Protein dynamics in Escherichia coli

活细胞的内部是一个复杂的环境，它被许多不同种类的分子密集地包裹着。其中包括许多蛋白质——执行细胞许多基本功能的生物大分子。有两种传统的方法来理解细胞中发生的过程。生化方法包括分离特定的细胞成分，在体外研究它们的行为，然后推断它们在完整细胞内的行为，通常假设细胞内部是一个相当简单的液体分子“袋”。第二种方法涉及对细胞内部组织和特定细胞成分的微观结构研究。这两种方法都不能完整地描述细胞内部的功能。生物化学方法忽略了细胞内部复杂、结构和拥挤的本质，而传统的显微镜方法显示了细胞结构，但对细胞中分子的运动提供了很少的信息。然而，有许多方法可以研究活细胞内蛋白质的运动。光漂白后荧光恢复是一项关键技术。对于FRAP，必须以某种方式用荧光标签标记感兴趣的蛋白质。当在荧光显微镜下观察细胞时，可以检测到蛋白质。通常，不能观察到单个的蛋白质分子，而荧光显微照片显示的是细胞内成百上千个蛋白质分子的分布。通过使用高度聚焦的激光束快速“漂白”细胞小区域内的荧光，可以测量蛋白质的扩散。在荧光显微照片中，漂白区域显示为暗区。如果蛋白质群是流动的，那么漂白区域就会以一种特有的方式扩散和填充。然后可以估计荧光蛋白的扩散速率。人们已经对不同种类细胞中的单个蛋白质进行了大量的FRAP研究，但令人惊讶的是，迄今为止还没有对控制蛋白质扩散速率的因素进行系统的研究。我们将以众所周知的肠道细菌大肠杆菌作为模式生物，进行这样一个系统的研究。大肠杆菌可以说是我们最了解的有机体。对于我们的研究来说，它的优点是细胞结构相对简单。此外，它很容易在基因上被操纵。在FRAP研究中，大肠杆菌的一个明显缺点是它的细胞相对较小，这使得基于光学显微镜的研究更加困难。然而，我们已经开发出克服这个问题的方法，并表明可以使用FRAP精确测量大肠杆菌细胞中的蛋白质扩散。我们将使用遗传方法为不同大小和性质的蛋白质添加荧光标签，这些蛋白质将在细胞内合成。根据蛋白质的性质，它们要么留在细胞质中，与围绕细胞质的内膜相联系，要么被输出到外周质中，即细胞内外膜之间的隔室。各种蛋白质的扩散速率将提供对细胞中这些环境的物理特性的深刻探测。我们将使用这些数据为这些环境构建物理模型。人们曾多次尝试构建大肠杆菌中蛋白质扩散过程的数学模型，但这些模型都不是基于对蛋白质扩散速率的精确实验测量。我们将利用我们的数据为细胞过程构建更现实的模型，包括细胞内传播的化学信息控制其游动方向的方式，以及细胞能够在其中点分裂的方式。这个项目将帮助我们理解细胞作为一个动态的物理系统是如何运作的。

The interior of a living cell is a complex environment which is densely packed with many different kinds of molecules. These include numerous proteins - biological macromolecules which carry out many essential functions of the cell. There are two traditional approaches to understanding the processes occurring in the cell. The biochemical approach involves isolating specific cell components, studying their behaviour in vitro, and then inferring their behaviour within the intact cell, usually on the assumption that the cell interior is a rather simple, fluid 'bag' of molecules. A second approach involves microscopic structural studies on the organisation of the cell interior and specific cell components. Neither approach gives a complete picture of the function of the cell interior. The biochemical approach neglects the complex, structured, and crowded nature of the cell interior, while the conventional microscopic approach shows cell structure but gives little information on the movement of molecules in the cell. However, there are number of ways to study the movement of proteins within living cells. A key technique is Fluorescence Recovery after Photobleaching (FRAP). For FRAP, the protein of interest must be somehow be labelled with a fluorescent tag. When the cell is observed in a fluorescence microscope, the protein can then be detected. Usually, individual protein molecules are not observed - instead the fluorescence micrograph shows the distribution of hundreds or thousands of protein molecules within the cell. The diffusion of the protein can be measured by using a highly-focussed laser beam to rapidly 'bleach' fluorescence in a small area of the cell. The bleached area appears as a dark area in the fluorescence micrograph. If the protein population is mobile, the bleached area spreads and fills in a characteristic way. The rate of diffusion of the fluorescent protein can then be estimated. There have been numerous FRAP studies on individual proteins in different kinds of cells, but surprisingly there has to date been no systematic study of the factors that control the rates of protein diffusion. We will carry out such a systematic study, using as a model organism the well-known gut bacterium Escherichia coli. E. coli is arguably the organism we understand best. For our study, it has the advantage of a relatively simple cell structure. Furthermore, it is very easy to genetically manipulate. A perceived disadvantage of E. coli for FRAP studies is that it has relatively small cells, which can make studies based on optical microscopy harder. However, we have developed ways to overcome this problem, and shown that it is possible to use FRAP to make accurate measurements of protein diffusion in E. coli cells. We will use genetic methods to add fluorescent tags to proteins of different sizes and properties, which will be synthesised inside the cell. Depending on the properties of the proteins, they will either remain in the cytoplasm, be associated with the inner membrane that surrounds the cytoplasm, or be exported into the periplasm, the compartment between the inner and outer membranes of the cell. The diffusion rates of the various proteins will provide an incisive probe of the physical properties of these environments in the cell. We will use the data to construct physical models for these environments. There have been several attempts to construct mathematical models for processes involving protein diffusion in E. coli, but these have not been based on accurate experimental measurements of the rates of protein diffusion. We will use our data to construct more realistic models for cell processes, including the way that chemical messages travelling within the cell control the direction in which it swims, and the way the cell is able to divide at its midpoint. The project will help to us to understand how a cell functions as a dynamic physical system.