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The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an ‘anti-Warburg’ effect.

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca 2+ 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 Ca 2+ 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. Proper mitochondrial structure is critical for normal function. Here, the authors show with SIM that the mitochondrial Ca 2+ uniporter complex and localization of component MICU1 is critical to maintaining cristae junction stability and overall mitochondrial membrane structure.

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 Ca 2+ -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. K + plays an important role in physiology and disease, but the lack of high specificity K + sensors limits our understanding of its spatiotemporal dynamics. Here the authors develop genetically-encoded FRET-based probes able to quantify K + concentration in body fluids, cells and specific organelles.

Alterations in the function of K + channels such as the voltage- and Ca 2+ -activated K + channel of large conductance (BK Ca ) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BK Ca splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBK Ca ). Through a combination of genetic knockdown and knockout along with a cell permeable BK Ca channel blocker, we show that mitoBK Ca modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the ‘Warburg effect’, thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBK Ca and BK Ca transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBK Ca could represent a treatment strategy for selected BC patients in future.

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 Ca 2+ sensor allowed us to visualize and Ca 2+ 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. Nitric oxide is a volatile free radical second messenger with a large number of biological effects. Here Eroglu et al . develop genetically encoded fluorescent biosensors for nitric oxide and use them to visualise subcellular nitric oxide dynamics in single cells.

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.

Mutations of large conductance Ca 2+ - and voltage-activated K + channels (BK) are associated with cognitive impairment. Here we report that CA1 pyramidal neuron-specific conditional BK knock-out (cKO) mice display normal locomotor and anxiety behavior. They do, however, exhibit impaired memory acquisition and retrieval in the Morris Water Maze (MWM) when compared to littermate controls (CTRL). In line with cognitive impairment in vivo, electrical and chemical long-term potentiation (LTP) in cKO brain slices were impaired in vitro. We further used a genetically encoded fluorescent K + biosensor and a Ca 2+ -sensitive probe to observe cultured hippocampal neurons during chemical LTP (cLTP) induction. cLTP massively reduced intracellular K + concentration ([K + ] i ) while elevating L-Type Ca 2+ channel- and NMDA receptor-dependent Ca 2+ oscillation frequencies. Both, [K + ] i decrease and Ca 2+ oscillation frequency increase were absent after pharmacological BK inhibition or in cells lacking BK. Our data suggest that L-Type- and NMDAR-dependent BK-mediated K + outflow significantly contributes to hippocampal LTP, as well as learning and memory.

Neuronal activity is accompanied by a net outflow of potassium ions (K + ) from the intra- to the extracellular space. While extracellular [K + ] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K + biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K + ] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K + ] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K + ] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K + ]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K + ] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials.

Ca 2+ -activated K + channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K + - and Ca 2+ - homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca 2+ - and K + -dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

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+.