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

细胞中约30%的蛋白质是膜蛋白，占所有已知药物靶点的60%以上，在感染和免疫中起着关键作用。已知膜蛋白组织成复合物、它们在脂质结构域中的分离以及它们对膜形状的影响影响诸如细胞内运输、细胞分裂、细胞迁移和信号转导的过程。尽管这一重要性，理解蛋白质的组织和功能的基本原则，直接在膜脂质环境是严重限制了缺乏合适的时空分辨率和灵敏度的非侵入性测量技术。我们最近开发了[1]一种称为干涉选通离轴反射（iGOR）显微镜的新型光学成像工具，适用于悬浮双层膜中单个未标记生物分子的快速跟踪。它将iSCAT [2]的灵敏度与离轴干涉测量[3]的现场检索相结合。值得注意的是，该技术能够通过测量形貌和波动来量化脂质膜的生物力学。在这个项目中，您将应用iGOR来推进我们的知识：（i）悬浮合成模型膜系统（例如巨型单层囊泡）中的相分离和结构域形成[4]的动力学，该系统包含形成液体有序和无序结构域的三元脂质混合物，重现脂筏。(ii)整合膜蛋白P2 X的功能，[5]细胞表面离子通道由细胞外ATP激活，导致影响神经传递，疼痛感觉，炎症的下游信号事件，使其成为重要的药物靶点。(iii)跨膜孔的研究。关于多聚体膜蛋白孔组装的机制以及随后的功能，仍然存在许多未解答的问题。这将在最小模型膜系统中使用iGOR进行研究[6]。(iv)病毒颗粒的对接和跨膜转运机制。细胞的病毒感染通过病毒包膜糖蛋白与细胞膜蛋白的附着以及随后的膜融合而发生。您将在未修改的病毒上研究此过程，揭示其实时动态。[1]Tual Monfort等人，在制备[2] Young等人中，Science 360，423-427（2018）DOI：10.1126/science.aar5839[3] James L. Flewellen等人，Rev. Sci.仪器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等.方法147，221-228（2018）DOI：10.1016/j.ymeth.2018.05.024

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