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

膜蛋白代表了目前市场上三分之二的药物的靶标[1]，并且在感染和免疫中都起着关键作用。尽管如此，膜蛋白在天然脂环境中的直接研究仍然是一个巨大的挑战。膜蛋白如何与脂双分子层动态相互作用，双分子层的组成如何影响膜蛋白的功能？膜蛋白-脂质相互作用能以高时空精度定量吗？本项目的目标是应用新的无标记光学成像技术在单个蛋白水平上定量膜蛋白在生理上相关的异质类‘RAFT’类脂质有序/无序结构域中的扩散。该项目将专门研究P2X7的扩散和脂质分配，这是一种已知划分为脂质微域的ATP门阳离子通道，在炎症信号转导中起重要作用[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(CARS)显微镜[3]、定量差分干涉对比(QDIC)[4]以及新型干涉门控离轴反射(IGOR)显微镜的开发。这是一个在物理-生命科学界面的高度交叉的项目，第一年的两个评估的旋转小项目将提供有针对性的培训。第一次轮换将在Borri和Langbein教授的生物光子学实验室举行，他们是物理学家，在开发先进的激光显微光谱技术方面具有丰富的经验。这个小型项目将专注于由CARS和qDIC成像的具有脂质有序/无序微域的合成膜。在项目结束时，学生将能够制造人工脂质双层，并对结构域的化学成分和厚度进行量化。第二个轮换项目将在杨博士的实验室进行，杨博士是真核膜蛋白的结构、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P2X受体家族。这个小型项目将致力于优化P2X7的表达和纯化，以便重新构建成合成膜，并使用纯化蛋白质的透射电子显微镜(TEM)和单颗粒分析(SPA)进行低到中分辨率的结构研究。到项目结束时，学生将能够提纯足够的P2X7以重新构建成合成膜，用于随后的扩散和脂质分配实验。参考文献：J.P.Overington等人，NAT Rev Drug Discov 5,993(2006).A.Surprenant和R.A.North，Annu。菲西奥尔牧师。71,333-59(2009).A.Zumbusch等人。脂类研究进展52,615(2013).C.McPhee等，生物物理杂志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,