Genome interrogation of homeostatic calcium activity using CaMP-screen

Genome interrogation of homeostatic calcium activity using CaMP-screen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication Genome interrogation of homeostatic calcium activity using CaMP-screen Amado Carreras-sureda, Sana Kouba, Xin Zhang, Cyril Castelbou, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4656479/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Calcium signals regulate crucial cellular functions yet many genes coding for Ca 2+ handling proteins remain unknown as their identification relies on low-throughput single-cell approaches. Here we describe a novel method to measure Ca 2+ activity in cells isolated by flow cytometry following pooled genome interrogation. Using a CRISPR/ CAMPARI2 screen, we identified enhancers and inhibitors of homeostatic Ca 2+ activity. Biological sciences/Cell biology/Cell signalling Biological sciences/Biological techniques/Sensors and probes/Fluorescent proteins Biological sciences/Biological techniques/Electrophysiology/Intracellular recording Biological sciences/Biological techniques/High-throughput screening Figures Figure 1 Figure 2 Main Ion channel research relies on single cell electrophysiological or imaging recordings, time-consuming approaches of high precision but limited throughput. Genomic screens involving ion channels rely on multiplexed plate readers targeting a single gene per well in an arrayed configuration 1 . Recent advancements on genome interrogation using pooled configuration have significantly lowered the costs and the time needed to screen for any biological function. The Ca 2+ -Activated Photoconvertible Ratiometric indicator 2 (CaMPARI2) 2 sensor is irreversibly photoconverted from green to red upon combined Ca 2+ binding and UV light exposure. This feature allows the rapid identification of Ca 2+ activity in single cells and has been used to report Ca 2+ transients in populations of neurons or in zebrafish 3–5 . Here, we describe how to use CaMPARI2 for genome interrogation of Ca 2+ activity by flow cytometry. CaMPARI2 green fluorescence decreases upon Ca 2+ binding and becomes irreversibly red only when UV light is applied in this Ca 2+ -bound state 2 (Figure 1a-b). To study large populations of cells, we built a UV photoconversion (PC) setup (Ø 0= 6 cm; 10-25 mW/cm 2 , Figure s1a) and used it to photoconvert mouse embryonic fibroblasts (MEF) cells treated with cyclopiazonic acid (CPA) or Yoda1 to activate store-operated or mechanosensitive channels, respectively (Figure 1C and s1b). 67% of CPA-treated and 32% of Yoda1-treated cells were photoconverted, validating the efficiency of the setup to identify Ca 2+ fluxes. Unexpectedly, approximately 10% of cells were photoconverted in the absence of chemicals, suggesting that Ca 2+ elevations occurred spontaneously during the 1 min UV exposure. Comparable basal activity was observed in three different cell lines and was absent in cells treated with CPA in Ca 2+ -free to deplete intracellular Ca 2+ stores and prevent Ca 2+ entry (Fig 1 c-e and s1c-d) indicating that it reflected homeostatic Ca 2+ activity. CaMPARI2 calibration using Fura-2 as reference 6 yielded a KD of 98 nM while a CaMPARI2 bearing mutations preventing Ca 2+ binding (Dead-CaMP2) did not report spontaneous or evoked Ca 2+ fluxes (Figure s1e-f). The Orai1 channel inhibitor GSK7975A 7 and the phospholipase C (PLC) inhibitor U73122 8 decreased the proportion of cells undergoing spontaneous PC (Figure 1f), linking the homeostatic activity to IP 3 -mediated Ca 2+ release fueled by store operated calcium entry (SOCE). To identify genes controlling this activity, we generated MEF cells stably expressing a whole genome knockout library with 4 sgRNAs per gene 9 . Cells were sorted for spontaneous PC and processed for next generation sequencing analysis (NGS) in two independent experiments (Figure 2a). Depletion and enrichment analysis using an FDR of 0.01 identified 468 genes sustaining (enhancers) and 918 genes mitigating (inhibitors) the spontaneous activity (Figure 2b). Gene ontology and enrichment analysis of the 468 enhancers revealed an enrichment of Ca 2+ ion binding and G-protein modulator on this dataset (Fig S2a). Notable hits are Stim1 , a major SOCE component, Kif23 identified in the first SOCE screening 1 , and gnas, encoding the alpha subunit of the stimulatory G protein driving cAMP signaling and PKA activity 10 . Analysis of the 918 inhibitors revealed an enrichment of phospholipase and ion channel proteins (Fig S2b). Notable hits are TMEM110, a known Stim1 inhibitor 11 and Trpm4 , a known SOCE blocker 12 , validating the link to SOCE. To validate the enhancers, we ectopically expressed STIM1 and Gɑs in HEK-293 cells lacking or not both STIM1 and STIM2 genes 13 . Gɑs and STIM1 expression both enhanced the proportion of cells undergoing spontaneous photoconversion, which was reduced by STIM1/2 ablation (Figure 2c, S2c-d). Pharmacological inhibition of Trpm4 increased spontaneous photoconversion (Fig s2e), validating this inhibitor. We then picked the top 3 inhibitors hits related to pathophysiology: Small nuclear ribonucleoprotein polypeptide N (Snpn), linked to Prader Willy syndrome 14 , Hepatitis A Virus Cellular Receptor (Havcr), involved in viral infection 15 and Plastin 1 (PLS1), encoding for fimbrin protein involved in deafness 16 . All these 3 top hits robustly enhanced spontaneous and SOCE-evoked PC when downregulated (Fig 2d and s2e). In summary, our current work presents the implementation of CaMPARI2 technology for pooled Ca 2+ screenings applied here to spontaneous/homeostatic Ca 2+ activity. Tailoring every protocol for the right ion channel will accelerate ion research in an unprecedent way. Targeting CaMPARI2 to other organelles and membranes for the study of microdomains and contact sites will also shed light on intracellular communication, critical to coordinate cellular outputs. This new technique will not only boost