An essential role for EROS in redox-dependent endothelial signal transduction.

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Sensory ataxia and cardiac hypertrophy caused by neurovascular oxidative stress in chemogenetic transgenic mouse lines.

Oxidative stress is associated with cardiovascular and neurodegenerative diseases. Here we report studies of neurovascular oxidative stress in chemogenetic transgenic mouse lines expressing yeast D-amino acid oxidase (DAAO) in neurons and vascular endothelium. When these transgenic mice are fed D-amino acids, DAAO generates hydrogen peroxide in target tissues. DAAO-TGCdh5 transgenic mice express DAAO under control of the putatively endothelial-specific Cdh5 promoter. When we provide these mice with D-alanine, they rapidly develop sensory ataxia caused by oxidative stress and mitochondrial dysfunction in neurons within dorsal root ganglia and nodose ganglia innervating the heart. DAAO-TGCdh5 mice also develop cardiac hypertrophy after chronic chemogenetic oxidative stress. This combination of ataxia, mitochondrial dysfunction, and cardiac hypertrophy is similar to findings in patients with Friedreich's ataxia. Our observations indicate that neurovascular oxidative stress is sufficient to cause sensory ataxia and cardiac hypertrophy. Studies of DAAO-TGCdh5 mice could provide mechanistic insights into Friedreich's ataxia.

Differential endothelial hydrogen peroxide signaling via Nox isoforms: Critical roles for Rac1 and modulation by statins.

Statins have manifold protective effects on the cardiovascular system. In addition to lowering LDL cholesterol levels, statins also have antioxidant effects on cardiovascular tissues involving intracellular redox pathways that are incompletely understood. Inhibition of HMG-CoA reductase by statins not only modulates cholesterol synthesis, but also blocks the synthesis of lipids necessary for the post-translational modification of signaling proteins, including the GTPase Rac1. Here we studied the mechanisms whereby Rac1 and statins modulate the intracellular oxidant hydrogen peroxide (H2O2) via NADPH oxidase (Nox) isoforms. In live-cell imaging experiments using the H2O2 biosensor HyPer7, we observed robust H2O2 generation in human umbilical vein endothelial cells (HUVEC) following activation of cell surface receptors for histamine or vascular endothelial growth factor (VEGF). Both VEGF- and histamine-stimulated H2O2 responses were abrogated by siRNA-mediated knockdown of Rac1. VEGF responses required the Nox isoforms Nox2 and Nox4, while histamine-stimulated H2O2 signals are independent of Nox4 but still required Nox2. Endothelial H2O2 responses to both histamine and VEGF were completely inhibited by simvastatin. In resting endothelial cells, Rac1 is targeted to the cell membrane and cytoplasm, but simvastatin treatment promotes translocation of Rac1 to the cell nucleus. The effects of simvastatin both on receptor-dependent H2O2 production and Rac1 translocation are rescued by treatment of cells with mevalonic acid, which is the enzymatic product of the HMG-CoA reductase that is inhibited by statins. Taken together, these studies establish that receptor-modulated H2O2 responses to histamine and VEGF involve distinct Nox isoforms, both of which are completely dependent on Rac1 prenylation.

Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells.

BACKGROUND: Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species. METHODS: CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed. RESULTS: An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα. CONCLUSIONS: Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.

MICU1 controls spatial membrane potential gradients and guides Ca2+ fluxes within mitochondrial substructures.

Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.

Metabolomic and transcriptomic signatures of chemogenetic heart failure.

The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.

Dissecting in vivo and in vitro redox responses using chemogenetics.

