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.

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.

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.

Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca²⁺ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca²⁺ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca²⁺ uptake upon intracellular Ca²⁺ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca²⁺ overload and subsequent cancer cell death.

Recent studies revealed that mitochondrial Ca²⁺ channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca²⁺ uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca²⁺ uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca²⁺ overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca²⁺ uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca²⁺ uptake. Our data demonstrate that mitochondrial Ca²⁺ uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca²⁺ sensitivity. UCP2/3 normalize Ca²⁺ sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca²⁺ uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca²⁺ uniporter by PRMT1 and UCP2/3.