Summary Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB1R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB1R-derived signaling, including Gi-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFκB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB1R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca2+ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether, these data demonstrate that the physiological effects of anandamide on endothelial cells depend on the status of integrin clustering.

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.

Nitric oxide () 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 probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca2+ sensor allowed us to visualize and Ca2+ signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macro-molecules. 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.

Cytosolic Ca2+ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca2+ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca2+/H+ antiporter that achieved mitochondrial Ca2+ sequestration at small Ca2+ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca2+ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca2+ but strongly diminished the transfer of entering Ca2+ into mitochondria, subsequently, resulting in a reduction of store-operated Ca2+ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca2+ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca2+ uptake at low Ca2+ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca2+ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca2+ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca2+ uptake pathways in intact endothelial cells.

Background: The molecular contributors of the mitochondrial Ca2+ uptake, which is essential for metabolism-secretion coupling in β-cells, are unknown.Results: Knockdown of MICU1 and MCU reduced agonist- and depolarization-induced mitochondrial Ca2+ sequestration, ATP production, and d-glucose-stimulated insulin secretion.Conclusion: MICU1 and MCU are integral to metabolism-secretion coupling in β-cells.Significance: The presented data identify MICU1 and MCU as important contributors to pancreatic β-cell function.

Summary 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.

Background: Accumulation of palmitic acid in endothelial cells induces cellular dysfunction and death.Results: Palmitic acid triggers Ca2+-dependent autophagy, which results in programmed necrotic death (necroptosis) of endothelial cells.Conclusion: Autophagy promotes lipotoxic signaling of palmitic acid in endothelial cells leading to necroptosis.Significance: Showing a new molecular mechanism of palmitic acid-induced cytotoxicity may reveal novel strategies in the treatment of diseases related to lipid overload.