mitoBKCa is functionally expressed in murine and human breast cancer cells and potentially contributes to metabolic reprogramming.

Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa 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 mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.

Genetically encoded fluorescent sensor to monitor intracellular arginine methylation.

Arginine methylation (ArgMet), as a post-translational modification, plays crucial roles in RNA processing, transcriptional regulation, signal transduction, DNA repair, apoptosis and liquid-liquid phase separation (LLPS). Since arginine methylation is associated with cancer pathogenesis and progression, protein arginine methyltransferases have gained interest as targets for anti-cancer therapy. Despite considerable process made to elucidate (patho)physiological mechanisms regulated by arginine methylation, there remains a lack of tools to visualize arginine methylation with high spatiotemporal resolution in live cells. To address this unmet need, we generated an ArgMet-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based biosensor, called GEMS, capable of quantitative real-time monitoring of ArgMet dynamics. We optimized these biosensors by using different ArgMet-binding domains, arginine-glycine-rich regions and adjusting the linkers within the biosensors to improve their performance. Using a set of mammalian cell lines and modulators, we demonstrated the applicability of GEMS for monitoring changes in arginine methylation with single-cell and temporal resolution. The GEMS can facilitate the in vitro screening to find potential protein arginine methyltransferase inhibitors and will contribute to a better understanding of the regulation of ArgMet related to differentiation, development and disease.

Nitric Oxide Sensing by a Blue Fluorescent Protein.

S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Aequorea victoria Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology. We have examined the response to nitric oxide of fluorescence emission by the blue-emitting fluorescent protein mTagBFP2. To our surprise, upon exposure to micromolar concentrations of nitric oxide, we observed a roughly 30% reduction in fluorescence quantum yield and lifetime. Recovery of fluorescence emission is observed after treatment with Na-dithionite. Experiments on related fluorescent proteins from different families show similar nitric oxide sensitivity of their fluorescence. We correlate the effect with S-nitrosylation of Cys residues. Mutation of Cys residues in mTagBFP2 removes its nitric oxide sensitivity. Similarly, fluorescent proteins devoid of Cys residues are insensitive to nitric oxide. We finally show that mTagBFP2 can sense exogenously generated nitric oxide when expressed in a living mammalian cell. We propose mTagBFP2 as the starting point for a new class of genetically encoded nitric oxide sensors based on fluorescence lifetime imaging.

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors.

Ion and analyte changes in the tumor microenvironment (TME) alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. Consequently, accurate determination and visualization of extracellular changes of analytes in real time is desired. In this study, we genetically combined FRET-based biosensors with nanobodies (Nbs) to specifically visualize and monitor extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these Nb-fused biosensors quantitatively visualized K+ alterations on cancer and non-cancer cell lines and primary neurons. By implementing a HER2-specific Nb, we generated functional K+ and pH sensors, which specifically stained HER2-positive breast cancer cells. Based on the successful development of several Nb-fused biosensor combinations, we anticipate that this approach can be readily extended to other biosensors and will open new opportunities for the study of extracellular analytes in advanced experimental settings.

Assessing K+ ions and K+ channel functions in cancer cell metabolism using fluorescent biosensors.

Cancer represents a leading cause of death worldwide. Hence, a better understanding of the molecular mechanisms causing and propelling the disease is of utmost importance. Several cancer entities are associated with altered K+ channel expression which is frequently decisive for malignancy and disease outcome. The impact of such oncogenic K+ channels on cell patho-/physiology and homeostasis and their roles in different subcellular compartments is, however, far from being understood. A refined method to simultaneously investigate metabolic and ionic signaling events on the level of individual cells and their organelles represent genetically encoded fluorescent biosensors, that allow a high-resolution investigation of compartmentalized metabolite or ion dynamics in a non-invasive manner. This feature of these probes makes them versatile tools to visualize and understand subcellular consequences of aberrant K+ channel expression and activity in K+ channel related cancer research.

Investigating the K+ sensitivity of cellular metabolism by extracellular flux analysis.

We have recently demonstrated that the activity of hexokinase 2 is dependent on the intracellular potassium ion (K+) concentration ([K+]). To analyze the K+ dependency of the cell metabolism in cell populations, we used an extracellular flux analyzer to assess oxygen consumption and acidification rates as well-established measures of oxidative- and glycolytic metabolic activities. This protocol describes in detail how a potential K+ sensitivity of the cell metabolism can be elucidated by extracellular flux analysis. For complete details on the use and execution of this protocol, please refer to Bischof et al. (2021).

Unveiling the K+-sensitivity of cell metabolism using genetically encoded, FRET-based K+, glucose, and ATP biosensors.

Investigating dynamic changes of mitochondrial ATP and cytosolic glucose levels of single living cells over time by genetically encoded biosensors provides an informative readout of their metabolic activities. Here, we describe how to monitor the metabolic K+-sensitivity of HEK293 cells exploiting ATP-, glucose-, and K+ probes. Fluorescence live-cell imaging of these Förster resonance energy transfer-based biosensors over time in response to gramicidin, an ionophoric peptide, indicated an absolute dependency of cellular ATP homeostasis on high intracellular K+ levels. For complete information on the generation and use of this protocol please refer to Bischof et al. (2021).

Potassium ions promote hexokinase-II dependent glycolysis.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism.

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca2+ and Energy Stresses.

One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca2+ and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca2+ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca2+ mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca2+ stress levels.

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

Tracking intra- and inter-organelle signaling of mitochondria.

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca2+ and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine-tuned by intra- and interorganelle H+ , K+ , Na+ , and Ca2+ signaling. In this review, we provide an up-to-date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting-edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.

Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2.

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.

Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes.

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+.