Dynamics and Function of the NF-kB Signalling System

生物学中的一个主要挑战是了解细胞如何识别外部信号并做出适当的反应。现在人类基因组的序列已经完成，重要的是要为每个基因分配功能，并确定控制关键细胞功能的相应蛋白质。怀特和他的同事率先开发了基于显微镜的方法，用于可视化和测量单个活细胞中的生物过程。我们使用了萤火虫、水母和荧光珊瑚中的天然发光蛋白质。这些蛋白质的合成(表达)导致哺乳动物细胞变得发光(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在控制目标基因的启动子旁边，我们可以使用活细胞的发光作为一种测量目标基因正常开启和关闭的方法。荧光蛋白也被用来对感兴趣的蛋白质进行基因标记，这样蛋白质的运动就可以在活细胞中可视化。怀特和他的同事之前使用了时间推移荧光和发光显微镜与计算机模拟相结合来研究细胞决策。我们发现，一组重要的信号蛋白，称为核因子-kappaB，反复进入和离开细胞核，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(如莫尔斯电码)。这是一个惊喜，因为最初的核因子-kappaB蛋白p65是20年前发现的，被认为是一个简单的开关，一旦进入细胞核就能激活基因。只有在单个活细胞中的时间流逝测量才能看到这一点。核因子-kappaB系统被广泛认为对控制重要的细胞过程至关重要，包括细胞分裂和细胞死亡。它与多种疾病有关，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件。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，使用数学模型进行计算机预测对于帮助我们直观地了解这个系统是如何工作的将是至关重要的。我们将对参与核因子-kappaB信号的(大得多的)一组蛋白质以及由这些信号控制的基因进行准确的测量。(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。该项目将以结构化和有组织的方式进行管理，以便数学模型可以用于预测和设计生物实验。一个由实验官员组成的中央团队将负责协调实验、数据和模型存储以及团队成员之间的信息交流。我们将研究每一种核因子-kappaB蛋白在细胞中的分子数量、它们的稳定性、化学状态以及彼此之间以及与其他蛋白的相互作用。我们还将详细研究它们与哪些基因结合并控制它们。我们还将致力于了解作用于单个基因的单个蛋白质分子如何控制细胞的生死决定。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、滨松光电子、相干和纳米成像设备公司对培训课程和研讨会的赞助。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