Cell-surface mechanism for stabilisation of plasma membrane protein dynamics

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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.