A Quad-Cistronic Fluorescent Biosensor System for Real-Time Detection of Subcellular Ca²⁺ Signals

A Quad-Cistronic Fluorescent Biosensor System for Real-Time Detection of Subcellular Ca²⁺ Signals | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL British Journal of Pharmacology This is a preprint and has not been peer reviewed. Data may be preliminary. 3 March 2025 V1 Latest version Share on A Quad-Cistronic Fluorescent Biosensor System for Real-Time Detection of Subcellular Ca²⁺ Signals Authors : Anna Lischnig , Yusuf Erdoğan , Benjamin Gottschalk , Wolfgang Graier 0000-0003-1871-3298 , and Roland Malli [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174098553.38904074/v1 571 views 255 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose The calcium ion (Ca²⁺) is a versatile cellular messenger regulating various biological processes. Compounds modulating subcellular Ca²⁺ signals hold substantial pharmacological potential. Advances in fluorescent biosensors have revolutionized Ca2+ imaging. Still, co-expression of targeted biosensors for simultaneous measurement of Ca2+ signals in multiple cellular compartments is complicated by heterogeneous expression levels of the various sensors. Experimental Approach Here, we introduce CARMEN, a ribosomal skipping-based quad-cistronic fluorescent biosensor system that enables high-content Ca²⁺ imaging across three compartments. CARMEN facilitates equal co-expression of spectrally distinct Ca²⁺ biosensors: the near-infrared Ca²⁺ biosensor for the cytosol (NIR-GECO2G-NES), the green Ca²⁺ biosensor for mitochondria (CEPIA3mt), the red Ca²⁺ biosensor for the endoplasmic reticulum (R-CEPIA1er), along with a Ca²⁺-insensitive blue fluorescent protein targeted to the nucleus (NLS-mTagBFP2), serving as a normalization reference. Key Results CARMEN allows spatiotemporal correlation of Ca²⁺ signals across the cytosol, ER, and mitochondria, revealing distinct dynamics. We noted delayed mitochondrial Ca²⁺ uptake compared to the other compartments. We validated CARMEN across three cell types and tested two recently identified mitochondrial Ca²⁺ uniporter inhibitors (MCUis), MCUi4 and MCUi11, showcasing the potential of CARMEN for its application in pharmacological research. Our results show that while both MCUi4 and MCUi11 inhibited mitochondrial Ca²⁺ uptake in HeLa S3 cells, MCUi4 reduced cytosolic Ca²⁺ signals and oscillations, whereas MCUi11 had opposing effects. Conclusions and Implications CARMEN is a powerful tool for real-time, multiplexed analysis of compartment-specific Ca²⁺ signals, with the potential for automation in high-content drug screening. Introduction Dysregulation of cellular Ca²⁺ homeostasis is implicated in a wide range of diseases, including cardiovascular (1,2) and metabolic disorders (3–5), neurodegeneration (6,7), and cancer (8,9). Subcellular Ca²⁺ fluxes, particularly between the endoplasmic reticulum (ER) and mitochondria (10,11), play essential roles in regulating cellular function and fate (12,13). Disruptions in these Ca²⁺ fluxes can activate stress responses, such as the unfolded protein response (UPR) (14), or trigger metabolic adaptations (15,16). Over time, chronic disturbances in Ca 2+ signals and signalling pathways may lead to self-reinforcing cycles that contribute to disease progression (13). Therapeutic targeting of Ca²⁺ signalling has long been a focus of drug development (17,18). For example, voltage-dependent Ca²⁺ channels (VDCCs) are well-established targets for treating hypertension and arrhythmias (19). L-type Ca²⁺ channel blockers, such as amlodipine, nifedipine, and verapamil, reduce vascular smooth muscle contraction by inhibiting Ca²⁺ influx (20). However, such drugs do not specifically target subcellular Ca²⁺ channels and transporters, which are increasingly recognized as critical for cellular function (21,22). Organelles such as the ER, lysosomes, and mitochondria harbour unique Ca²⁺ transport systems that regulate localized Ca²⁺ dynamics, yet selective chemical modulators for these organelle-specific signals remain scarce (23). Developing tools for the investigation of targeted modulation of subcellular Ca²⁺ signals (24) expands therapeutic possibilities. Recent advances in genetically encoded Ca 2+ indicators (GECIs) have revolutionized the study of subcellular Ca²⁺ dynamics (25). The high sensitivity and specificity of GECIs enabled detailed mapping of Ca 2+ dynamics with high spatial and temporal resolution (26–28). These biosensors, engineered from Ca²⁺-binding domains like calmodulin (29,30) or troponin C (31) fused to fluorescent proteins (FPs), allow precise visualization of Ca²⁺ fluxes (29). Single FP-based biosensors, in particular, are efficiently targetable and well-suited for monitoring Ca²⁺ in specific organelles (32). The