A label-free tool to unravel the dynamics of lipid bilayers containing single membrane proteins: iGOR microscopy

在一个给定的生物体中，大约30%的蛋白质是膜蛋白。它们占所有已知药物靶点的60%以上，并在感染和免疫方面发挥关键作用。众所周知，膜蛋白组成复合物的组织、它们在脂质结构域的分离以及它们对膜形状的影响会影响细胞内运输、细胞分裂、细胞迁移和信号转导等过程。尽管具有这种重要性，但由于缺乏合适的具有足够时空分辨率和灵敏度的非侵入性技术，直接了解膜脂环境中蛋白质组织和功能的基本原理受到严重限制。光学成像与基于接触的技术（如原子力显微镜）相比，在非侵入性方面具有明显的优势，并且可以提供超过光学衍射极限的纳米级定位精度。为了获得足够的灵敏度和特异性，光学方法主要依赖于荧光标记，其缺点是由于光漂白，光毒性，最重要的是需要化学或基因样品操作/修饰，这就提出了观察到的行为是真实的还是人为的问题。在这里，我们提出了一种新的光学成像方法，我们称之为干涉门控离轴反射（iGOR）显微镜，适用于通过悬浮双层膜中的固有散射光，以毫秒时间分辨率快速跟踪单个未标记的生物分子。值得注意的是，该技术将能够量化脂质膜的弹性力学特性，通过精确测量层形貌及其波动动力学，同时在毫秒时间尺度上跟踪单个蛋白质的3D扩散，从而揭示膜-蛋白质相互作用的新见解。iGOR将作为光学设置硬件和相关定量数据分析工具包开发，以前所未有的灵敏度和精度提取与时间相关的膜轴向位置和厚度图相关的单个蛋白质的时间相关位置坐标。作为iGOR能力的生物学相关测试，我们将研究整体膜蛋白（P2X受体）在悬浮脂质膜中的扩散。P2X受体是由细胞外ATP激活的细胞表面离子通道。激活导致下游信号事件，对神经传递、疼痛感觉、炎症和平滑肌张力的控制有重要影响。因此，P2X受体是镇痛或抗炎作用的重要药物靶点。值得注意的是，尽管是基于间接的生物化学分析，但越来越多的证据表明，它们分为脂质有序的区室。因此，iGOR是直接解决P2X受体如何扩散和分裂到脂质微域的问题的理想技术。我们预计，插入蛋白质将导致膜的局部变形，这可以通过我们的技术灵敏地测量。最近有人提出，这种变形导致蛋白质之间有效的排斥和吸引相互作用，由膜介导，类似于电荷之间的库仑相互作用。因此，我们的iGOR方法提供的独特可能性将为理解膜生物物理学的这些基本方面打开令人兴奋的前景，这些方面吸引了很多人的兴趣。在未来的工作中，iGOR可以升级为包括电生理分析来测量膜电压和离子通量，这将为解决膜蛋白研究领域的长期问题铺平道路，例如，离子通道功能是否取决于这些蛋白质是在脂筏还是非筏舱室中。

Approximately 30% of the proteins in a given organism are membrane proteins. These 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 suitable non-invasive techniques with sufficient spatio-temporal resolution and sensitivity.Optical imaging has distinct advantages compared to contact-based techniques such as atomic force microscopy in terms of non-invasiveness, and can offer localisation precision at the nanoscale beyond the optical diffraction limit. To achieve enough sensitivity and specificity, optical methodologies mostly rely on fluorescence labelling with the drawbacks of limited observation periods due to photobleaching, phototoxicity, and most importantly the need for chemical or genetic sample manipulation/modification which raises the question if the observed behaviour is real or artifactual. Here, we propose the development of a novel optical imaging method which we have called interferometric gated off-axis reflectance (iGOR) microscopy suitable for fast tracking of single unlabelled biomolecules with millisecond time resolution, via their intrinsic scattered light, in suspended bilayer membranes. Notably, the technique will enable to quantify the elasto-mechanical properties of the lipid membrane, through precise measurement of the layer topography and its fluctuation dynamics, simultaneously with tracking single protein diffusion in 3D on the millisecond time scale, thus revealing new insights into membrane-protein interactions. iGOR will be developed as an optical set-up hardware and associated quantitative data analysis toolkits, to extract time-dependent position coordinates of single proteins correlated with time-dependent membrane axial position and thickness maps, with unprecedented sensitivity and precision. As a biologically-relevant test of iGOR's capabilities, we will investigate the diffusion of integral membrane proteins (P2X receptors) into a suspended lipid membrane. P2X receptors are cell-surface ion channels which are activated by extracellular ATP. Activation leads to downstream signalling events which have important consequences for nerve transmission, pain sensation, inflammation and control of smooth muscle tone. Therefore, P2X receptors are important drug targets for analgesic or anti-inflammatory actions. Notably, growing evidence, albeit based on indirect biochemistry assays, indicates that they partition into lipid-ordered compartments. iGOR is therefore an ideal technology to directly address the question of how P2X receptors diffuse and partition into lipid microdomains.We expect that inserting the protein will lead to a local deformation of the membrane, which can be sensitively measured by our technique. It was recently suggested that this deformation results in effective repulsive and attractive interactions between proteins, mediated by the membrane, similar to a Coulomb interaction between charges. The unique possibilities offered by our iGOR method will thus open the exciting prospect of understanding these fundamental aspects of membrane biophysics, which are attracting a lot of interest.In future work, iGOR could be upgraded to include an electrophysiology assay to measure membrane voltages and ion fluxes, which will pave the way toward addressing long-standing questions in the membrane-protein research field, e.g. whether ion channel function varies depending if these proteins are in lipid rafts or non-raft compartments.