Cell-surface mechanism for stabilisation of plasma membrane protein dynamics

细胞被脂质和蛋白质组成的膜所包围。许多细胞过程，如与环境的沟通，防御病原体的攻击，和分子的摄取是由膜蛋白介导的。最近的几项发现表明，并非所有蛋白质都能在细胞膜平面内自由扩散。膜蛋白分布的不均匀性被称为膜亚结构，并以对细胞功能至关重要的方式浓缩蛋白质和蛋白质复合物，如孔。我们实验室的工作表明，在植物细胞中，细胞骨架（细胞内的支架结构）和细胞壁（细胞外的支撑结构）都在膜亚结构中发挥作用。我们不知道大多数膜蛋白如何与细胞壁相互作用，也不知道细胞骨架结构的改变是否会影响膜蛋白的分布。我们研究膜蛋白分布和扩散的方法是用荧光颜色标记单个蛋白质，以便使用高分辨率显微镜在活细胞中观察它们。最近开发的一种技术现在可以让我们观察细胞膜内的单个分子。这项技术的改进应该使我们能够确定分子在扩散时是否遵循轨道或仅限于膜的小区域。为了确定细胞壁的哪些成分与膜蛋白相互作用，我们将研究植物中蛋白质的扩散，这些蛋白质在细胞壁结构的不同方面被改变或缺乏。可能影响膜蛋白组织和扩散的细胞壁组分包括纤维素、果胶和半纤维素。为了研究细胞骨架重排对膜蛋白运动的影响，我们将生产出改变了FORMIN1含量的植物。当这种蛋白质过多时，它会导致细胞骨架发生非常剧烈的、高度分支化的重排。由于这种现象会杀死幼苗，我们将改变FORMIN1水平，使幼苗在诱导FORMIN1水平降低或升高之前可以生长到适当的研究阶段。最后，我们最近观察到一种名为VAP 36的蛋白质在稳定细胞膜中的点中起作用，这些点对于构建称为内质网的内部膜系统至关重要。VAP 36的分布和移动将使用所描述的技术在改变的细胞壁和细胞骨架条件下进行研究，以便我们可以确定这些结构的改变是否影响细胞内以及细胞表面的过程。作为本研究的实际应用，我们将使用这些技术来研究植物防御机制被激活时细胞膜蛋白的扩散。当植物细胞受到病原体攻击时，它们会利用复杂的信号传导机制进行感知，但病原体攻击的早期阶段对细胞膜亚结构的影响尚未研究。这项工作将提供有关细胞膜在抵御细菌和真菌病原体攻击中的作用的见解，这些病原体每年破坏很大比例的粮食作物。

Cells are surrounded by membranes composed of lipids and proteins. Many cellular processes such as communication with the environment, defense from pathogen attack, and uptake of molecules are mediated by membrane proteins. Several recent discoveries show that not all proteins diffuse freely within the plane of the cell membrane. Inhomogeneity in membrane protein distribution is called membrane sub-structuring and concentrates proteins and protein complexes such as pores in a way that is vital for cell function. Work in our laboratories has demonstrated that, in plant cells, both the cytoskeleton, a scaffolding structure inside cells, and the cell wall, a supporting structure outside of cells, play roles in membrane sub-structuring. We do not know how the majority of membrane proteins interact with the cell wall or whether alterations in cytoskeleton structure will affect membrane protein distribution. Our approach to the study of membrane protein distribution and diffusion is to tag individual proteins with a fluorescent colour so that they are observable in living cells using high resolution microscopes. One recently developed technique now lets us observe single molecules within the cell membrane. Refinement of this technique should allow us to determine if molecules follow tracks or are restricted to small regions of the membrane as they diffuse. To determine what components of the cell wall interact with membrane proteins, we will examine diffusion of proteins in plants that are altered or deficient in different aspects of cell wall structure. Cell wall components that might affect membrane protein organisation and diffusion include cellulose, pectin, and hemicellulose. To examine the effect of cytoskeleton rearrangement on membrane protein movement, we will produce plants that have altered amounts of FORMIN1. This protein causes a very drastic, highly branching rearrangement of the cytoskeleton when it is overly abundant. Because this phenomenon kills seedlings, we will alter FORMIN1 levels in such a way that seedlings can grow to an appropriate stage for study before inducing a reduction or increase in FORMIN1 levels. Finally, we have recently observed that a protein called VAP36 plays a role in stabilising points in the cell membrane that are important for structuring of an internal membrane system known as the endoplasmic reticulum. The distribution and movement of VAP36 will be studied in altered cell wall and cytoskeleton conditions using the techniques described so that we can determine whether alterations in these structures affect processes within cells as well as those at the cell surface.As a practical application of this research, we will use these techniques to study the diffusion of cell membrane proteins when plant defense mechanisms are activated. Plant cells sense when they are under attack by pathogens using elaborate signalling mechanisms but the effect of the early stages of pathogen attack on cell-membrane sub-structuring have not been studied. This work will provide insight about the role of cell membranes in fending off attack from bacterial and fungal pathogens that destroy a large percentage of food crops annually.