development of appropriate GECIs with customized affinity (K d ) for specific applications is crucial, as this ensures optimal sensitivity for the targeted organelle (33–38). However, most studies using these biosensors examine Ca²⁺ dynamics in only one cellular compartment and rely on separate experiments to study different organelles (39). This limits our understanding of how Ca²⁺ signals in one organelle influence those in others (24,28). Practical tools and protocols that are capable of simultaneously visualizing compartment-specific Ca²⁺ alterations within a single cell hold significant potential for elucidating these intricate interactions (24–27). Such approaches enable a more comprehensive evaluation of pharmacological compounds on subcellular Ca²⁺ signals. In this study, we developed a biosensor reporter system, named CARMEN, that enables simultaneous imaging of Ca²⁺ dynamics in the ER, cytosol, and mitochondria within single cells in vitro , leveraging an optimized ribosomal skipping approach (40). We used this system to monitor Ca 2+ dynamics at subcellular resolution and examined known small-molecule inhibitors of the mitochondrial Ca²⁺ uniporter (MCU) (41,42), demonstrating CARMEN’s utility for studying how these compounds specifically affect distinct subcellular Ca²⁺ signals. Plasmid design The Ca 2+ biosensors CEPIA3mt (37), NIR-GECO2G-NES(38), and R-CEPIA1er (37) and the FP NLS-mTagBFP2 (43) were linked by 2A sequences T2A, P2A, E2A (the full sequence is shown in Figure S2a). Following the design, the reporter system sequence was commercially synthesized and cloned into pcDNA 3.1 (+) (Gene Universal Inc, Newark DE). Cell culture and transfection HeLa S3 (RRID:CVCL_0058) and EA.hy926 (RRID:CVCL_3901) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM D5523, Sigma-Aldrich) supplemented with 10% FCS, 10 mM NaHCO 3 , 50 U/mL penicillin-streptomycin, 1.25 μg/mL amphotericin B and 25 mM HEPES; pH 7.45 with NaOH. INS-1 832/13 (RRID:CVCL_7226) cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.05 mM beta-mercaptoethanol, 50 U/mL penicillin-streptomycin and 1.25 μg/mL amphotericin B. Cell culture materials were obtained from Greiner Bio-One (Kremsmünster, Austria). Cells were grown in a humified incubator (5% CO 2 , 37 °C). Cells were seeded on 30 mm glass coverslips (Co. KG, Lauda-Königshofen) in 6-well plates (Paul Marienfeld GmbH, Germany). For transient transfection, the transfection reagent PolyJet (SignaGen Laboratories, Rockville, USA) was used according to the manufacturer’s instructions. Briefly, to transfect one well of a 6-well plate, 3 μL of PolyJet reagent in 100 μL of DMEM (no additives) was mixed with 1 μg of CARMEN DNA in 100 μL of DMEM (no additives). The transfection mixture was added to 1 mL of culture medium for 8-10 h and was then replaced with 2 mL of culture medium. Live-cell imaging was performed 36-48 hours after CARMEN transfection. Chemicals and imaging buffers Before all imaging experiments, cells were equilibrated in cell storage buffer for at least 20 minutes (2 mM CaCl 2 , 138 mM NaCl, 1 mM MgCl 2 , 5 mM KCl, 10 mM HEPES, 2.6 mM NaHCO 3 , 0.44 mM KH 2 PO 4 , 0.34 mM Na 2 HPO 4 , 1X MEM Amino Acids Solution (Gibco), 1X MEM Vitamin Solution (Gibco), 10 mM D-glucose, 2 mM L-glutamine, 1% penicillin-streptomycin and 1% Amphotericin B; pH 7.45 with NaOH). During dynamic microscopy experiments, a physiological imaging buffer (2 mM CaCl 2 , 141 mM NaCl, 1mM MgCl 2 , 5mM KCl, 10 mM HEPES and 10 mM D-glucose; pH 7.4 with NaOH) and a Ca 2+ -free imaging buffer (141 mM NaCl, 1 mM MgCl 2 , 5 mM KCl, 10mM HEPES, 1 mM EGTA, and 10 mM D-glucose; pH 7.4 with NaOH) were used. Modifications on the imaging buffers are indicated in the figures, figure legends, and text (e.g. treatment or stimulant). Adenosine 5′-triphosphate disodium salt (ATP) (Carl Roth, Graz, Austria), 2,5-di-tert-butylhydroquinone (BHQ) (Sigma-Aldrich, Massachusetts, United States) were employed to stimulate distinct Ca 2+ signals (Sigma-Aldrich). Structured Illumination Microscopy Structured illumination microscopy (SIM) was performed using a Zeiss Elyra 7 system equipped with a 63x/1.4 NA oil-immersion objective (Plan-Apochromat DIC M27, Zeiss) and two sCMOS pco.edge 4.2 CLHS cameras with a pixel size of approximately 6.5 µm. Lasers at 488 nm and 561 nm, were used for the excitation of the GFP and RFP, respectively. Imaging was conducted in Lattice SIM mode with 13 phases using a lattice SIM pattern. Exposure times were 10 and 30ms for the channels, respectively. Image reconstruction was performed using Zeiss Zen software. Confocal and epifluorescence microscopy Nikon Eclipse Ti2 microscope (Nikon, Austria) was used for CARMEN visualization, for endpoint and dynamic time-lapse imaging. Imaging was performed using a 100×/1.45 