ion channel drug research but also enhance our understanding of the underlying pathologies related to intracellular communication, including those associated with cancer, aging, neurodegeneration, and diabetes. Methods Cell lines, reagents and plasmids Mouse embryonic fibroblasts (MEFs) or Human Embryonic Kidney (HEK293) or HeLA cells were maintained in their recommended media supplemented with 5 and 10% fetal bovine serum (FBS) respectively and non-essential amino acids, and grown at 37°C with 5% CO2. STIM1 and 2 Knockout were also a Kind gift from Dr. Trebak 13 . MEF cells expressing PLX311-Cas9i720 and Plenti Campari2, which were generated in 5 . All sorted cells in this study were generated using a Beckman Coulter MoFlo Astrios integrated in PSL2 hood. All cell lines from this study were tested negative for mycoplasma contamination. HEK 293, Hela and MEF cells are listed as commonly misidentified cell lines maintained by the International Cell Line Authentication Committee. In our hands, HEK 293T cells were genetically confirmed (by genomic profiling [STRs]) prior to stockage. HeLa cells were tested in 2018 (Microsynth, HeLa_Mic_001597) MEF cells were not tested. The following reagents were used in this manuscript; Thapsigargin (T9033/CAY10522, Sigma); Fura2-AM, (F1201, Invitrogen); Cyclopiazonic acid from Penicillium cyclopium, (c1530, Sigma), Yoda (Sigma SML1558), GSK7975A (Sigma, AOB 4124), U-73122 (Cayman, Item id: 70740), Histamine (Tci-chemicals, H0146), Adenosine 5'-triphosphate disodium salt hydrate (NaATP), (Sigma A6419), TRPM4 inhibitor, 9-Phenanthrol (Sigma, 648492). Si-RNAs were purchased from: Dharmacon, On targetplus mouse siRNA for Snrpn, Pls1, Havcr1 and Non targeting RNA pool (Control). Briefly, siRNA was transfected on MEF cells (40nM final) and assayed for CaMPARI2 after 72h. CAMPARI2 was a kind gift from prof. Eric R Schreiter 2 Mutant CaMPARI2 bearing T394D and L398T was generated using site directed mutagenesis based on previous works 2 . Other plasmids used in this this study involve Galpha s, a kind gift form Prof. Vladimir Katanaev lab and Dr. Gonzalo Solis cDNA Resource center, (#GNA0SL000). STIM1 plasmids were a kind gift form Dr. Stéphane Konig. CAMPARI calcium measurements CAMPARI2 was first used in single-cell microscopy transiently transfected or stably expressed in HEK-WT cells. Cells were seeded into 35 mm coverslips the day before experiment and imaged using GFP (488ex/520 em) and RFP (560ex/600em) settings. Cells were bathed in modified Ringer’s and Photoactivation was achieved by exposing the field of view for the indicated times with 405 light. GFP and RFP channels were measured before and after PC during the indicated protocols to establish PC rate. In Fig. 1 we developed SOCE in a Ca 2+ -containing solution using Thapsigargin 1 µM. For the rest of the work, cyclopiazonic acid 10 µM in a Ca 2+ -free solution containing 1 mM EGTA was used before re-addition (or not) of 2 mM final Ca 2+ concentration. CaMPARI2 calibration was achieved using Fura-2 as an internal control as in 6 . Briefly, wells were transfected with CaMPARI2 and seeded into 35 mm coverslips with Poly-L-lysine for 24 to 48 hours. Fura2 3 µM loading was performed at room temperature for 25 minutes followed with a 10-minute washout. For Fura2 a 340/380 nm excitation (30 and 10 ms) and 510 ± 40 nm emission ratiometric imaging was performed while for GFP a 480 excitation and same emission with 100 ms every 20 seconds was performed. Cells were bathed in Ca 2+ -containing solution then replaced for Ca 2+ -free in 2 µM Digitonin solution. Extracellular Ca 2+ increasing solutions were slowly replaced every 5 minutes to equilibrate extracellular and intracellular compartments. Fura2 ratios were normalized with Rmin = 0 and Rmax = 1 while CaMPARI2 initial values were set to 1 and minimal to 0. Calibration was calculated using the Kd of Fura of 225nm. FACS measurement of CAMPARI2 in MEF stable expressing cells was achieved by exposing cells to PC light using a homemade CaMPARI setup (see Figure S1b) composed of: a CHOLIS 6-LED High power Source (ThorLabs) with 360, 385 and 420 nm lines at 100% rendering 150mW/cm 2 maximal output. When indicated cells were exposed to CPA 10 µM in Ca 2+ -free solution (1 mM EGTA) for 8–10 minutes, or stimulated with the indicated agonists with the light on, or left untreated in a Ringler Ca 2+ -containing solution (2 mM) for an integration time of 3 minutes. Wells were then washed in 1x trypsin with Sytox Blue (1:5000) or DAPI (1:5000) and placed into FACS tubes with DMEM with Serum on ice before measurement of the DAPI, GFP, RFP and Elfuor 700 (when needed) lines in a BDLSR Fortessa unit. Not PC and/or PC in 1mM EGTA with CPA 10 µM for 10 minutes were always used for each experiment in order to define PC. CAMPARI2 CRISPR screening MEF cells for CRISPR screening were previous described in 5 . Briefly, MEF cells were single cloned for Cas9-i720 and CAMPARI2. A clone with high CaMPARI2 and Cas9 signal was used to integrate the De Brie CRISPR library (Addgene #73633) 9 . We spin-infected 180 million cells with a MOI of 0.4 (10 µg/ml Polybrene), expanded and froze 9 million/vial. For this work, 6 vials were thawed and expanded for 1 passage before PC and FACS sorting. CAMPARI2 PC for screening was performed in 60 mm plates by lighting with 365, 385 420 nm Led light (Cholis High-Power Led source (Thorlabs) for 3 minutes at 25mW/cm 2 . CPA CF 10 minutes was used to set the negative control. 