Developing functional and structural imaging of PMF determinants

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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.