NA oil objective (CFI Apochromat, Nikon), standard filter sets, and a back-illuminated Kinetix Scientific CMOS camera (TELEDYNE PHOTOMETRICS, USA, Tucson) mounted to the Nipkow based Crest optics X Light V3 with 70 µm pinhole spinning disc system (Crestoptics, Italy, Rome). For confocal images, we used the spinning disc and laser excitation (Celesta, Light Engine) at 405 nm, 477 nm, 546 nm, and 638 nm for BFP, GFP, RFP, NIR, respectively. Emission signals were captured through corresponding emission filters at 438 nm, 511 nm, 595 nm, or 685 nm. For epifluorescence imaging, the CoolLED pE800 light source (CoolLED, UK, Andover) with wavelengths at 400 nm, 470 nm, 550 nm, and 635 nm, managed through a turret (Turret-Lo: MXR00724, LED Full Multiband Penta) for efficient switching was used. Emission detection was consistent with the confocal setup (438 nm, 511 nm, 595 nm, or 685 nm), allowing seamless integration between confocal and EPI imaging modalities. NIS Elements software (NIS-Elements AR 5.42.06 (Build 1821) LO 64bit, Nikon, Austria) was used for microscope control and image acquisition. Image acquisition, data analysis and presentations Z-stack confocal images were acquired with a step size of 200 nm, capturing a series of optical sections through the cells. These z-stack images were analysed and processed using NIS Elements software (Nikon). The images were subjected to 3D blind-deconvolution, before creating the volume view. The video was then animated, starting from the xy plane, with a 360° rotation around the x-axis to provide a comprehensive, dynamic view of the three-dimensional structure. Images shown in the figures were either background-subtracted using the rolling ball method or through arithmetic subtraction of the background. Line profiles were measured and presented in graphs, with the respective measurement lines indicated in the corresponding images. For endpoint measurements using CARMEN, all four fluorescence channels (BFP, GFP, RFP, and NIR) were acquired in epifluorescence mode at multiple positions before and after a 12 min incubation with 15 µM BHQ. Image analysis was performed using Fiji software (version 2.14.0/1.54f). In this study, we opted for manual ROI annotation of the whole cell and the entire nucleus to analyse Ca 2+ -mediated intensity changes and to normalise the signals. For the time-lapse recording of CARMEN, all four fluorescence channels (BFP, GFP, RFP, NIR) were acquired from individual cells using epifluorescence microscopy at 2-second intervals. The buffers, stimulant and inhibitor concentrations, and pre-incubation times used in time-lapse imaging are provided in the figure legends. During imaging, buffers containing stimulants and/or inhibitors were perfused using a gravity-based perfusion system (NGFI, Graz, Austria). Regions of interest (ROIs) for the whole cell and nucleus were manually defined, and mean intensities were measured using Fiji. The intensity data was background-subtracted (background ROI), and exponential fitting was used to calculate individual bleaching functions (F 0 ). These data were normalized by using the BFP NT reference signal or scaled to percentage using range normalization, with the latter applied for comparative analysis. The exponential decay function was fitted to each intensity curve using two different approaches: A script that removes outliers using a local window approach, and fits an exponential decay model to the data or the One-Phase decay function in GraphPad Prism version 10.2.3 (GraphPad Software, Boston, Massachusetts USA). For further analysis of these intensity curves a script was compiled. The script smooths signal data (e.g. GFP, RFP, NIR) using Savitzky-Golay filtering (44) (window length and polynomial order were adjusted according to the data) and calculates the first derivative to identify significant changes. Signal emergence times were detected by annotating peaks in the derivative. The script was developed with the assistance of an AI-based coding tool ChatGPT (GPT-4, OpenAI, GPT-4o, and o1 models), to streamline the analysis process. The data obtained were visualized in GraphPad Prism. The spectral data of mTagBFP2, mIFP for NIR-GECO2G-NES, mApple for R-CEPIA1er, eGFP for CEPIA3mt were retrieved from FPbase (https://www.fpbase.org/) on 15 October 2024 (45) and plotted on a graph, including the emission filter set of the epifluorescence part of the Nikon Eclipse Ti2 microscope used in this study. Schematics representing cells expressing CARMEN were designed using Servier Medical Art (http://www. servier .com/Powerpointimage-bank) under a Creative Commons attribution 3.0 Unported License. Statistical analysis Data were visualized and statistically analysed using GraphPad Prism. The statistical tests used are indicated in the figure