5 to 10% (on n1 and n2 respectively) spontaneously positive cells (250.000 to 500.000 cells) were expanded for 3 days and isolated for their genomic DNA using JetQuick™ Blood and Cell Culture DNA Midiprep Kit (A30703, Thermo). Of note, Unsorted cells were (approximately 20 million) were also isolated for every n. gDNA was used in a two-step PCR protocol. First PCR used 2 µg of DNA in 80 µL final reactions using Fw: AAT GGA CTA TCA TAT GCT TAC CGT AAC TTG AAA GTA TTT CG and Rv: TCT GCT GTC CCT GTA ATA AAC CCG AAA ATT TTG AA primers. PCR products from all the reactions were pooled and 10 µL were used in a 100 µL final reaction PCR using barcoded P5 and P7 primers as described on 9 and sequenced using paired ending 100 bp reader (Illumina, San Diego, CA, USA) at the iGE3 Genomics platform at the University of Geneva. Enrichment and depletion analyses were done with Pynaple.py platform using a sgRNA ranking based on sidak, p-value 0.05/0.01, clustered by highest variance. Read count was normalized using counts per million/median. Declarations Acknowledgements We thank prof. Eric R Schreiter 2 for CaMPARI constructs, Prof. Vladimir Katanaev lab and Dr. Gonzalo Solis for G alpha s constructs and Dr. Stéphane Konig for sharing STIM1 constructs. We also thank all members of the Demaurex lab for their insightful discussions and the Bioimaging, READS, Proteomics, Genomics, and Flow Cytometry platforms/facilities (Geneva Medical Center). This work was funded by Swiss National Science Foundation (310030_189042 [to N.D), the Sir Jules Thorn Foundation (2022), the FSRMM, and the Novartis Young investigation Grant (22B082) and SPARK Swiss National Science Foundation (SPARK: CRSK-3_221284) to (A.C.-S.). References Zhang, S. L. et al. Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. Proc Natl Acad Sci U S A 103 , 9357–62 (2006). Moeyaert, B. et al. Improved methods for marking active neuron populations. Nat Commun 9 , (2018). Hammer, A. et al. Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights. Biomedicines 10 , 138 (2022). Kanyo, R. et al. Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ9-tetrahydrocannabinol. Sci Rep 11 , 11515 (2021). Carreras-Sureda, A. et al. The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation. Cell Rep 42 , 113540 (2023). Dong, T. X. et al. T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse. Elife 6 , (2017). Ashmole, I. et al. CRACM/Orai ion channel expression and function in human lung mast cells. Journal of Allergy and Clinical Immunology 129 , 1628-1635.e2 (2012). Bleasdale, J. E. et al. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther 255 , 756–68 (1990). Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34 , 184–191 (2016). Ramms, D. J. et al. G α s–Protein Kinase A (PKA) Pathway Signalopathies: The Emerging Genetic Landscape and Therapeutic Potential of Human Diseases Driven by Aberrant G α s-PKA Signaling. Pharmacol Rev 73 , 1326–1368 (2021). Quintana, A. et al. TMEM110 regulates the maintenance and remodeling of mammalian ER–plasma membrane junctions competent for STIM–ORAI signaling. Proceedings of the National Academy of Sciences 112 , (2015). Holzmann, C. et al. Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells. Oncotarget 6 , 41783–41793 (2015). Emrich, S. M. et al. Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity. Journal of Biological Chemistry 294 , 6318–6332 (2019). Özçelik, T. et al. Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader–Willi syndrome critical region. Nat Genet 2 , 265–269 (1992). Feigelstock, D., Thompson, P., Mattoo, P., Zhang, Y. & Kaplan, G. G. The Human Homolog of HAVcr-1 Codes for a Hepatitis A Virus Cellular Receptor. J Virol 72 , 6621–6628 (1998). Morgan, A. et al. Mutations in PLS1 , encoding fimbrin, cause autosomal dominant nonsyndromic hearing loss. Hum Mutat 40 , 2286–2295 (2019). Additional Declarations There is NO Competing Interest. Supplementary Files FigureS1.png Figure S1 CaMPARI fused to Flow cytometry reveals a spontaneous calcium positivity. (a) Picture and description of the components of the CaMPARI2 photoconversion setup. (b) diagram of the FACS profiles expected after PC and analysis with the flow cytometer. (c-d) Related to Figure 1 e. MEF stably expressing CaMPARI2 or Hela cells transiently expressing CaMPARI2 treated with Cyclopiazonic acid (10µM) in Ca 2+ -free (CPA CF) for 10 minutes or untreated. Graph bars represent the mean and SEM % of positive cells, using CPA CF as reference gating (n = 7 and 6) biological replicated from 3 independent experiments. (e) CaMPARI2 or dead CaMPARI were transiently expressed in HEK cells recorded for Green and Fura2 dye in live cell imaging. Cells were bathed in Ca 2+ -containing media, shifted to Ca 2+ -free and treated with digitonin 2 µM solution with increasing extracellular Ca 2+ solutions as indicated. Changes were perfused every 5 minutes, images acquired every 20 seconds. Graphs show the normalized GFP or Fura-2 ratio (min is 0 max is 1) for both conditions. Intracellular Ca 2+ concentrations are plotted for Fura2 and WT CaMAPRI2 to calculate the Ca 2+ Kd (98 nM) using Fura2 Kd (225 nM) as reference. Dead CaMPARI2 did not fit any curve. (f) HEK cells transiently expressing CaMPARI2 or Dead CaMPARI2 photoconverted under the same conditions as (d-e) or treated with ATP 100 µM in Ca 2+ -containing medium. Graph bars represent the mean and SEM % of photoconversion (n = 7 to 16 biological replicates from 4 independent experiments). T test were achieved using unpaired t test (d-e) or ANOVA sidak multiple comparison test (f). FigureS2.png Figure S2. CaMPARI screening identifies genes involved in spontaneous Calcium activity. (a-b) Protein classification using Panther Gene ontology enrichment analysis for the depleted (enhancers) genes or the enriched (inhibitors) from figure 2b. (c-d) related to figure 2c, WT or DKO cells expressing CaMPARI2 and the indicated constructs photoconverted in CPA CF 10 minutes preincubation or with ATP or SOCE protocol. Graph bars show the mean and SEM photoconverted population (n= 4-9 biological replicates form 3 independent experiments). (e) MEF cells expressing CaMPARI2 treated for 10 minutes with indicated concentrations of 9-Phenanthrol (Trpm4 inhibitor) before PC. Graph bars present the mean and SEM % of PC. Unpaired t test (c and d) ANOVA multiple comparison t-test (f) related to Figure 2d. siRNA transfection of the indicated siRNAs on MEF cells stably expressing CaMPARI2. Graph bars shows the mean and SEM % of photoconversion for CF CPA (10µM) negative control and SOCE (re-addition of 2mM Ca 2+ ) PC (n = 18 biological replicates form 3 independent experiments. