Implementation of a Bayesian Segmentation Algorithm to the analysis of receptor conformational changes in multidimensional single-molecule data

细胞间的通讯是通过向血液中分泌化学信使来实现的，其中重要的一类是生长因子。生长因子的主要功能是促进细胞增殖。没有生长因子，生命是不可生存的，因为它们是协调细胞生长以维持每个器官的完整性和功能所必需的。为了检测生长信号，细胞插入其表面受体蛋白，其功能是结合血液中的生长因子，然后可能通过构象的改变和/或与其他受体的相互作用，将信号通过质膜传递到细胞内部。当细胞内蛋白质在受体中识别到信号的开始时，它们就会启动导致信号放大的反应，最终导致DNA复制和细胞分裂。近年来，生长因子受体的发现爆炸式增长，包括直接的临床应用，如赫赛汀，因为这些受体与许多人类肿瘤的发展有关。这种联系并不令人惊讶，因为恶性肿瘤只是不受控制的细胞增殖的产物。了解生长促进和生长抑制事件之间微妙平衡背后的分子机制，从而导致不受控制的细胞生长和恶性肿瘤，是目前科学界最大的努力之一。这些事件需要在分子水平和活细胞的背景下被理解，因为受体信号本质上是一个非同步的事件，其细胞的结果取决于细胞环境的特定特征，以及哪些受体伴侣被允许或不被允许在附近。为此，我们在之前的拨款中开发了独特的成像仪器，可以通过荧光显微镜观察活细胞中单个受体分子的信号行为。在这个应用程序中，我们的目标是实施一个工作计划，通过这个计划，我们将能够使用最先进的图像分析算法来分析我们的单分子受体数据，该算法最初是为了从极其微弱、嘈杂和模糊的天文数据中提取定量信息而开发的。在单分子研究中，来自单个受体的微弱信号与活细胞荧光背景的不可避免的存在相结合，意味着数据中的基本信息可能被背景噪声模糊和/或隐藏。在我们寻求实现的成像分析方法中，关于受体数据的预先存在的信息可以明确地包含在图像分析算法中，从而创建一个更强大的算法，并证明其结果值得信赖。结合这些新颖的图像分析方法，我们所建立的仪器将成为分子尺度上精确的光谱尺和量角器，以报告自然细胞环境中受体构象动力学。希望对正常和致癌生长因子受体的行为机制的定量阐明将最终有助于设计新的抑制剂分子来对抗突变或过表达受体在癌症中的作用。

Intercellular communication is achieved via secretion into the blood of chemical messengers, an important class of which are the growth factors. The main function of growth factors is the promotion of cell multiplication. Without growth factors life is not viable, as they are required to orchestrate cell growth in a manner that maintains the integrity and function of each organ. To detect growth signals cells insert into their surface receptor proteins whose function is to bind the growth factors in the blood and then transduce, probably via a change in conformation and/or interaction with other receptors, the signal across the plasma membrane to the cell interior. When intracellular proteins recognise in the receptor the onset of signalling, they initiate the reactions that lead to the amplification of this signal, an ultimately to DNA replication and cell division. Recent years have seen an explosion of discoveries in growth factor receptors, including direct clinical applications such as Herceptin, as these receptors are linked to the development of a number of human tumours. This link is hardly surprising, as a malignant tumour is just the product of uncontrolled cell proliferation. Understanding the molecular mechanisms behind the delicate balance between growth-promoting and growth-inhibiting events giving rise to uncontrolled cell growth and malignancy is currently one of the biggest endeavours in science. Such events need to be understood at the molecular level and in the context of the living cell, as receptor signalling is by nature an unsynchronised event whose outcome for the cell depends on the particular characteristics of the cell environment and on which receptor partners are allowed or not in the vicinity. For this purpose we have developed during previous grants unique imaging instrumentation that allows the visualisation through a fluorescence microscope of the signalling behaviour of individual receptor molecules in a living cell. In this application we aim to implement a programme of work by which we will be able to analyse our single molecule receptor data using a state-of-the art image analysis algorithm originally developed to extract quantitative information from extremely faint, noisy and blurred astronomical data. In single molecule research, the combination of weak signals from single receptors with the unavoidable presence of the fluorescence background from living cells implies that essential information in the data can be blurred and/or hidden by the background noise. In the imaging analysis method we seek to implement, pre-existing information on the receptor data can be explicitly included in the image analysis algorithms, creating a much more powerful algorithm with demonstrated trustworthy results. In combination with these novel image analysis methods the instrument we have built will become an accurate spectroscopic ruler and protractor at the molecular scale to report on receptor conformational dynamics in the natural cell environment. It is hoped that the quantitative elucidation of the mechanisms of behaviour of normal and oncogenic growth factor receptors will ultimately help in the designing of new inhibitor molecules that antagonise the action of mutant or overexpressed receptor in cancer.