Plasma-membrane protein / actin cytoskeleton interactions

动物和植物细胞能够利用它们的蛋白质信号网络对周围环境的信号做出反应。来自细胞外的化学物质或物理感觉会导致细胞内生化机制的变化。蛋白质的相互作用是细胞用来对环境信号做出反应的分子开关。动物细胞的主要信号通路之一依赖于细胞外膜上的蛋白质与肌动蛋白细胞骨架之间的连接。肌动蛋白在细胞膜内形成一系列细丝，支撑细胞，其他蛋白质和细胞器可以在这些细丝上移动。当膜上的受体蛋白接收到来自细胞外的信号时，它通过改变形状向肌动蛋白发出信号，进而导致激活许多细胞过程中的任何一个。植物细胞，就像动物细胞一样，能够对环境的刺激做出反应。它们的外膜也有蛋白质和肌动蛋白细胞骨架。然而，我们不知道这些膜蛋白是如何与肌动蛋白相互作用的。许多蛋白质在实验室中已经被证明具有肌动蛋白结合的特性，但这还没有在活细胞中得到证实。我们将如何确定蛋白质和肌动蛋白之间是否存在物理相互作用？我开发了一种使用共聚焦显微镜监测蛋白质运动的技术。这种类型的显微镜使用激光使细胞膜上的蛋白质发出荧光。通过将这种被称为绿色荧光蛋白(GFP)的蛋白质融合到膜蛋白上，我可以可视化膜蛋白的运动。GFP扮演着“市场标记者”的角色。我使用的绿色荧光蛋白可以被短脉冲激光激活，使我能够激活一小块膜，并在较长时间内(长达几个小时)跟踪蛋白质的运动。用数学，我可以描述蛋白质的运动，即它的运动速度和方向。我的假设是，如果蛋白质与肌动蛋白细胞骨架相互作用，那么如果我破坏细胞骨架，它的移动就会不同。有一些化学物质会导致细胞骨架的分解，这些化学物质可以应用于细胞，这样当细胞骨架缺失时，就可以测量蛋白质的运动。此外，还有一些突变植物的肌动蛋白细胞骨架存在缺陷。这些植物的外观异常是由于肌动蛋白结构不正确造成的。我预测，如果膜蛋白与异常的肌动蛋白细胞骨架联系在一起，它们在这些植物中的运动方式将会不同。这项工作将是了解质膜和肌动蛋白之间的联系的第一步，肌动蛋白在植物细胞信号传递中是如此重要。

Animal and plant cells are able to respond to cues from their surroundings using their protein signalling networks. Chemicals or physical sensations from outside the cell result in changes in the biochemical machinery inside the cell. Interactions of proteins are the molecular switches used by cells to respond to environmental cues. One of the primary signalling pathways in animal cells relys on the connection between proteins in the outer cell membrane and the actin cytoskeleton. Actin forms a series of filaments inside the cell membrane that support the cell and over which other proteins and organelles can move. When a receptor protein in the membrane receives a signal from outside the cell, it signals this to the actin by changing shape and this, in turn, results in activation of any of a number of cellular processes. Plant cells, like animal cells, are capable of responding to stimuli from their environment. They also have proteins in their outer membrane and an actin cytoskeleton. We don't, however, know how these membrane proteins interact with the actin. Many of the proteins have been shown in the lab to have actin binding properties but this has not been demonstrated in living cells. How will we determine if there is a physical interaction between the proteins and the actin? I have developed a technique to monitor protein movement using a confocal microscope. This type of microscope uses lasers to make a protein in the membrane of cells fluorescent. By fusing this protein - which is called Green Fluorescent Protein (GFP) - to a membrane protein I can visualise movement of the membrane protein. GFP acts as a 'marker.' The GFP I use can be activated by a short pulse of laser light enabling me to activate a small region of membrane and follow movement of the protein for longish periods of time (up to several hours). Using mathematics, I can describe the movement of the protein, i.e. its speed and direction of movement. My hypothesis is that if the protein interacts with the actin cytoskeleton then it will move differently if I destroy the cytoskeleton. There are chemicals that cause the cytoskeleton to breakdown and these can be applied to the cells so that protein movement can be measure when the cytoskeleton is absent. In addition, there are mutant plants that have defects in their actin cytoskeletons. These plants are abnormal looking as a result of improper actin structure. I predict that membrane proteins will move differently in these plants if they associagte with the abnormal actin cytoskeleton. This work will be the first step towards understanding the connection between plasma membrane and actin that is so essential in plant cell signalling.