Unravelling the dynamics of lipid bilayers, membrane proteins and their reaction to toxins and viral infection on the single molecule level using labe

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Approximately 30% of the proteins in a cell are membrane proteins, which represent more than 60% of all known drug targets and play a critical role in both infection and immunity. The organization of membrane proteins into complexes, their segregation in lipid domains, and their effect on membrane shape is known to influence processes such as intracellular transport, cell division, cell migration, and signal transduction. Despite this importance, understanding the underlying principles of protein organization and function directly in the membrane lipid environment is severely limited by the lack of non-invasive measurement techniques with suited spatio-temporal resolution and sensitivity. We have recently developed [1] a novel optical imaging tool called interferometric gated off-axis reflectance (iGOR) microscopy, suitable for fast tracking of single unlabelled biomolecules in suspended bilayer membranes. It combines the sensitivity of iSCAT [2] with the field retrieval of off-axis interferometry [3]. Notably, the technique enables the quantification of the elastomechanics of the lipid membrane, through measurement of topography and fluctuations. In this project you will apply iGOR to advance our knowledge regarding:(i) The dynamics of phase separation and domain formation [4] in suspended synthetic model membrane systems (e.g. giant unilamellar vesicles) containing ternary lipid mixtures forming liquid ordered and disordered domains, recapitulating lipid rafts. (ii) The function of the integral membrane protein P2X, [5] a cell-surface ion channel activated by extracellular ATP, leading to downstream signaling events affecting nerve transmission, pain sensation, inflammation, making them important drug targets.(iii) Study of membrane spanning pores. There remain many unanswered questions about the mechanisms of multimeric membrane protein pore assembly, and subsequent function. This will be investigated using iGOR in minimal model membrane systems [6].(iv) Docking and trans-membrane transport mechanisms of viral particles. Viral infection of cells occurs via attachment of the virus envelope glycoproteins to cell membrane proteins, and subsequent membrane fusion. You will study this process on un-modified viruses, revealing its real-time dynamics.[1] Tual Monfort et al., in preparation[2] Young et al., Science 360, 423-427 (2018) DOI: 10.1126/science.aar5839[3] James L. Flewellen et al. Rev. Sci. Instrum. 90, 023705 (2019) DOI: 10.1063/1.5066556[4] D. Regan et al, Langmuir 35, 13805 (2019) DOI: 10.1021/acs.langmuir.9b02538[5] Pasqualetto G et al., Frontiers in Pharmacology 9, 58 (2018) DOI: 10.3389/fphar.2018.00058.[6] Castell et al. Methods 147, 221-228 (2018) DOI: 10.1016/j.ymeth.2018.05.024