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4656479","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":324252243,"identity":"4f47cf1d-2188-46bf-9b1b-444c1ce2f162","order_by":0,"name":"Amado Carreras-sureda","email":"data:image/png;base64,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","orcid":"","institution":"Department of Cell Physiology and Metabolism, University of Geneva","correspondingAuthor":true,"prefix":"","firstName":"Amado","middleName":"","lastName":"Carreras-sureda","suffix":""},{"id":324252244,"identity":"edfe39fd-536d-4a31-bd20-7441c0fbf4a2","order_by":1,"name":"Sana Kouba","email":"","orcid":"","institution":"University of Geneva, Department of Cell Physiology and Metabolism","correspondingAuthor":false,"prefix":"","firstName":"Sana","middleName":"","lastName":"Kouba","suffix":""},{"id":324252245,"identity":"5d6f516d-bc1f-4691-b390-20e01c5ceaca","order_by":2,"name":"Xin Zhang","email":"","orcid":"","institution":"University of Geneva, Department of Cell Physiology and Metabolism","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":324252246,"identity":"549b2368-599d-492e-8c6f-77f85c3b3b89","order_by":3,"name":"Cyril Castelbou","email":"","orcid":"","institution":"University of Geneva, Department of Cell Physiology and Metabolism","correspondingAuthor":false,"prefix":"","firstName":"Cyril","middleName":"","lastName":"Castelbou","suffix":""},{"id":324252247,"identity":"c5929a9f-2a92-438b-8c19-fc69c3c34750","order_by":4,"name":"Nicolas Demaurex","email":"","orcid":"https://orcid.org/0000-0002-9933-6772","institution":"University of Geneva","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Demaurex","suffix":""}],"badges":[],"createdAt":"2024-06-28 19:10:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4656479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4656479/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60783670,"identity":"f58585ea-15d6-42b6-97c5-436fbb6a3c79","added_by":"auto","created_at":"2024-07-22 04:14:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaMPARI fused to Flow cytometry reveals a spontaneous calcium positivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Scheme of a CaMPARI2-expressing cell before and after photoconversion. (b) HEK cells transiently expressing CaMPARI2 recorded in live cell imaging (Green = GFP, Red = RFP) bathed in 2 mM Ca\u003csup\u003e2+\u003c/sup\u003e media, treated with Tg 1 µM. Violet section = Photoconversion (1 minute). Left graph: ratio of Red over Green channels (orange). Images from kinetic graphs. (c) Microscopy-based Ca\u003csup\u003e2+\u003c/sup\u003e measuring with the indicated agonists/protocols (Top) and FACS-based CaMPARI2 measurements showing GFP to RFP contour scatter plot (Bottom). Graph bars represent the mean and SEM of the % of positive photoconverted population (n = 4-15 independent experiments). (d) HEK and (e) HeLa cells transiently expressing CaMPARI2 not photoconverted, photoconverted with no agonist (spont), ATP (50 µM) or Histamine (10 µM) during UV (3 minutes). Scatter contour plots for GFP and RFP intensities are presented. Graph bars indicate the mean and SEM % of photoconverted cells (n = 3-8 biological replicated form 3 independent experiments) (f) MEF cells stably expressing CaMPARI2, exposed or not to UV light for 3 minutes pre-treated with GSK7975A (2 µM) and U-73122 (2 µM) for 8 minutes before photoconversion. Graph bars plot the mean and SEM % of photoconversion (n = 4 independent experiments). Statistical analyses were performed using ANOVA test Uncorrected Fisher's LSD, (c); Dunnett's multiple comparisons test for d-f).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4656479/v1/92ebcd1539eaca14386957a1.png"},{"id":60783673,"identity":"f4d8472a-3028-46cf-8814-98a5328cf1c5","added_by":"auto","created_at":"2024-07-22 04:14:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaMPARI screening identifies genes involved in spontaneous calcium activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic representation of the screening strategy. (b) Sigma FC score for Enriched (Blue dots) and depleted (red dots) Genes after PC compared to unsorted cells n = 2 independent Screens. Orange non-targeting sgRNAS (1000x), Green TOP3 inhibitrs, Violet, known inhibitors, dark red, known enhancers (c) HEK WT and Stim1 and 2 Double knockout (HEK DKO) cells transiently expressing CaMPARI2 and the indicated constructs were photoconverted during the indicated treatments. Left, representative FACS plots for the indicated conditions. Graph bars shows the mean and SEM % of photoconversion for every condition (n = 9-11 biological replicates form 4 independent experiments). (d) siRNA transfection of the indicated conditions on MEF cells stably expressing CaMPARI2. Left, representative FACS plots for the indicated conditions. Graph bars shows the mean and SEM % of photoconversion for spontaneous PC (n = 18 biological replicates form 3 independent experiments. (e) Cartoon highlighting the major hits uncovered using CaMPARI2 screening.