legends. p-values of ≤0.05 were considered as significant, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns (not significant). The number of replicate experiments ‘r,’ and the number of single cells analyzed ‘c’ are provided in the format n = r/c. For example, n = 6/62 indicates that 6 biological replicates were performed, analyzing a total of 62 cells. Results Design of a quad-cistronic Ca 2+ reporter for multiplexing and NCC We designed a quad-cistronic Ca 2+ reporter for multiplexing and Nucleo Cytoplasmic Compartmentalisation (NCC) (46,47), termed CARMEN ( Figure 1 and Figure S1 ) to enable simultaneous monitoring of Ca²⁺ signals in different cellular compartments in one given cell. CARMEN consists of four FP-based components: CEPIA3mt, mTagBFP2, NIR-GECO2G, and R-CEPIA1er targeted to the mitochondria, the nucleus, the cytosol, and the ER, respectively. These components are connected by three different 2A peptide sequences (40), ensuring efficient and coordinated polycistronic expression ( Figure 1a and Figure S2a ). The nuclear-targeted mTagBFP2 (NLS-mTagBFP2) serves as a reference protein for normalization and provides a secondary readout for senescence (46,47). CARMEN, thus, provides two critical functionalities: i. simultaneous, compartment-specific measurements of Ca²⁺ dynamics by targeted intensiometric Ca²⁺ biosensors ( Figure S1b ) and ii. the ability to correlate these measurements with cellular senescence through the NCC module ( Figure S1c ). We used high-resolution live cell imaging to visualize the CARMEN components in the respective cellular compartments in different cell types, including cervical carcinoma cell line HeLa S3, immortalized human umbilical vein endothelial cells EA.hy926, and the rat pancreatic beta cell line INS-1. All cells transiently expressing CARMEN were positive for all four of the targeted components ( Figure 1b, Figure S1a ), demonstrating the efficiency of the quad-cistronic approach. We highlighted mitochondria- and ER-targeting of the green and red CEPIA variants of CARMEN using superresolution structural illumination microscopy ( Figure S3 ). Moreover, we performed 3D rendering of z-stack images of a HeLa S3 ( Video S1 ) and a EA.hy926 ( Video S2 ) cell to show the correct targeting of all CARMEN components. We noted some single-cell variances in the fluorescence distribution of CARMEN components, which could be attributed to cellular morphological heterogeneity ( Figure S4 ). Furthermore, HeLa S3 cells exhibited the highest overall fluorescence intensity among the tested cell types, with an mean fluorescence signal 4.2-fold higher than in EA.hy926 cells and 4.9-fold higher than in INS-1 cells. This suggests that HeLa S3 cells were particularly efficient at producing and processing the components of the CARMEN system under the chosen experimental conditions. Next, we examined NCC patterns in the cultured cells transiently expressing CARMEN by screening for BFP distribution between the nucleus and cytosol. Interestingly, all HeLa S3 cells showed stable NCC, implying the absence of senescence in this particular cell population ( Figure S5 ). In contrast, in a small fraction of EA.hy926 ( Figure S5 ) and INS-1 cells ( Figure S5c,d ), we observed perturbed NCC, which was analysed either by linescan ( Figure S5a and b ) or based on cell to nucleus ratios of the respective FP fluorescence intensity ( Figure S5c and d ). CARMEN reveals cell-type-specific subcellular Ca 2+ redistribution in response to a SERCA inhibitor Using CARMEN we imaged subcellular Ca 2+ signals before and after treatment with 2,5-di-tert-butylhydroquinone (BHQ) ( Figure 2a and Figure S6 ), a reversible sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor (48). SERCA inhibition mobilizes Ca²⁺ from the ER and strongly activates store-operated Ca²⁺ entry (SOCE) (49), which led to significant alterations in the absolute fluorescence intensities of the three targeted Ca²⁺ biosensors within CARMEN ( Figure 2b and Figure S7 ). It should be noted that, in contrast to the ER- and mitochondria-targeted Ca 2+ biosensors, the cytosolic-targeted Ca 2+ biosensor NIR-GECO2G-NES (38) gives a reduction in fluorescence intensity upon Ca 2+ binding. In the majority of untreated INS-1 cells, the fluorescence intensity of NIR-GECO2G-NES was lower compared to HeLa S3 and EA.hy926 cells ( Figure 2b and Figure S7 ). This may stem from poor bioavailability of the NIR-IR FP chromophore biliverdin for NIR-GECO2G-NES (38) in INS-1 cells. Furthermore, we used the Ca 2+ -insensitive NLS-mTagBFP2 of CARMEN for ratiometric normalization, accounting for heterogeneities in expression levels ( Figure 2b and Figure S7 ), performed paired analysis ( Figure S8 ), and calculated changes in fluorescence signals of individual cells before and