Changes in intra- and extracellular potassium ion (K⁺) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K⁺ changes is severely limited by the lack of practicable detection methods. We developed K⁺-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K⁺ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K⁺ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K⁺ signals. Hitherto unknown Ca²⁺-triggered, organelle-specific K⁺ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K⁺ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K⁺ assays and open new avenues for live-cell K⁺ imaging.

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca²⁺ uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca²⁺ elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.

Reprogramming of metabolic pathways determines cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resolution in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor – referred to as pH-Lemon – optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.

Nitric oxide ("Equation missing") is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based "Equation missing"probes by fusing a bacteria-derived "Equation missing"-binding domain close to distinct fluorescent protein variants. These genetically encoded "Equation missing"probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular "Equation missing"dynamics and, hence, open a new era of "Equation missing"bioimaging. The combination of geNOps with a Ca²⁺sensor allowed us to visualize "Equation missing"and Ca²⁺signals simultaneously in single endothelial cells. Moreover, targeting of the "Equation missing"probes was used to detect "Equation missing"signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of "Equation missing"signalling on the single (sub)cellular level.

Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K⁺, alkaline earth metals including Mg²⁺ and Ca²⁺, and transition metals including Cu⁺/Cu²⁺ and Zn²⁺. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe²⁺/Fe³⁺, Mn²⁺ and Na⁺.