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4656479/v1/40a17b44adce6bb736499cc5.png"},{"id":60784637,"identity":"9ab0a604-a366-4d98-933b-78e52b4d52b9","added_by":"auto","created_at":"2024-07-22 04:38:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":604345,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4656479/v1/2c1b0bfb-55a2-425f-8cf6-7d16566c165c.pdf"},{"id":60783672,"identity":"e6fa1e70-06ab-4044-a40f-2a79202dd711","added_by":"auto","created_at":"2024-07-22 04:14:27","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":170740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1 CaMPARI fused to Flow cytometry reveals a spontaneous calcium positivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Picture and description of the components of the CaMPARI2 photoconversion setup. (b) diagram of the FACS profiles expected after PC and analysis with the flow cytometer. (c-d) Related to Figure 1 e. MEF stably expressing CaMPARI2 or Hela cells transiently expressing CaMPARI2 treated with Cyclopiazonic acid (10µM) in Ca\u003csup\u003e2+\u003c/sup\u003e-free (CPA CF) for 10 minutes or untreated. Graph bars represent the mean and SEM % of positive cells, using CPA CF as reference gating (n = 7 and 6) biological replicated from 3 independent experiments. (e) CaMPARI2 or dead CaMPARI were transiently expressed in HEK cells recorded for Green and Fura2 dye in live cell imaging. Cells were bathed in Ca\u003csup\u003e2+\u003c/sup\u003e-containing media, shifted to Ca\u003csup\u003e2+\u003c/sup\u003e-free and treated with digitonin 2 µM solution with increasing extracellular Ca\u003csup\u003e2+\u003c/sup\u003e solutions as indicated. Changes were perfused every 5 minutes, images acquired every 20 seconds. Graphs show the normalized GFP or Fura-2 ratio (min is 0 max is 1) for both conditions. Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentrations are plotted for Fura2 and WT CaMAPRI2 to calculate the Ca\u003csup\u003e2+\u003c/sup\u003e Kd (98 nM) using Fura2 Kd (225 nM) as reference. Dead CaMPARI2 did not fit any curve. (f) HEK cells transiently expressing CaMPARI2 or Dead CaMPARI2 photoconverted under the same conditions as (d-e) or treated with ATP 100 µM in Ca\u003csup\u003e2+\u003c/sup\u003e-containing medium. Graph bars represent the mean and SEM % of photoconversion (n = 7 to 16 biological replicates from 4 independent experiments). T test were achieved using unpaired t test (d-e) or ANOVA sidak multiple comparison test (f).\u003c/p\u003e","description":"","filename":"FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-4656479/v1/c40573174f78543d2d391610.png"},{"id":60783978,"identity":"468a7c13-172d-4a5f-b07a-10d035fd8e43","added_by":"auto","created_at":"2024-07-22 04:22:27","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":67150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. CaMPARI screening identifies genes involved in spontaneous Calcium activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-b) Protein classification using Panther Gene ontology enrichment analysis for the depleted (enhancers) genes or the enriched (inhibitors) from figure 2b. (c-d) related to figure 2c, WT or DKO cells expressing CaMPARI2 and the indicated constructs photoconverted in CPA CF 10 minutes preincubation or with ATP or SOCE protocol. Graph bars show the mean and SEM photoconverted population (n= 4-9 biological replicates form 3 independent experiments). (e) MEF cells expressing CaMPARI2 treated for 10 minutes with indicated concentrations of 9-Phenanthrol (Trpm4 inhibitor) before PC. Graph bars present the mean and SEM % of PC. Unpaired t test (c and d) ANOVA multiple comparison t-test (f) related to Figure 2d. siRNA transfection of the indicated siRNAs on MEF cells stably expressing CaMPARI2. Graph bars shows the mean and SEM % of photoconversion for CF CPA (10µM) negative control and SOCE (re-addition of 2mM Ca\u003csup\u003e2+\u003c/sup\u003e) PC (n = 18 biological replicates form 3 independent experiments.\u003c/p\u003e","description":"","filename":"FigureS2.png","url":"https://assets-eu.researchsquare.com/files/rs-4656479/v1/5ae4c722244a61483a963d1b.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Genome interrogation of homeostatic calcium activity using CaMP-screen","fulltext":[{"header":"Main","content":"\u003cp\u003eIon channel research relies on single cell electrophysiological or imaging recordings, time-consuming approaches of high precision but limited throughput. Genomic screens involving ion channels rely on multiplexed plate readers targeting a single gene per well in an arrayed configuration\u003csup\u003e1\u003c/sup\u003e. Recent advancements on genome interrogation using pooled configuration have significantly lowered the costs and the time needed to screen for any biological function. The Ca\u003csup\u003e2+\u003c/sup\u003e-Activated Photoconvertible Ratiometric indicator 2 (CaMPARI2)\u003csup\u003e2\u003c/sup\u003e sensor is irreversibly photoconverted from green to red upon combined Ca\u003csup\u003e2+\u003c/sup\u003e binding and UV light exposure. This feature allows the rapid identification of Ca\u003csup\u003e2+\u003c/sup\u003e activity in single cells and has been used to report Ca\u003csup\u003e2+\u003c/sup\u003e transients in populations of neurons or in zebrafish\u003csup\u003e3\u0026ndash;5\u003c/sup\u003e. Here, we describe how to use CaMPARI2 for genome interrogation of Ca\u003csup\u003e2+\u003c/sup\u003e activity by flow cytometry.\u003c/p\u003e\n\u003cp\u003eCaMPARI2 green fluorescence decreases upon Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003ebinding and becomes irreversibly red only when UV light is applied in this Ca\u003csup\u003e2+\u003c/sup\u003e-bound state\u003csup\u003e2\u003c/sup\u003e (Figure 1a-b). To study large populations of cells, we built a UV photoconversion (PC) setup (\u0026Oslash; 0= 6 cm; 10-25 mW/cm\u003csup\u003e2\u003c/sup\u003e, Figure s1a) and used it to photoconvert mouse embryonic fibroblasts (MEF) cells treated with cyclopiazonic acid (CPA) or Yoda1 to activate store-operated or mechanosensitive channels, respectively (Figure 1C and s1b). 