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1. Plasma membrane SK2 channel activity regulates migration and chemosensitivity of high‐grade serous ovarian cancer cells

Author

Olivier Romito, Aude Lemettre, Aurélie Chantôme, Ophélie Champion, Noémie Couty, Lobna Ouldamer, Nadine Hempel, Mohamed Trebak, Caroline Goupille, and Marie Potier‐Cartereau

Subjects
cell migration, chemosensitivity, high‐grade serous ovarian cancer, LPA, SK2 channel, Taxol®, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

No data are currently available on the functional role of small conductance Ca2+‐activated K+ channels (SKCa) in ovarian cancer. Here, we characterized the role of SK2 (KCa2.2) in ovarian cancer cell migration and chemosensitivity. Using the selective non‐cell‐permeant SK2 inhibitor Lei‐Dab7, we identified functional SK2 channels at the plasma membrane, regulating store‐operated Ca2+ entry (SOCE) in both cell lines tested (COV504 and OVCAR3). Silencing KCNN2 with short interfering RNA (siRNA), or blocking SK2 activity with Lei‐Dab7, decreased cell migration. The more robust effect of KCNN2 knockdown compared to Lei‐Dab7 treatment suggested the involvement of functional intracellular SK2 channels in both cell lines. In cells treated with lysophosphatidic acid (LPA), an ovarian cancer biomarker of progression, SK2 channels are a key player of LPA pro‐migratory activity but their role in SOCE is abolished. Concerning chemotherapy, SK2 inhibition increased chemoresistance to Taxol® and low KCNN2 mRNA expression was associated with the worst prognosis for progression‐free survival in patients with serous ovarian cancer. The dual roles of SK2 mean that SK2 activators could be used as an adjuvant chemotherapy to potentiate treatment efficacy and SK2 inhibitors could be administrated as monotherapy to limit cancer cell dissemination.

Published
2024
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2. Store-Operated Ca Entry in Fibrosis and Tissue Remodeling

Author

Ahmed Emam Abdelnaby and Mohamed Trebak

Subjects
Biology (General), QH301-705.5, Biochemistry, QD415-436
Abstract

Fibrosis is a pathological condition characterized by excessive tissue deposition of extracellular matrix (ECM) components, leading to scarring and impaired function across multiple organ systems. This complex process is mediated by a dynamic interplay between cell types, including myofibroblasts, fibroblasts, immune cells, epithelial cells, and endothelial cells, each contributing distinctively through various signaling pathways. Critical to the regulatory mechanisms involved in fibrosis is store-operated calcium entry (SOCE), a calcium entry pathway into the cytosol active at the endoplasmic reticulum-plasma membrane contact sites and common to all cells. This review addresses the multifactorial nature of fibrosis with a focus on the pivotal roles of different cell types. We highlight the essential functions of myofibroblasts in ECM production, the transformation of fibroblasts, and the participation of immune cells in modulating the fibrotic landscape. We emphasize the contributions of SOCE in these different cell types to fibrosis, by exploring the involvement of SOCE in cellular functions such as proliferation, migration, secretion, and inflammatory responses. The examination of the cellular and molecular mechanisms of fibrosis and the role of SOCE in these mechanisms offers the potential of targeting SOCE as a therapeutic strategy for mitigating or reversing fibrosis.

Published
2024
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3. Calcium oscillations in HEK293 cells lacking SOCE suggest the existence of a balanced regulation of IP3 production and degradation

Author

Clara Octors, Ryan E. Yoast, Scott M. Emrich, Mohamed Trebak, and James Sneyd

Subjects
calcium oscillation, calcium influx, HEK293 cells, SOCE, PLC regulation, IP3, Physiology, QP1-981
Abstract

The concentration of free cytosolic Ca2+ is a critical second messenger in almost every cell type, with the signal often being carried by the period of oscillations, or spikes, in the cytosolic Ca2+ concentration. We have previously studied how Ca2+ influx across the plasma membrane affects the period and shape of Ca2+ oscillations in HEK293 cells. However, our theoretical work was unable to explain how the shape of Ca2+ oscillations could change qualitatively, from thin spikes to broad oscillations, during the course of a single time series. Such qualitative changes in oscillation shape are a common feature of HEK293 cells in which STIM1 and 2 have been knocked out. Here, we present an extended version of our earlier model that suggests that such time-dependent qualitative changes in oscillation shape might be the result of balanced positive and negative feedback from Ca2+ to the production and degradation of inositol trisphosphate.

Published
2024
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4. Orai3 and Orai1 mediate CRAC channel function and metabolic reprogramming in B cells

Author

Scott M Emrich, Ryan E Yoast, Xuexin Zhang, Adam J Fike, Yin-Hu Wang, Kristen N Bricker, Anthony Y Tao, Ping Xin, Vonn Walter, Martin T Johnson, Trayambak Pathak, Adam C Straub, Stefan Feske, Ziaur SM Rahman, and Mohamed Trebak

Subjects
Orai1, Orai3, B cells, CRAC channels, SOCE, metabolism, Medicine, Science, Biology (General), QH301-705.5
Abstract

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes.