Hydrogen peroxide (H2O2) is the most abundant reactive oxygen species (ROS) within mammalian cells. At low concentrations, H2O2 serves as a versatile cell signaling molecule that mediates vital physiological functions. Yet at higher concentrations, H2O2 can be a toxic molecule by promoting pathological oxidative stress in cells and tissues. Within normal cells, H2O2 is differentially distributed in a variety of subcellular locales. Moreover, many redox-active enzymes and their substrates are themselves differentially distributed within cells. Numerous reports have described the biological and biochemical consequences of adding exogenous H2O2 to cultured cells and tissues, but many of these observations are difficult to interpret: the effects of exogenous H2O2 do not necessarily replicate the cellular responses to endogenous H2O2. In recent years, chemogenetic approaches have been developed to dynamically regulate the abundance of H2O2 in specific subcellular locales. Chemogenetic approaches have been applied in multiple experimental systems, ranging from in vitro studies on the intracellular transport and metabolism of H2O2, all the way to in vivo studies that generate oxidative stress in specific organs in living animals. These chemogenetic approaches have exploited a yeast-derived d-amino acid oxidase (DAAO) that synthesizes H2O2 only in the presence of its d-amino acid substrate. DAAO can be targeted to various subcellular locales, and can be dynamically activated by the addition or withdrawal of its d-amino acid substrate. In addition, recent advances in the development of highly sensitive genetically encoded H2O2 biosensors are providing a better understanding of both physiological and pathological oxidative pathways. This review highlights several applications of DAAO as a chemogenetic tool across a wide range of biological systems, from analyses of subcellular H2O2 metabolism in cells to the development of new disease models caused by oxidative stress in vivo.

The importance of aquaporin-8 for cytokine-mediated toxicity in rat insulin-producing cells.

Aquaporin-8 (AQP8) is a peroxiporin, a transmembrane water and hydrogen peroxide (H2O2) transport protein expressed in the mitochondrial and plasma membranes of pancreatic ß-cells. AQP8 protein expression is low under physiological conditions, but it increases after cytokine exposure both, in vitro and in vivo, possibly related to a NF-κB consensus sequence in the promoter. AQP8 knockdown (KD) insulin-producing RINm5F cells are particularly susceptible to cytokine-mediated oxidative stress. Cytokine (a mixture of IL-1ß, TNF-α, and IFN-γ) treated AQP8 KD cells exhibited pronounced sensitivity to reactive oxygen and nitrogen species (ROS and RNS), resulting in a significant loss of ß-cell viability due to enhanced toxicity of the increased concentrations of H2O2 and hydroxyl radicals (●OH) in mitochondria of AQP8 KD cells. This viability loss went along with increased caspase activities, reduced nitrite concentration (representative of nitric oxide (NO●) accumulation) and increased lipid peroxidation. The explanation for the increased toxicity of the proinflammatory cytokines in AQP8 KD cells resides in the fact that efflux of the H2O2 generated during oxidative stress in the ß-cell mitochondria is hampered through the loss of the peroxiporin channels in the mitochondrial membranes of the AQP8 KD cells. The increased proinflammatory cytokine toxicity due to loss of AQP8 expression in the KD ß-cell mitochondria is thus the result of increased rates of apoptosis. This decreased cell viability is caused by increased levels of oxidative stress along with a ferroptosis-mediated cell death component due to decreased NO● generation.

Assessment of Mitochondrial Ca2+ Uptake.

Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.

AQP8 is a crucial H2O2 transporter in insulin-producing RINm5F cells.

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H2O2) across cellular membranes. H2O2 serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic ß-cells, H2O2 has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H2O2 homeostasis in pancreatic ß-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H2O2 specific biosensor HyPer together with endogenous generation of H2O2 using d-amino acid oxidase, live cell imaging revealed enhanced H2O2 flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H2O2 sensor HyPer7.2 clearly unveiled AQP8 as a H2O2 transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H2O2 pore in insulin-producing RINm5F cells involved in the transport of H2O2 through the mitochondria and cell membrane and may help to explain the H2O2 transport and toxicity in pancreatic ß-cells.

The contribution of uncoupling protein 2 to mitochondrial Ca2+ homeostasis in health and disease - A short revisit.

Considering the versatile functions attributed to uncoupling protein 2 (UCP2) in health and disease, a profound understanding of the protein's molecular actions under physiological and pathophysiological conditions is indispensable. This review aims to revisit and shed light on the fundamental molecular functions of UCP2 in mitochondria, with particular emphasis on its intricate role in regulating mitochondrial calcium (Ca2+) uptake. UCP2's modulating effect on various vital processes in mitochondria makes it a crucial regulator of mitochondrial homeostasis in health and disease.

Differential endothelial signaling responses elicited by chemogenetic H2O2 synthesis.

