Cell-surface mechanism for stabilisation of plasma membrane protein dynamics

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.

This project will utilise some of the latest developments in bioimaging technology to investigate the mechanisms of protein localisation at the plant cell surface. A new observation from our laboratories is that the cell wall constrains diffusion of PM proteins.

Several scientific questions will be addressed using fluorescence recovery after photobleaching (FRAP) and total internal reflection - single molecule tracking (TIRF-SMT) techniques. We are seeking to refine the TIRF-SMT analysis technique by generating a large amount of data using TIRF-SMT on photoactivatable-GFP labelled PM proteins so that we can unambiguously describe molecular diffusion constraints by mean square diffusion analysis.

Most important of the project goals is to study PM protein diffusion when either the actin cytoskeleton or the cell wall are perturbed. We have fluorescently-tagged PM proteins that fall into several different classes based on membrane anchoring type and protein function. These include FORMIN1, LTI6b, PINs, PIPs, SNAREs, CESA, AGP4, FLS2, GPA1 and various others. Overexpression of the Arabidopsis PM protein FORMIN1 results in an almost mat-like cortical actin morphology so this protein will be co-expressed with the others and their diffusion characteristics will be monitored to assess the effect of actin morphology modification.

Similarly, the set of PM proteins will be expressed in cell-wall structure mutant lines of Arabidopsis to assess the effect of altered cell wall on protein dynamics. Cell wall mutant lines will include those deficient in structural components such as CESA6, EXP1, XTH2, BGAL1.

We will also seek to evaluate the effect of treatment with pathogen elicitors such as flagellin (FLG22) or chitin. This will help us understand whether early stages of the pathogen response include or are mediated by alterations in protein diffusion and association within the PM.

This project will seek to determine the cellular mechanisms behind our recent discovery that the plant cell wall affects plasma membrane protein distribution and dynamics. We will investigate interactions between components of the ER, actin and microtubule cytoskeletons, plasma membrane, and cell wall. Results of this work will be important for both plant and animal cell biologists as they will highlight key regulatory mechanisms at the cell surface. This type of research in our laboratories has recently attracted the interest of those doing research in diverse fields including development and response to environmental change. As part of this project, we will study plasma-membrane based pathogen response. Knowledge generated from the research will therefore be important for not only the cell biology community but will be adoptable by those in academia and industry who work on food and biofuel production. Food security and biofuels have recently been high-priority research areas as we seek to feed the growing population and develop cleaner energy sources. Finally, a component of the project will be in technology development as we seek to improve our current techniques of single molecule tracking and this work will benefit those in the physics and bioimaging communities because of the potential for this kind of technology in medical research.

Intellectual property: In the event of any exploitable IP being generated during the course of the project the Research and Business Development Office at Oxford Brookes will ensure a timely protection of IP and will direct any exploitation.

Outreach: The Brookes plant cell biology group are actively involved in science outreach programmes, including organising events for the Oxfordshire Science Festival, hosting school teachers in the laboratory, organising equipment loan schemes for Schools, presenting School talks, writing articles for various blogs and using social media to disseminate educational videos and plant cell biology breakthroughs. Outcomes from this project will, when appropriate, be disseminated via these activities.

Training: The Postdoctoral Research Associate on this project should start with a good understanding of plant cell biology and molecular biology but will learn about the technology and analyses required for single molecule tracking by working with our collaborators at the Rutherford Appleton Laboratories.