In recent years, my group has specialized in developing techniques ranging from time-resolved fluorescence spectroscopy to advanced spectromicroscopic methods to bridge the gap between the molecular and cellular levels [1-5]. As a prime example, we use cytochrome c oxidase (CcO). As part of the respiratory chain, CcO contributes to the proton driving force (pmf) that drives ATP synthesis. At the molecular level, a clear picture of the proton escape pathway or pathways is still unclear. At the cellular level, CcO dysfunction appears to be associated with increased reactive oxygen species production and cellular toxicity, although a molecular picture is lacking. Using CcO from P. denitrificans, we start to answer these open questions using selected CcO variants that contain surface mutations [6-7], including proton collecting antennas and newly discovered surface switch residues. A voltage-sensitive fluorescent protein sensor [3] for pmf detection at the single enzyme level was developed to extend our research to the cellular level. Surface switch residues influence the proton escape and thus the generation of a pmf. Along these lines, the molecular action of the cancer drug doxorubicin (DOX) has also been studied, although the mechanistic details of its adverse cytotoxic effects are disputed. We show that DOX directly affects electron-coupled proton transfer. To transfer these findings to the cellular level, we developed cluster fluorescence lifetime imaging (FLIM) [1,4] and FLIM-ROX [2] to quantify minute amounts of possible oxidative stress caused by DOX. If time permits, I will also present how Cluster-FLIM facilitates visualization of nanocarrier and drug action in 2D and 3D tissue models.
[1] Brodwolf, R. et al., Theranostics 10, 6322 (2020)
[2] Balke, J. et al., Small 14, 1800310 (2018)
[3] Silapetere, A. et al., Nat. Communications 13, 5501 (2022)
[4] Alexiev, U. and Rühl, E., Handbook of Experimental Pharmacology: Visualization of Nanocarriers and Drugs in Cells and Tissue (2023)
[5] Wolf, A. et al., Small 19, 2206722 (2023)
[6] Wolf, A. et al., FEBS J. 287, 1232 (2019)
[7] Wolf, A. et al., Chemical Science 11, 3804 (2020)