after BHQ treatment ( Figure 2c ). These analyses demonstrated that the majority of HeLa S3, EA.hy926, and INS-1 cells showed reduced ER Ca 2+ levels and an increase of Ca 2+ within the cytosol upon SERCA inhibition ( Figure 2c , Figure S8 ). Despite cell-to-cell heterogeneities, most HeLa S3 and EA.hy926 cells showed elevated Ca 2+ within mitochondria after BHQ treatment ( Figure 2c and Figure S8 ). However, in INS-1 cells, BHQ treatment yielded mixed mitochondrial Ca²⁺ alterations, with a minority of cells showing elevated mitochondrial Ca²⁺ signals ( Figure 2c and Figure S8 ). Overall, with this set of experiments, we demonstrated that CARMEN can provide comprehensive insights into subcellular Ca 2+ redistribution of individual cells using endpoint fluorescence microscopy. Visualisation of IP₃-induced compartment-specific Ca²⁺ oscillations by CARMEN time-lapse imaging Next, we used CARMEN in time-lapse imaging experiments to visualize organelle-specific propagations of Ca 2+ signals ( Figure S9 ). To evoke oscillatory Ca 2+ signals, HeLa S3 cells were treated with a submaximal concentration of the physiological inositol-trisphosphate (IP 3 ) -generating agonist ATP in the presence of extracellular Ca 2+ ( Figure 3 ). Multispectral imaging of the localized Ca²⁺ biosensors revealed that ATP triggers an immediate elevation in cytosolic Ca²⁺, synchronized with a decrease in ER Ca²⁺, while mitochondrial Ca²⁺ uptake followed with a clear delay ( Figure 3a-c ). CARMEN analysis of numerous HeLa S3 and EA.hy926 cells under identical experimental conditions revealed distinct differences in signal emergence ( Figure 3c ). We quantified the delay between cytosolic and mitochondrial Ca²⁺ rises, measuring 11.1 ± 0.9 s (n = 60) in HeLa S3 cells and 29.0 ± 3.8 s (n = 21) in EA.hy926 cells. Delving deeper into the signals of a single HeLa S3 cell shown in Figure 3a, we observed that cytosolic Ca²⁺ levels stabilize in a brief plateau phase before gradually declining ( Figure 3b and d ). Simultaneously, ER Ca²⁺ levels replenished ( Figure 3b ), suggesting enhanced SERCA activity counteracting IP₃ receptor (IP 3 R)-mediated Ca²⁺ release. Interestingly, mitochondrial Ca²⁺ uptake was initiated only during this plateau phase of elevated cytosolic Ca²⁺ ( Figure 3b ), consistent with findings that a certain threshold of Ca²⁺ is required to activate the mitochondrial calcium uniporter (MCU) complex (50–52). In this particular HeLa S3 cell, mitochondrial Ca²⁺ accumulation was temporarily halted as ER refilling progressed, suggesting that SERCA activity counteracts further mitochondrial Ca²⁺ uptake. Subsequently, ER Ca²⁺ levels dropped again, triggering another cytosolic Ca²⁺ elevation, and a delayed mitochondrial Ca²⁺ uptake ( Figure 3a-d ). As the oscillations continued, cytosolic and ER Ca²⁺ remained tightly synchronized, while mitochondrial Ca²⁺ exhibited a lagged response, mirroring these fluctuations with lower amplitude and gradually returning toward baseline ( Figure 3a and d ). Using the CARMEN tool, we found that individual HeLa S3 cells exhibited highly heterogeneous Ca²⁺ responses to 30 µM ATP ( Figure S10 and Supplementary file 1 ), with ER and cytosolic Ca²⁺ signals always oscillating in synchronicity. In contrast, EA.hy926 cells displayed more homogeneous, non-oscillatory, and long-lasting cytosolic and organellar Ca²⁺ changes under the same experimental conditions ( Figure S11 and Supplementary file 2 ). CARMEN uncovers distinct effects of MCU inhibitors on cytosolic and ER Ca²⁺ dynamics We tested two different MCU inhibitors (MCUis), MCUi4 and MCUi11 (41,42), and demonstrated the use of CARMEN in high-content pharmacological profiling. We pre-treated HeLa S3 cells for 90 minutes with either 30 µM MCUi4 or 10 µM MCUi11 and compared CARMEN signals with untreated control cells. Cells were stimulated with 100 µM ATP in the absence of extracellular Ca 2+ to examine the inhibitory effects of MCUis on mitochondrial Ca²⁺ uptake solely sourced from Ca²⁺ released by the ER ( Figure 4a ). Under these conditions, around 98% of control cells showed clear cytosolic and ER Ca²⁺ signal changes ( Figure 4 and Figure S12a ; strong and moderate responders), whereas only 60% of these cells exhibited clear mitochondrial Ca²⁺ elevations, with 40% of control cells remaining mitochondrial non-responders ( Figure 4 and Figure S12a ). With CARMEN, we identified a link between weak cytosolic Ca 2+ signals and mitochondrial non-responders ( Figure 4b ). This aligns again with the assumption that activation of mitochondrial Ca 2+ uptake requires high Ca 2+ levels (50,51). MCUi4 strongly reduced mitochondrial Ca²⁺ signals and attenuated cytosolic Ca 2+ elevations, while ER Ca 2+ release remained unaffected ( Figure 4 and Figure S12 ). MCUi11 also considerably hampered