67% of CPA-treated and 32% of Yoda1-treated cells were photoconverted, validating the efficiency of the setup to identify Ca\u003csup\u003e2+\u003c/sup\u003e fluxes. Unexpectedly, approximately 10% of cells were photoconverted in the absence of chemicals, suggesting that Ca\u003csup\u003e2+\u003c/sup\u003e elevations occurred spontaneously during the 1 min UV exposure. Comparable basal activity was observed in three different cell lines and was absent in cells treated with CPA in Ca\u003csup\u003e2+\u003c/sup\u003e-free to deplete intracellular Ca\u003csup\u003e2+\u003c/sup\u003e stores and prevent Ca\u003csup\u003e2+\u003c/sup\u003e entry (Fig 1 c-e and s1c-d) indicating that it reflected homeostatic Ca\u003csup\u003e2+\u003c/sup\u003e activity. CaMPARI2 calibration using Fura-2 as reference\u003csup\u003e6\u003c/sup\u003e yielded a KD of 98 nM while a CaMPARI2 bearing mutations preventing Ca\u003csup\u003e2+\u003c/sup\u003e binding (Dead-CaMP2) did not report spontaneous or evoked Ca\u003csup\u003e2+\u003c/sup\u003e fluxes (Figure s1e-f). The Orai1 channel inhibitor\u0026nbsp;GSK7975A\u003csup\u003e7\u003c/sup\u003e and the phospholipase C (PLC) inhibitor U73122\u003csup\u003e8\u003c/sup\u003e\u0026nbsp; \u0026nbsp;decreased the proportion of cells undergoing spontaneous PC (Figure 1f), linking the homeostatic activity to IP\u003csub\u003e3\u003c/sub\u003e-mediated Ca\u003csup\u003e2+\u003c/sup\u003e release fueled by\u0026nbsp;store operated calcium entry\u0026nbsp;(SOCE).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo identify genes controlling this activity, we generated MEF cells stably expressing a whole genome knockout library with 4 sgRNAs per gene\u003csup\u003e9\u003c/sup\u003e. Cells were sorted for spontaneous PC and processed for next generation sequencing analysis (NGS) in two independent experiments (Figure 2a). Depletion and enrichment analysis using an FDR of 0.01 identified 468 genes sustaining (enhancers) and 918 genes mitigating (inhibitors) the spontaneous activity (Figure 2b). Gene ontology and enrichment analysis of the 468 enhancers revealed an enrichment of\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e ion binding and G-protein modulator on this dataset (Fig S2a). Notable hits are \u003cem\u003eStim1\u003c/em\u003e, a major SOCE component, \u003cem\u003eKif23\u003c/em\u003e identified in the first SOCE screening\u003csup\u003e1\u003c/sup\u003e, and\u0026nbsp;\u003cem\u003egnas,\u0026nbsp;\u003c/em\u003eencoding the alpha subunit of the stimulatory G protein driving cAMP signaling and PKA activity\u003csup\u003e10\u003c/sup\u003e. Analysis of the 918 inhibitors revealed an enrichment of phospholipase and ion channel proteins (Fig S2b). Notable hits are \u003cem\u003eTMEM110,\u003c/em\u003e a known Stim1 inhibitor\u003csup\u003e11\u003c/sup\u003e and \u003cem\u003eTrpm4\u003c/em\u003e, a known SOCE blocker\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e,\u0026nbsp;validating the link to SOCE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the enhancers, we ectopically expressed STIM1 and Gɑs in HEK-293 cells lacking or not both STIM1 and STIM2 genes\u003csup\u003e13\u003c/sup\u003e. Gɑs and STIM1 expression both enhanced the proportion of cells undergoing spontaneous photoconversion, which was reduced by \u003cem\u003eSTIM1/2\u003c/em\u003e ablation (Figure 2c, S2c-d). Pharmacological inhibition of Trpm4 increased spontaneous photoconversion (Fig s2e), validating this inhibitor. We then picked the top 3 inhibitors hits related to pathophysiology: Small nuclear ribonucleoprotein polypeptide N (Snpn), linked to Prader Willy syndrome\u003csup\u003e14\u003c/sup\u003e,\u0026nbsp;Hepatitis A Virus Cellular Receptor (Havcr), involved in viral infection\u003csup\u003e15\u003c/sup\u003e and Plastin 1 (PLS1), encoding for fimbrin protein involved in deafness\u003csup\u003e16\u003c/sup\u003e. All these 3 top hits robustly enhanced spontaneous and SOCE-evoked PC when downregulated (Fig 2d and s2e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, our current work presents the implementation of CaMPARI2 technology for pooled Ca\u003csup\u003e2+\u003c/sup\u003e screenings applied here to spontaneous/homeostatic Ca\u003csup\u003e2+\u003c/sup\u003e activity. Tailoring every protocol for the right ion channel will accelerate ion research in an unprecedent way. Targeting CaMPARI2 to other organelles and membranes for the study of microdomains and contact sites will also shed light on intracellular communication, critical to coordinate cellular outputs. This new technique will not only boost ion channel drug research but also enhance our understanding of the underlying pathologies related to intracellular communication, including those associated with cancer, aging, neurodegeneration, and diabetes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines, reagents and plasmids\u003c/h2\u003e \u003cp\u003eMouse embryonic fibroblasts (MEFs) or Human Embryonic Kidney (HEK293) or HeLA cells were maintained in their recommended media supplemented with 5 and 10% fetal bovine serum (FBS) respectively and non-essential amino acids, and grown at 37\u0026deg;C with 5% CO2. STIM1 and 2 Knockout were also a Kind gift from Dr. Trebak\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMEF cells expressing PLX311-Cas9i720 and Plenti Campari2, which were generated in\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. All sorted cells in this study were generated using a Beckman Coulter MoFlo Astrios integrated in PSL2 hood. All cell lines from this study were tested negative for mycoplasma contamination. HEK 293, Hela and MEF cells are listed as commonly misidentified cell lines maintained by the International Cell Line Authentication Committee. In our hands, HEK 293T cells were genetically confirmed (by genomic profiling [STRs]) prior to stockage. HeLa cells were tested in 2018 (Microsynth, HeLa_Mic_001597) MEF cells were not tested.\u003c/p\u003e \u003cp\u003eThe following reagents were used in this manuscript; Thapsigargin (T9033/CAY10522, Sigma); Fura2-AM, (F1201, Invitrogen); Cyclopiazonic acid from Penicillium cyclopium, (c1530, Sigma), Yoda (Sigma SML1558), GSK7975A (Sigma, AOB 4124), U-73122 (Cayman, Item id: 70740), Histamine (Tci-chemicals, H0146), Adenosine 5'-triphosphate disodium salt hydrate (NaATP), (Sigma A6419), TRPM4 inhibitor, 9-Phenanthrol (Sigma, 648492). Si-RNAs were purchased from: Dharmacon, On targetplus mouse siRNA for Snrpn, Pls1, Havcr1 and Non targeting RNA pool (Control). Briefly, siRNA was transfected on MEF cells (40nM final) and assayed for CaMPARI2 after 72h.