Total Internal Reflection Fluorescence (TIRF) and Multidimensional Single Molecule Fluorescence (MSMM) microscopy of plant proteins

This interdisciplinary project will pioneer implementation of state-of-the-art multidimensional single molecule fluorescence microscopy (MSMM) into the Plant developmental Research field. Using this technique our colleagues at Daresbury have been able to quantify the oligomerisation state and conformational changes of the epidermal growth factor receptor in its constitutive and active states, in living cells under normal physiological conditions. We propose to use MSMM and Bayesian segmentation analysis to analyse plant proteins labelled with fluorescent tags. This technique also uses a state-of/the art image analysis algorithm originally developed for extracting quantitative information from extremely faint, noisy and blurred astronomical data. In single molecule research, the combination of weak signals from single receptors with the unavoidable presence of the fluorescence background from living cells implies that essential information in the data can be blurred and/or hidden by the background noise. The imaging analysis method seeks to implement, pre-existing information on the receptor data, which can be explicitly included in the image analysis algorithms, creating a more powerful algorithm with demonstrated trustworthy results. In combination with these image analysis methods this instrument will serve as an accurate spectroscopic ruler and protractor at the molecular scale to provide data on the conformation and binding characteristics of fluorescently labelled proteins in the native environment. Fluorescently labelled proteins will be transiently expressed in onion epidermal cells, protoplasts and cell suspension cultures and used for TIRFM. MSMM requires extremely low levels of fluorescence, which are unlikely to be generated using native promoters or CaMV35S. The use of SNAP-tags will therefore be tested. This system relies upon creating a fusion protein between the protein of interest and a small 180 amino-acid protein 'SNAP-tagTM (Covalys) which can be covalently labelled in vivo with a wide range of fluorescent or affinity substrates. This has the advantage that varying levels of signal can be generated independent of expression levels, by altering the amount of labelling, which allows the generation of low-levels of fluorescence needed for MSMM. The SNAP-tag system will be used to generate fusion proteins that can be covalently labelled in vivo for low-levels of fluorescence required for MSMM. This project will provide the opportunity to integrate this technology into the Plant Sciences Research field.

Confocal Microscopy is used extensively in plant development, however to date TIRFM excitation Multidimensional single molecule microscopy (MSMM) has not been applied to plant cells. The technique holds great potential since it can provide data on the conformation and oligomerisation state of interacting proteins and stoichiometry of networks in vivo. Nevertheless there are significant technical difficulties that need to be overcome, including distance of imaging planes to the glass-sample interface and sample adherence. This interdisciplinary project will pioneer implementation of state-of-the-art multidimensional single molecule fluorescence microscopy and Bayesian segmentation analysis into Plant Research. Fluorescently labelled proteins will be transiently expressed in onion epidermal cells, protoplasts and cell suspension cultures and used for TIRFM. The SNAP-tag system will be used to generate fusion proteins that can be covalently labelled in vivo for low-levels of fluorescence required for MSMM. MSMM uses an algorithm originally developed for extracting quantitative information from extremely faint, noisy and blurred astronomical data. The combination of weak signals from single receptors with the unavoidable presence of the fluorescence background from living cells implies that essential information in the data can be blurred and/or hidden by the background noise. The image analysis method seeks to implement, pre-existing information on the receptor data, which can be explicitly included in the image analysis algorithms, creating a more powerful algorithm with demonstrated trustworthy results. In combination with these analysis methods this instrument will serve as an accurate spectroscopic ruler and protractor at the molecular scale to provide data on the conformation and binding characteristics of fluorescently labelled proteins in the native environment. This project will provide an opportunity to integrate this technology into Plant Research.