Developing functional and structural imaging of PMF determinants

高分辨率成像的快速发展和体内生物传感的扩展正在给生物学研究带来革命性的变化。这两种方法相结合的力量允许在原位量化细胞生理学和可视化(亚)细胞器和分子动力学。最近，这些发展也开始改变我们对质子推动力(PMF)的理解。线粒体中pH动态和Δψ的可视化一直在描绘一幅意想不到的图景，展示了PMF是如何组织的，以及它在单个细胞器中是如何动态变化的。对于跨光合膜的PMF还没有取得类似的进展，还需要专门的方法开发。了解PMF在活体内的动态变化将进一步需要对参与PMF分配的离子(如K+)以及与叶绿体PMF紧密相连的相关代谢产物(如NADPH：NADP+、NADH：NAD+和ATP)进行实时传感的能力。由于许多现有的生物传感器都是基于蛋白质的，因此在生物能膜中对蛋白质进行特异性亚细胞靶向和基因融合是可行的，并具有最高的局部特异性。然而，听起来直截了当的是，需要在隔离和原位对生物传感器的特定生化和生物物理性质进行广泛的表征，以确保特异性和避免人工制品。需要专门的技术知识，而每个用户获取这些知识是不切实际的。Z-项目将汇集现有的生物传感专业知识，开发监测光合作用PMF所需的生物传感策略，并优化叶绿体和光合膜的高分辨率光学显微镜。具体地说，将开发H+的间质和管腔生物传感，以获得ΔpH的直接读数。NADPH：NADP+生物传感将是先驱，针对光合作用活动变化的生物传感定制设置将得到优化。建立光合膜亚细胞器分辨率的超分辨率成像系统。这将不仅包括采用最先进的去卷积和AiryScan检测的共聚焦显微镜的实时成像，而且还将建立用于超分辨率成像的先进的蛋白质标记协议、单颗粒定位显微镜和类囊体膜蛋白的迁移性研究。在与该联盟成员的密切互动中，PMF决定因素的光学分析工具箱将不断得到扩展、改进和实施。

Rapid advances in high-resolution imaging and the expansion of in vivo biosensing have been revolutionizing biological research. The power of both approaches combined allows for the quantification of cellular physiology in situ and the visualization of (sub-)organellar and molecular dynamics. Those developments have recently also started changing our understanding of the proton motive force (PMF). Visualization of pH dynamics and Δψ in mitochondria has been painting an unexpected picture of how the PMF is organized and how dynamically it changes in single organelles. Comparable advances for the PMF across photosynthetic membranes are yet to be made and dedicated method development will be needed. Understanding PMF dynamics in vivo will further require capabilities for live sensing of ions, such as K+, that contribute to PMF partitioning, and associated metabolites, such as NADPH:NADP+, NADH:NAD+ and ATP, that are tightly linked with the PMF of chloroplasts. Since many of the now available biosensors are protein-based, specific subcellular targeting and genetic fusion to proteins in bioenergetic membranes is feasible to measure with highest local specificity. However, what sounds straightforward, requires extensive characterization of the specific biochemical and biophysical properties of the biosensors in isolation and in situ to ensure specificity and to avoid artefacts. Specialized technical knowledge is required that is unrealistic to be acquired by each individual user. The Z-project will bring together the available expertise on biosensing, develop biosensing strategies needed for photosynthetic PMF monitoring, and optimize high resolution light microscopy of chloroplasts and photosynthetic membranes. Specifically, stromal and lumenal biosensing of H+ will be developed to obtain a direct readout of ΔpH. NADPH:NADP+ biosensing will be pioneered and bespoke setups for biosensing under changeable photosynthetic activity will be optimized. The establishment of super-resolution imaging with sub-organellar resolution will be developed for photosynthetic membranes. That will not only include live imaging by confocal microscopy with state-of-the-art deconvolution and Airyscan detection but also the establishment of advanced protocols of protein labelling for super-resolution imaging, single particle localization microscopy and mobility studies of thylakoid membrane proteins. In close interaction with members of the consortium, the tool-box for the optical analysis of PMF determinants will be continuously extended, refined and implemented.