mitochondrial Ca 2+ elevations in most cells, while cytosolic and ER signals were enhanced in contrast to MCUi4 ( Figure 4a and b ). Notably, leveraging the multiparametric readout of CARMEN, we observed that both MCUis abolished mitochondrial Ca 2+ uptake even in cells with pronounced cytosolic and ER Ca 2+ signals ( Figure 4a and b ). This corroborates the inhibitory effects of MCUi4 and MCUi11 on the mitochondrial Ca 2+ uptake machinery. Single cell analysis revealed other key differences between MCUi4 and MCUi11: MCUi4 consistently reduced mitochondrial Ca²⁺ uptake across all cells. However, some cells treated with MCUi11 still displayed strong mitochondrial Ca²⁺ signals, despite an overall reduction in mitochondrial responders ( Figure 4b and Figure S12a ). Furthermore, we classified cells into two groups - oscillators and non-oscillators – and tested if MCUis changed this distribution. Interestingly, cells treated with MCUi11 showed more oscillators than untreated cells, while MCUi4 reduced the number of oscillating cells ( Figure 4 and Figure S12b ). Discussion and Conclusion In this study, we developed CARMEN, a quad-cistronic reporter system that enables real-time monitoring of Ca²⁺ dynamics across three distinct compartments in individual cells. By simultaneously examining cytosolic, mitochondrial, and ER Ca²⁺ alterations, we identified significant differences in subcellular Ca²⁺ signals of individual cells and between different cell types. CARMEN facilitates the development of targeted interventions that modulate specific subcellular Ca²⁺ signals, deepening our understanding of Ca²⁺ regulation and highlighting CARMEN’s potential for high-content pharmacological investigations. Although numerous Ca²⁺ biosensors have been developed over the years and continue to be improved, the simultaneous use of multiple fluorescent biosensors with distinct colours and targeting properties remains relatively rare. Previous studies have employed GECOs or CEPIAs and performed triple or dual colour imaging (26,37). Our new reporter system incorporates three well-established fluorescent Ca²⁺ biosensors (CEPIA3mt (37), NIR-GECO2G (38), and R-CEPIA1er (37)) along with a blue fluorescent reference protein (43), connected with 2A sequences, to enable efficient and balanced expression of the four protein components in a single transcript. This strategy helps circumvent unpredictable expression patterns typically observed with standard co-transfection approaches (40). CARMEN employs biosensors with non-overlapping excitation and emission spectra, ensuring robust spectral resolution across compartments. Specifically, green CEPIA3mt, targeted to the mitochondrial matrix, has a K d of ~11 µM, making it highly sensitive to physiological Ca²⁺ fluctuations of this particular organelle. R-CEPIA1er, localized to the ER, features a higher K d of ~565 µM, optimizing it for resolving ER Ca²⁺ dynamics. NIR-GECO2G, with a K d of levels. Most mammalian cells should work well with these K d values; however, certain cell types, such as neurons and muscle cells, may require different sensitivities. The modular design of CARMEN enables its components to be easily exchanged to accommodate different Ca 2+ biosensors with preferred sensitivities or other biosensors to correlate diverse signalling activities in multiparametric experiments (53). The nuclear BFP in CARMEN serves two purposes: first, as a reference protein, and second, for the NCC assay (46,47). We observed clear heterogeneities in the fluorescence intensities of the CARMEN components, likely pointing to variances in expression levels between cells, which we confirmed and considered using the Ca 2+ -insensitive BFP NT for normalisation. The differences in fluorescence intensities may also reflect intrinsic morphological variations between cell types, including organelle size and density, cellular volume, and the bioavailability of biliverdin for the near-IR Ca 2+ biosensor (38). We, thus, recommend considering these aspects for correct data interpretations and signal normalization particularly when CARMEN is used in endpoint measurements. Targeting of the constructs to the mitochondria and ER was consistent and accurate across all three cell lines, while the nuclear and cytosolic targeting in EA.hy926 and INS-1 cells were more variable compared to HeLa S3 cells. The NCC assay is a well-established method for assessing cellular senescence (46,47). Although we have not yet specifically evaluated the reporter system in a senescence context, the observed mislocalization of the cytosolic and nuclear FPs of EA.hy926 and INS-1 cells suggests a potential link to cellular senescence or stress. Further studies are needed to determine the extent and underlying causes of this variability in NCC. Taken