Quantitative spatio-temporal dynamics of membrane proteins and lipid domains measured by label-free super-resolution optical microscopy

膜蛋白是目前市售药物中约三分之二的靶点[1]，并且在感染和免疫中发挥着关键作用。尽管这一重要性，在其天然脂质环境中的膜蛋白的直接研究仍然是一个重大的挑战。膜蛋白如何动态地与脂质双层相互作用，以及双层的组成如何影响膜蛋白的功能？膜蛋白-脂质相互作用能否以高时空精度定量？本项目的目的是应用新的无标记光学成像技术在单个蛋白质水平上定量膜蛋白在生理相关的异质性“筏状”脂质有序/无序结构域中的扩散。该项目将专门解决P2 X7的扩散和脂质分配问题，P2 X7是一种ATP门控阳离子通道，已知可分配到脂质微区，并且在炎症信号传导中起重要作用[2]。无标记成像将涉及线性和非线性光学方法的组合，包括相干反斯托克斯拉曼散射（汽车）显微镜[3]，定量微分干涉衬度（qDIC）[4]和一种新的干涉选通离轴反射（iGOR）显微镜的发展。这是一个在物理学-生命科学界面上高度交叉学科的项目，第一年的两个评估轮换小型项目将提供重点培训。第一轮将在Borri和Langbein教授的生物光子学实验室举行，他们是在先进激光显微光谱技术开发方面拥有丰富经验的物理学家。这个小型项目将集中在合成膜与脂质有序/无序微区成像汽车和qDIC。到项目结束时，学生将能够制造人工脂质双层，并量化域的化学成分和厚度。第二个轮换项目将在Young博士的实验室进行，Young博士是真核细胞膜蛋白结构、功能、表达和纯化方面的专家，特别关注ATP门控阳离子通道的P2 X受体家族。该小型项目将专注于优化P2 X7的表达和纯化，以重建成合成膜，并使用透射电子显微镜（TEM）和纯化蛋白的单颗粒分析（SPA）进行低至中分辨率结构研究。到项目结束时，学生将能够纯化足够的P2 X7，用于重建成合成膜，用于随后的扩散和脂质分配实验。Nat Rev Drug Discov 5，993（2006）. Surprenant和R. A.北边，安奴。71，333-59（2009）. A. Zumbusch等人，Progress in Lipid Research 52，615（2013）.麦克菲等人，生物物理学杂志105，

Membrane proteins represent the target of about two thirds of currently marketed pharmaceuticals[1] and play a critical role in both infection and immunity. Despite this importance, the direct study of membrane proteins in their native lipid environment remains a significant challenge. How do membrane proteins dynamically interact with the lipid bilayer, and how does bilayer composition affect membrane protein function? Can the membrane protein-lipid interaction be quantified with high spatio-temporal precision?The aim of this project is to apply novel label-free optical imaging techniques to quantify membrane protein diffusion at the single protein level, in physiologically relevant heterogeneous 'raft-like' lipid ordered/disordered domains.The project will specifically address the diffusion and lipid partitioning of P2X7, an ATP-gated cation channel which is known to partition into lipid microdomains, and which plays an important role in inflammatory signaling[2]. Label-free imaging will involve a combination of linear and nonlinear optical methods including Coherent Antistokes Raman Scattering (CARS) microscopy[3], quantitative Differential Interference Contrast (qDIC)[4] and the development of a novel interferometric Gated Off-axis Reflectance (iGOR) microscopy. Both synthetic lipid membrane bilayers and cellular membranes will be investigated.This is a highly cross-disciplinary project at the physics-life science interface and two assessed rotation mini-projects in year 1 will provide focused training. The first rotation will be held in the Biophotonics lab of Professors Borri and Langbein who are physicists with extensive experience in the development of advanced laser micro-spectroscopy techniques. This mini-project will focus on synthetic membranes with lipid ordered/disordered microdomains imaged by CARS and qDIC. By the end of the project the student will be able to fabricate artificial lipid bilayers and quantify the chemical composition and thickness of the domains. The second rotation project will be held in the lab of Dr Young who is an expert in the structure, function, expression and purification of eukaryotic membrane proteins, with particular focus on the P2X receptor family of ATP-gated cation channels. This mini-project will focus on the optimization of expression and purification of P2X7 for reconstitution into synthetic membranes, and for low- to medium-resolution structural studies using transmission electron microscopy (TEM) and single particle analysis (SPA) of purified protein. By the end of the project the student will be able to purify sufficient P2X7 for reconstitution into synthetic membranes for subsequent diffusion and lipid partitioning experiments.References:J. P. Overington et al., Nat Rev Drug Discov 5, 993 (2006).A. Surprenant and R. A. North, Annu. Rev. Physiol. 71, 333-59 (2009).A. Zumbusch et al. Progress in Lipid Research 52, 615 (2013).C. McPhee et al., Biophysical Journal 105,