Hydrogen peroxide (H2O2) modulates critical phosphorylation pathways in vascular endothelial cells, many of which affect endothelial nitric oxide synthase (eNOS) signal transduction. Both intracellular and extracellular sources of H2O2 have been implicated in eNOS regulation, yet the specific endothelial pathways remain incompletely understood. Here we exploited chemogenetic approaches and live-cell imaging methods to both generate and detect H2O2 in different subcellular compartments (cytosol, nucleus, and caveolae) of cultured EA.hy926 human endothelial cells. We developed novel recombinant constructs encoding differentially-targeted yeast d-amino acid oxidase (DAAO), which generates H2O2 only when its d-amino acid substrate is provided. DAAO was expressed as a fusion protein with the new H2O2 biosensor HyPer7.2, which allowed us to quantitate intracellular H2O2 levels by ratiometric imaging in living endothelial cells following the activation of DAAO by d-alanine. The addition of extracellular H2O2 to the HyPer-DAAO-transfected cells led to increases in H2O2 throughout different regions of the cell, as measured using the differentially-targeted HyPer biosensor for H2O2. The sensor response to extracellular H2O2 was more rapid than that quantitated following the addition of d-alanine to transfected cells to activate differentially-targeted DAAO. The maximal intracellular levels of H2O2 observed in response to the addition of extracellular H2O2 vs. intracellular (DAAO-generated) H2O2 were quantitatively similar. Despite these similarities in the measured levels of intracellular H2O2, we observed a remarkable quantitative difference in the activation of endothelial phosphorylation pathways between chemogenetically-generated intracellular H2O2 and the phosphorylation responses elicited by the addition of extracellular H2O2 to the cells. Addition of extracellular H2O2 had only a nominal effect on phosphorylation of eNOS, kinase Akt or AMP-activated protein kinase (AMPK). By contrast, intracellular H2O2 generation by DAAO caused striking increases in the phosphorylation of these same key signaling proteins. We also found that the AMPK inhibitor Compound C completely blocked nuclear H2O2-promoted eNOS phosphorylation. However, Compound C had no effect on eNOS phosphorylation following H2O2 generation from cytosol- or caveolae-targeted DAAO. We conclude that H2O2 generated in the cell nucleus activates AMPK, leading to eNOS phosphorylation; in contrast, AMPK activation by cytosol- or caveolae-derived H2O2 does not promote eNOS phosphorylation via AMPK. These findings indicate that H2O2 generated in different subcellular compartments differentially modulates endothelial cell phosphorylation pathways, and suggest that dynamic subcellular localization of oxidants may modulate signaling responses in endothelial cells.

Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation.

Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic ß-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.

Development and Application of Sub-Mitochondrial Targeted Ca2 + Biosensors.

Mitochondrial Ca2+ uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca2+ kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca2+ biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca2+-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca2+ levels inside the CL but not the IMS in response to IP3-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca2+ through the OMM while the cytosolic Ca2+ signal remained unchanged. The novel sub-mitochondrially targeted Ca2+ biosensors proved to be suitable for Ca2+ imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca2+ signaling under (patho)physiological conditions.

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and ß-Cells upon Elevated Glucose.

BACKGROUND/AIMS: In our recent work, the importance of GSK3ß-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in ß-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3ß-phosphorylated presenilin-1 for responsiveness of pancreatic islets and ß-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic ß-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in ß-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3ß in the pancreatic ß-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3ß in the pancreatic ß-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and ß-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3ß, a phenomenon that might be involved in the development of type 2 diabetes.

MICU1 controls cristae junction and spatially anchors mitochondrial Ca2+ uniporter complex.

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.

Live cell imaging of signaling and metabolic activities.

The interplay of metabolic and signaling processes is prerequisite for the functionality of cells. Any disturbances may have severe consequences, resulting in the development of diseases. However, the complex coordination of metabolism and signaling events makes it difficult to decipher the link between molecular irregularities and pathogenesis. An excellent way to provide more clarity is to see into the living cell and watch cellular processes in real-time, with the add-on of being able to manipulate certain processes. Live cell imaging enables us to do exactly that, with steadily improving spatial and temporal resolution. Modern genetically encoded fluorescent probes in combination with state-of-the-art high-resolution imaging devices have proven themselves as a valuable approach for monitoring, manipulating and ultimately understanding the interaction of cell metabolism and signaling. These probes also represent powerful tools for detecting biomarkers of disease, identifying new drug targets and elucidating drug actions at the cellular to the molecular level.

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+.