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，在感染和免疫中都发挥着关键作用。膜蛋白被组织成复合体，它们在脂类结构域中的分离，以及它们对膜形状的影响，都会影响细胞内运输、细胞分裂、细胞迁移和信号转导等过程。尽管这一点很重要，但由于缺乏合适的时空分辨率和灵敏度的非侵入性测量技术，直接在膜脂环境中理解蛋白质组织和功能的潜在原理受到严重限制。我们最近开发了一种新的光学成像工具，称为干涉门控离轴反射(IGOR)显微镜，适用于快速跟踪悬浮双层膜中的单个未标记生物分子。它结合了iSCAT[2]的灵敏度和离轴干涉测量法的场恢复[3]。值得注意的是，该技术能够通过测量地形和波动来量化脂膜的弹性力学。在本项目中，您将应用IGOR来提高我们在以下方面的知识：(I)悬浮合成模型膜系统(例如巨大的单层囊泡)中的相分离和结构域形成的动力学[4]，其中包含形成液体有序和无序结构域的三元脂肪混合物，重述脂筏。(Ii)完整的膜蛋白P2X的功能，[5]由细胞外ATP激活的细胞-表面离子通道，导致下游信号事件影响神经传递、痛觉、炎症，使它们成为重要的药物靶点。(Iii)跨孔膜的研究。关于多聚体膜蛋白的孔组装机制及其后续功能，仍有许多未解之谜。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传输机制。病毒对细胞的感染是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发生的。您将在未修改的病毒上研究这一过程，揭示其实时动态。[1]Tual Monfort等人，在准备中[2]Young等人，Science 360,423-427(2018年)DOI：10.1126/Science.aar5839[3]James L.Flewell en等人。科西牧师。Instrum。90,023705(2019年)DOI：10.1063/1.5066556[4]D.里根等人，朗缪尔35,2019(2019年)DOI：10.1021/acs.langmuir.9b02538[5]Pasquetto G等人，药理学前沿9，58(2018年)DOI：10.3389/fphar.2018.00058.[6]Castell等人.2018年)DOI：10.1016/j.yome.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