\u003c/p\u003e \u003cp\u003eCAMPARI2 was a kind gift from prof. Eric R Schreiter\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Mutant CaMPARI2 bearing T394D and L398T was generated using site directed mutagenesis based on previous works \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Other plasmids used in this this study involve Galpha s, a kind gift form Prof. Vladimir Katanaev lab and Dr. Gonzalo Solis cDNA Resource center, (#GNA0SL000). STIM1 plasmids were a kind gift form Dr. St\u0026eacute;phane Konig.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCAMPARI calcium measurements\u003c/h2\u003e \u003cp\u003eCAMPARI2 was first used in single-cell microscopy transiently transfected or stably expressed in HEK-WT cells. Cells were seeded into 35 mm coverslips the day before experiment and imaged using GFP (488ex/520 em) and RFP (560ex/600em) settings. Cells were bathed in modified Ringer\u0026rsquo;s and Photoactivation was achieved by exposing the field of view for the indicated times with 405 light. GFP and RFP channels were measured before and after PC during the indicated protocols to establish PC rate. In Fig.\u0026nbsp;1 we developed SOCE in a Ca\u003csup\u003e2+\u003c/sup\u003e-containing solution using Thapsigargin 1 \u0026micro;M. For the rest of the work, cyclopiazonic acid 10 \u0026micro;M in a Ca\u003csup\u003e2+\u003c/sup\u003e-free solution containing 1 mM EGTA was used before re-addition (or not) of 2 mM final Ca\u003csup\u003e2+\u003c/sup\u003e concentration.\u003c/p\u003e \u003cp\u003eCaMPARI2 calibration was achieved using Fura-2 as an internal control as in\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Briefly, wells were transfected with CaMPARI2 and seeded into 35 mm coverslips with Poly-L-lysine for 24 to 48 hours. Fura2 3 \u0026micro;M loading was performed at room temperature for 25 minutes followed with a 10-minute washout. For Fura2 a 340/380 nm excitation (30 and 10 ms) and 510\u0026thinsp;\u0026plusmn;\u0026thinsp;40 nm emission ratiometric imaging was performed while for GFP a 480 excitation and same emission with 100 ms every 20 seconds was performed. Cells were bathed in Ca\u003csup\u003e2+\u003c/sup\u003e-containing solution then replaced for Ca\u003csup\u003e2+\u003c/sup\u003e-free in 2 \u0026micro;M Digitonin solution. Extracellular Ca\u003csup\u003e2+\u003c/sup\u003e increasing solutions were slowly replaced every 5 minutes to equilibrate extracellular and intracellular compartments. Fura2 ratios were normalized with Rmin\u0026thinsp;=\u0026thinsp;0 and Rmax\u0026thinsp;=\u0026thinsp;1 while CaMPARI2 initial values were set to 1 and minimal to 0. Calibration was calculated using the Kd of Fura of 225nm.\u003c/p\u003e \u003cp\u003eFACS measurement of CAMPARI2 in MEF stable expressing cells was achieved by exposing cells to PC light using a homemade CaMPARI setup (see Figure S1b) composed of: a CHOLIS 6-LED High power Source (ThorLabs) with 360, 385 and 420 nm lines at 100% rendering 150mW/cm\u003csup\u003e2\u003c/sup\u003e maximal output. When indicated cells were exposed to CPA 10 \u0026micro;M in Ca\u003csup\u003e2+\u003c/sup\u003e-free solution (1 mM EGTA) for 8\u0026ndash;10 minutes, or stimulated with the indicated agonists with the light on, or left untreated in a Ringler Ca\u003csup\u003e2+\u003c/sup\u003e-containing solution (2 mM) for an integration time of 3 minutes. Wells were then washed in 1x trypsin with Sytox Blue (1:5000) or DAPI (1:5000) and placed into FACS tubes with DMEM with Serum on ice before measurement of the DAPI, GFP, RFP and Elfuor 700 (when needed) lines in a BDLSR Fortessa unit. Not PC and/or PC in 1mM EGTA with CPA 10 \u0026micro;M for 10 minutes were always used for each experiment in order to define PC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCAMPARI2 CRISPR screening\u003c/h2\u003e \u003cp\u003eMEF cells for CRISPR screening were previous described in\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Briefly, MEF cells were single cloned for Cas9-i720 and CAMPARI2. A clone with high CaMPARI2 and Cas9 signal was used to integrate the De Brie CRISPR library (Addgene #73633)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We spin-infected 180\u0026nbsp;million cells with a MOI of 0.4 (10 \u0026micro;g/ml Polybrene), expanded and froze 9\u0026nbsp;million/vial. For this work, 6 vials were thawed and expanded for 1 passage before PC and FACS sorting. CAMPARI2 PC for screening was performed in 60 mm plates by lighting with 365, 385 420 nm Led light (Cholis High-Power Led source (Thorlabs) for 3 minutes at 25mW/cm\u003csup\u003e2\u003c/sup\u003e. CPA CF 10 minutes was used to set the negative control. 5 to 10% (on n1 and n2 respectively) spontaneously positive cells (250.000 to 500.000 cells) were expanded for 3 days and isolated for their genomic DNA using JetQuick\u0026trade; Blood and Cell Culture DNA Midiprep Kit (A30703, Thermo). Of note, Unsorted cells were (approximately 20\u0026nbsp;million) were also isolated for every n. gDNA was used in a two-step PCR protocol. First PCR used 2 \u0026micro;g of DNA in 80 \u0026micro;L final reactions using Fw: AAT GGA CTA TCA TAT GCT TAC CGT AAC TTG AAA GTA TTT CG and Rv: TCT GCT GTC CCT GTA ATA AAC CCG AAA ATT TTG AA primers. PCR products from all the reactions were pooled and 10 \u0026micro;L were used in a 100 \u0026micro;L final reaction PCR using barcoded P5 and P7 primers as described on\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and sequenced using paired ending 100 bp reader (Illumina, San Diego, CA, USA) at the iGE3 Genomics platform at the University of Geneva.\u003c/p\u003e \u003cp\u003eEnrichment and depletion analyses were done with Pynaple.py platform using a sgRNA ranking based on sidak, p-value 0.05/0.01, clustered by highest variance. Read count was normalized using counts per million/median.