and death. This multidisciplinary approach is essential in order to understand this complex system. A further aim of the project is to provide training for post-docs and students. In this respect, we will benefit from sponsorship of training courses and symposia by the instrumentation companies Carl Zeiss, Hamamatsu Photonics, Coherent and Nano Imaging Devices. The project will also benefit from ongoing collaborations with Genetix and AstraZeneca