together, CARMEN can assess both compartmental integrity (i.e. NCC) and organelle-specific Ca 2+ alterations, underlining its utility as a tool for exploring the interplay between Ca 2+ signals and cellular aging. We proceeded to successfully use CARMEN in time-lapse imaging experiments that allowed spatiotemporal correlations of Ca 2+ signals from three important cellular compartments. To our knowledge, CARMEN is the first reporter system that allows multiparametric co-imaging of Ca 2+ signals within the cytosol, mitochondria matrix, and the ER lumen by the co-expression of distinct Ca 2+ biosensors from a single vector, leveraging optimized ribosomal skipping sequences (40). Using this approach, we confirmed delayed mitochondrial Ca 2+ signals in HeLa S3 and EA.hy926 cells, which was recently reported using fura-2 imaging combined with red-shifted Förster resonance energy transfer (FRET)-based Ca 2+ biosensors targeted to mitochondria (27). Here, we additionally correlated ER Ca 2+ alterations with the cytosolic and mitochondrial Ca 2+ signals. Our data highlight synchronicity between oscillatory ER and cytosolic Ca 2+ signals, evoked by the IP 3 -generating agonist ATP. CARMEN also enables investigations of cell-to-cell variations in compartmentalized Ca 2+ signals. Given that any aspect of a signal – its amplitude, frequency, duration, delay, and subcellular localization, determines biological outcomes (24), CARMEN fills an important methodological gap in high-content investigations of subcellular Ca 2+ signals and signalling. One exciting aspect of this study is the differences observed in the mitochondrial Ca²⁺ handling , specifically in the response to MCUi (41,42). The MCU is a complex of several components that control mitochondrial Ca²⁺ uptake (5,54), which is crucial for cellular energy metabolism (55,56), signal transduction (57), and induction of cell death pathways (58). We examined the inhibitory effects of MCUi4 and MCUi11 following a 90-minute pre-incubation, as this duration was previously reported to ensure strong inhibition of mitochondrial Ca²⁺ uptake (41). Consistent with prior findings (41), we found that both compounds showed a certain potency to reduce mitochondrial Ca²⁺ uptake in HeLa S3 cells, the same cell type used for their initial identification from high-throughput screening. MCUi4 and MCUi11 exert their effects via binding to MICU1 (41), a key regulatory subunit of the MCU complex that prevents mitochondrial Ca²⁺ entry at low extra mitochondrial Ca²⁺ concentrations, thereby acting as a negative regulator of the mitochondrial Ca²⁺ uniporter in most cells (59–62). However, it remains unclear how compounds targeting a negative regulator of MCU can ultimately inhibit MCU activity—an effect that, at first glance, appears paradoxical. This paradox may be explained by the dynamic structural role of MICU1 in regulating mitochondrial Ca²⁺ uptake. Under low Ca²⁺ conditions, MICU1 forms hexamers that stabilize cristae junctions, restricting MCU to the cristae membrane and preventing Ca²⁺ influx (61–63). When intermembrane space Ca²⁺ levels rise sufficiently to bind MICU1’s EF-hand domains, this Ca²⁺ binding triggers the disassembly of MICU1 hexamers into dimers, leading to partial cristae junction opening (61–63). This structural change allows MCU to diffuse to the inner boundary membrane, where it interacts with MICU1 dimers, ultimately enabling mitochondrial Ca²⁺ uptake (61,62,64). In light of these novel insights into how mitochondrial Ca²⁺ uptake is regulated, MCUi4 and MCUi11 may either stabilize MICU1 in a conformation that further restricts MCU function or disrupt MICU1’s dynamic role in Ca²⁺ sensing and gating, thereby suppressing mitochondrial Ca²⁺ uptake. Further studies will be necessary to elucidate the precise mechanisms underlying their inhibitory effects. Interestingly, we observed striking differences from the initial study (41), particularly in how MCUi4 and MCUi11 influenced cytosolic Ca²⁺ signals. Despite both compounds effectively inhibiting mitochondrial Ca²⁺ uptake, they had opposing effects on cytosolic Ca²⁺ signals. MCUi4 not only reduced cytosolic Ca²⁺ signals but also decreased the fraction of cells exhibiting Ca²⁺ oscillations. In contrast, MCUi11 significantly enhanced cytosolic Ca²⁺ levels and increased the number of oscillatory responders. Several factors may underlie these divergent effects. Differences in compound concentrations could contribute, as we applied MCUi4 at 30 µM—likely to induce mitochondrial depolarization—while MCUi11 was used at a lower concertation of 10 µM. It is conceivable that prolonged exposure to the higher concentration of MCUi4 compromised cell viability, suppressing the IP₃ pathway. Conversely, MCUi11 at lower concentrations may have selectively prevented mitochondrial Ca²⁺ uptake while preserving the integrity of ER-mitochondria contact sites (MAMs) (65). This could result in increased Ca²⁺ accumulation within MAMs, allowing for greater cytosolic Ca²⁺ diffusion and potentially enhancing IP₃R-mediated Ca²⁺ release through Ca²⁺-induced Ca²⁺ release (CICR) mechanisms (66,67). 