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank prof. Eric R Schreiter\u003csup\u003e2\u003c/sup\u003e for CaMPARI constructs, Prof. Vladimir Katanaev lab\u0026nbsp;and Dr. Gonzalo\u0026nbsp;Solis for G alpha s constructs and Dr. Stéphane Konig for sharing STIM1 constructs. We also thank all members of the Demaurex lab for their insightful discussions and the Bioimaging, READS, Proteomics, Genomics, and Flow Cytometry platforms/facilities (Geneva Medical Center). This work was funded by Swiss National Science Foundation (310030_189042 [to N.D), the Sir Jules Thorn Foundation (2022), the FSRMM, and the Novartis Young investigation Grant (22B082) and SPARK Swiss National Science Foundation (SPARK: CRSK-3_221284) to (A.C.-S.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZhang, S. L. \u003cem\u003eet al.\u003c/em\u003e Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e103\u003c/strong\u003e, 9357\u0026ndash;62 (2006).\u003c/li\u003e\n \u003cli\u003eMoeyaert, B. \u003cem\u003eet al.\u003c/em\u003e Improved methods for marking active neuron populations. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2018).\u003c/li\u003e\n \u003cli\u003eHammer, A. \u003cem\u003eet al.\u003c/em\u003e Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 138 (2022).\u003c/li\u003e\n \u003cli\u003eKanyo, R. \u003cem\u003eet al.\u003c/em\u003e Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and \u0026Delta;9-tetrahydrocannabinol. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 11515 (2021).\u003c/li\u003e\n \u003cli\u003eCarreras-Sureda, A. \u003cem\u003eet al.\u003c/em\u003e The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 113540 (2023).\u003c/li\u003e\n \u003cli\u003eDong, T. X. \u003cem\u003eet al.\u003c/em\u003e T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, (2017).\u003c/li\u003e\n \u003cli\u003eAshmole, I. \u003cem\u003eet al.\u003c/em\u003e CRACM/Orai ion channel expression and function in human lung mast cells. \u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e \u003cstrong\u003e129\u003c/strong\u003e, 1628-1635.e2 (2012).\u003c/li\u003e\n \u003cli\u003eBleasdale, J. E. \u003cem\u003eet al.\u003c/em\u003e Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. \u003cem\u003eJ Pharmacol Exp Ther\u003c/em\u003e \u003cstrong\u003e255\u003c/strong\u003e, 756\u0026ndash;68 (1990).\u003c/li\u003e\n \u003cli\u003eDoench, J. G. \u003cem\u003eet al.\u003c/em\u003e Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 184\u0026ndash;191 (2016).\u003c/li\u003e\n \u003cli\u003eRamms, D. J. \u003cem\u003eet al.\u003c/em\u003e G \u003cem\u003e\u0026alpha;\u003c/em\u003e s\u0026ndash;Protein Kinase A (PKA) Pathway Signalopathies: The Emerging Genetic Landscape and Therapeutic Potential of Human Diseases Driven by Aberrant G \u003cem\u003e\u0026alpha;\u003c/em\u003e s-PKA Signaling. \u003cem\u003ePharmacol Rev\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 1326\u0026ndash;1368 (2021).\u003c/li\u003e\n \u003cli\u003eQuintana, A. \u003cem\u003eet al.\u003c/em\u003e TMEM110 regulates the maintenance and remodeling of mammalian ER\u0026ndash;plasma membrane junctions competent for STIM\u0026ndash;ORAI signaling. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, (2015).\u003c/li\u003e\n \u003cli\u003eHolzmann, C. \u003cem\u003eet al.\u003c/em\u003e Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells. \u003cem\u003eOncotarget\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 41783\u0026ndash;41793 (2015).\u003c/li\u003e\n \u003cli\u003eEmrich, S. M. \u003cem\u003eet al.\u003c/em\u003e Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, 6318\u0026ndash;6332 (2019).\u003c/li\u003e\n \u003cli\u003e\u0026Ouml;z\u0026ccedil;elik, T. \u003cem\u003eet al.\u003c/em\u003e Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader\u0026ndash;Willi syndrome critical region. \u003cem\u003eNat Genet\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 265\u0026ndash;269 (1992).\u003c/li\u003e\n \u003cli\u003eFeigelstock, D., Thompson, P., Mattoo, P., Zhang, Y. \u0026amp; Kaplan, G. G. The Human Homolog of HAVcr-1 Codes for a Hepatitis A Virus Cellular Receptor. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 6621\u0026ndash;6628 (1998).\u003c/li\u003e\n \u003cli\u003eMorgan, A. \u003cem\u003eet al.\u003c/em\u003e Mutations in \u003cem\u003ePLS1\u003c/em\u003e , encoding fimbrin, cause autosomal dominant nonsyndromic hearing loss. \u003cem\u003eHum Mutat\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 2286\u0026ndash;2295 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4656479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4656479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCalcium signals regulate crucial cellular functions yet many genes coding for Ca\u003csup\u003e2+ \u003c/sup\u003ehandling proteins remain unknown as their identification relies on low-throughput single-cell approaches. Here we describe a novel method to measure Ca\u003csup\u003e2+\u003c/sup\u003e activity in cells isolated by flow cytometry following pooled genome interrogation. Using a CRISPR/ CAMPARI2 screen, we identified enhancers and inhibitors of homeostatic Ca\u003csup\u003e2+\u003c/sup\u003e activity.\u003c/p\u003e","manuscriptTitle":"Genome interrogation of homeostatic calcium activity using CaMP-screen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 04:14:23","doi":"10.21203/rs.3.rs-4656479/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"29cb911a-62da-46ea-9c05-9ced713a0403","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34293674,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":34293675,"name":"Biological sciences/Biological techniques/Sensors and probes/Fluorescent proteins"},{"id":34293676,"name":"Biological sciences/Biological techniques/Electrophysiology/Intracellular recording"},{"id":34293677,"name":"Biological sciences/Biological techniques/High-throughput screening"}],"tags":[],"updatedAt":"2024-07-22T04:14:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 04:14:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4656479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4656479","identity":"rs-4656479","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}