Fluorescence microscopy allows the dynamic observation of phenomena in living cells. Until recently, however, the wavelike nature of light and its associated diffraction, limited the resolution of optical microscopy to ~200 nm. New ‘super-resolution’ fluorescence microscopy methods, have been developed to overcome the diffraction limit of ‘traditional’ microscopy. The enormous impact of super resolution microscopy methods was recognized by its inventors being awarded the Nobel Prize in Chemistry (2014). Super resolution methods can increase the resolution of fluorescence microscopy down to ~5 nm, thus enabling the observation of molecular processes in great detail.
Despite these advances in optical methods, labelling the biomolecule of interest remains a challenge, especially within live cells. Direct fusion of the protein of interest to a fluorescent protein is common strategy, because it is genetically encodable, can be used for live cell imaging, and is amenable to super-resolution microscopy (by judicious choice of fluorescent protein). Unfortunately, fluorescent proteins are quite large (>27 kDa) and a direct fusion can perturb the function, assembly, and/or location of the protein of interest.
To address these issues, the student will develop a novel super-resolution imaging method based on a labelling strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the biomolecule of interest. Rather than fusing the fluorescent protein itself to the protein of interest, a short peptide tag will be genetically encoded onto it. This tag can then be linked either covalently or non-covalently to a partner protein that is also expressed, in a controlled fashion, within the cell. Such a method has been demonstrated to work well to label proteins within bacteria and yeast1,2. The student will build on this work, adapting the strategy for use in mammalian cells. Most importantly, they will develop this approach using the photoswitchable fluorescent protein, mEoS, which will enable super-resolution images to be generated using photo-activated localization microscopy3. We are particularly interested in applying this novel strategy to visualize amyloid protein formed within human-derived neurons.
Training: the supervisors of this project will combine their expertise in protein structure and design (Regan), neuroscience (Horrocks) and super-resolution microscopy techniques (Horrocks). The training will be achieved primarily through practical application of the research techniques within the supervisors’ and collaborators’ laboratories. Skills that will be developed include cell and molecular biology techniques, cell culture (including differentiated induced pluripotent stem cells), gene editing, advanced microscopy (single-molecule and super-resolution), data analysis and coding. The student will be encouraged to participate in training workshops, to present in various multi-group meetings at the University, and to participate in public engagement activities. Science is a global endeavour, and the supervisors will ensure that the student has the opportunity to attend and present at international conferences.
(1) Pratt, S. E.; Speltz, E. B.; Mochrie, S. G. J.; Regan, L. Designed Proteins as Novel Imaging Reagents in Living Escherichia Coli. Chembiochem Eur. J. Chem. Biol. 2016, 17 (17), 1652–1657.
(2) Hinrichsen, M.; Lenz, M.; Edwards, J. M.; Miller, O. K.; Mochrie, S. G. J.; Swain, P. S.; Schwarz-Linek, U.; Regan, L. A New Method for Post-Translationally Labeling Proteins in Live Cells for Fluorescence Imaging and Tracking. Protein Eng. Des. Sel. PEDS 2017, 30 (12), 771–780.
(3) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313 (5793), 1642–1645.
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