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Main figure legends Figure 1: CARMEN: Quad-cistronic Ca 2+ Reporter for Multiplexing and nucleocytoplasmic compartmentalization assay (NCC) (a) Schematic of CARMEN design using the single FP based Ca 2+ indicators NIR-GECO2G, CEPIA3mt and R-CEPIA1er as well as the reference FP, mTagBFP2, separated by 2A sequences, these FPs are targeted to the cytosol (CT, dark red), the mitochondria (MT, green), the endoplasmic reticulum (ER, beige) and the nucleus (NT, blue), respectively. NCC refers to nuclear-cytoplasmic compartmentalization assay. (b) Representative confocal microscopy images and corresponding magnification images of HeLa S3 transiently expressing CARMEN. The columns from left to right show individual fluorescence channels for GFP (CEPIA3mt), BFP (NLS-mTagBFP2), near infrared (NIR-GECO2G-NES), RFP (R-CEPIA1er) and the merged image, illustrating the spatial distribution of CARMEN components. Scale bar is 10µM for all images. Figure 2: Effect of BHQ on Cellular Signals in Different Organelles (a) Schematic of the two-point measurement: baseline (T 0min ) and after 12 min of 15 µM BHQ treatment (T 12min ) (b) Absolute intensity values before and 12 min after 15µM BHQ incubation in HeLa S3 (n=3/345), displayed are median and interquartile range (IQR). Kruskal-Wallis test with Dunn’s multiple comparisons test. Statistical significance indicated as *p < 0.05, **p < 0.01, ****p < 0.0001, ns (not significant). (c) Δ(T 12min - T 0min ) values for HeLa S3 (n=3/345), EA.hy926 (n=5/257) and INS-1 (n=2/174), representing changes in cytosolic (top), mitochondrial (middle), and ER (bottom) signals, displayed as mean with SEM. Prior to Δ-calculation, all values were normalized as F/F0. All data used to calculate the Δ(T 12min - T 0min ) values in Figure 2c are shown in Figure S8. Figure 3: CARMEN reveals distinct temporal dynamics of Ca 2+ signals in organelles following ATP stimulation (a) Time-course live-cell imaging of HeLa S3 cells transiently expressing CARMEN, showing fluorescence changes in response to 30 µM ATP stimulation. The x-y graphs and single-cell heat maps depict normalized fluorescence intensity changes ΔF/F₀ (%) over time in different subcellular compartments: cytosol (top, NIR-GECO2G), mitochondria (middle, CEPIA3mt), and ER (bottom, R-CEPIA1er). (b) The graph presents a zoomed-in view of the initial response phase, focusing on the first detectable reaction to ATP stimulation. Fluorescence traces from all three biosensors overlaid to highlight the timing in compartment-specific Ca²⁺ dynamics. (c) Signal emergence times in CT, MT, and ER were displayed as violin plots with paired values connected by grey lines for HeLa S3 (n=6/60) and Ea.hy926 (n=6/21). (d) Time-course of Ca²⁺ signals showing distinct oscillations dynamics in subcellular compartments. Figure 4: Effects of MCU inhibitors MCUi4 and MCUi11 on cellular Ca²⁺ dynamics (a,b) HeLa S3 cells were pre-incubated for 90 minutes with either with 30µM MCUi4, 10µM MCUi11 or left untreated. (a) Representative single-cell Ca²⁺ curves in CT, MT and ER upon 100µM ATP stimulation. Statistical evaluation of MCUi treated and control cells (control, n=3/30; MCUi4, n=3/34; MCUi11, n=3/42) in (b) ΔF Max represents the maximum signal amplitude (baseline to peak) and is compared between MCUi and the control condition, displayed as individual points with the mean and SEM. Grey lines indicate paired signals from the same cell. Information & Authors Information Version history V1 Version 1 03 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection British Journal of Pharmacology Keywords ion channels mitochondria translational pharmacology Authors Affiliations Anna Lischnig Medical University of Graz View all articles by this author Yusuf Erdoğan Medical University of Graz View all articles by this author Benjamin Gottschalk Medical University of Graz View all articles by this author Wolfgang Graier 0000-0003-1871-3298 Medical University of Graz View all articles by this author Roland Malli [email protected] Medical University of Graz View all articles by this author Metrics & Citations Metrics Article Usage 571 views 255 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Anna Lischnig, Yusuf Erdoğan, Benjamin Gottschalk, et al. A Quad-Cistronic Fluorescent Biosensor System for Real-Time Detection of Subcellular Ca²⁺ Signals. Authorea . 03 March 2025. 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