Published
2023
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6. Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death

Author

Nolan T. Carew, Heidi M. Schmidt, Shuai Yuan, Joseph C. Galley, Robert Hall, Helene M. Altmann, Scott A. Hahn, Megan P. Miller, Katherine C. Wood, Bethann Gabris, Margaret C. Stapleton, Sean Hartwick, Marco Fazzari, Yijen L. Wu, Mohamed Trebak, Brett A. Kaufman, Charles F. McTiernan, Francisco J. Schopfer, Placido Navas, Patrick H. Thibodeau, Dennis M. McNamara, Guy Salama, and Adam C. Straub

Subjects
Cardiology, Medicine
Abstract

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

Published
2022
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7. The native ORAI channel trio underlies the diversity of Ca2+ signaling events

Author

Ryan E. Yoast, Scott M. Emrich, Xuexin Zhang, Ping Xin, Martin T. Johnson, Adam J. Fike, Vonn Walter, Nadine Hempel, David I. Yule, James Sneyd, Donald L. Gill, and Mohamed Trebak

Subjects
Science
Abstract

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, but the roles of ORAI2 and ORAI3 remained obscure. Here authors show that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation.

Published
2020
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8. STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

Author

Vivek Krishnan, Sher Ali, Albert L Gonzales, Pratish Thakore, Caoimhin S Griffin, Evan Yamasaki, Michael G Alvarado, Martin T Johnson, Mohamed Trebak, and Scott Earley

Subjects
STIM1, BK channels, TRPM4 channels, cerebral artery, peripheral coupling, membrane junctions, Medicine, Science, Biology (General), QH301-705.5
Abstract

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 – independent of SR Ca2+ store depletion – is critically important for stable peripheral coupling in contractile VSMCs.

Published
2022
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9. A protocol for detecting elemental calcium signals (Ca2+ puffs) in mammalian cells using total internal reflection fluorescence microscopy

Author

Vikas Arige, Scott M. Emrich, Ryan E. Yoast, Mohamed Trebak, and David I. Yule

Subjects
Cell Biology, Cell culture, Microscopy, Signal Transduction, Molecular/Chemical Probes, Science (General), Q1-390
Abstract

Summary: This protocol outlines steps to visualize and detect Ca2+ puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP3) from HEK-293 cells expressing only the native IP3R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with a higher signal-to-background ratio. Additionally, we shed light on commonly encountered pitfalls, which should be considered while recording Ca2+ puffs using TIRF microscopy.For complete details on the use and execution of this protocol, please refer to Emrich et al. (2021) and Lock et al. (2015a).

Published
2021
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10. A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactivation to shape NFAT induction

Author

Xuexin Zhang, Trayambak Pathak, Ryan Yoast, Scott Emrich, Ping Xin, Robert M. Nwokonko, Martin Johnson, Shilan Wu, Céline Delierneux, Maxime Gueguinou, Nadine Hempel, James W. Putney, Donald L. Gill, and Mohamed Trebak

Subjects
Science
Abstract

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel, but how this channel is turned off through Ca2+-dependent inactivation (CDI) remained unclear. Here the authors identify a spatially-restricted Ca2+/cAMP signaling crosstalk critical for mediating CDI which in turn regulates cellular Ca2+ signals and NFAT activation.

Published
2019
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11. Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation

Author

James H. Baraniak, Jr., Yandong Zhou, Robert M. Nwokonko, Michelle R. Jennette, Sarah A. Kazzaz, Jazmin M. Stenson, Abigale L. Whitsell, Youjun Wang, Mohamed Trebak, and Donald L. Gill

Subjects
Biology (General), QH301-705.5
Abstract

Summary: Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals.

Published
2021
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12. Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium‐dependent, non‐D2 receptor‐dependent, manner

Author

Jillian S. Weissenrieder, Jessie L. Reed, George‐Lucian Moldovan, Martin T. Johnson, Mohamed Trebak, Jeffrey D. Neighbors, Richard B. Mailman, and Raymond J. Hohl

Subjects
antipsychotic, calcium signaling, dopamine, glioblastoma, Therapeutics. Pharmacology, RM1-950
Abstract

Abstract Dopamine D2‐like receptor antagonists have been suggested as being potential anticancer therapeutics with specific utility for central nervous system cancers due to their ability to cross the blood‐brain barrier. Despite a plethora of data reporting anticancer effects for D2R antagonists in cell or animal studies, the ligand concentrations or doses required to achieve such effects greatly exceed the levels known to cause high degrees of occupancy of the D2 receptor. To resolve this conundrum, we interrogated a panel of glioblastoma multiforme (GBM) cell lines using D2 antagonists of varying chemotype. We studied the cytotoxic effects of these compounds, and also ascertained the expression of D2 receptors (D2R) on these cells. Although several chemotypes of D2R antagonists, including phenothiazines and phenylbutylpiperidines, were effective against GBM cell line cultures, the highly selective antagonist remoxipride had no anticancer activity at biologically relevant concentrations. Moreover the D2R antagonist‐induced cytotoxicity in monolayer cultures was independent of whether the cells expressed D2R. Instead, cytotoxicity was associated with a rapid, high‐magnitude calcium flux into the cytoplasm and mitochondria, which then induced depolarization and apoptosis. Blocking this flux protected the GBM cell lines U87MG, U251MG, and A172. Together, these data suggest that the cytotoxicity of these D2R antagonists involves calcium signaling mechanisms, not D2R antagonism. Repurposing of existing drugs should focus on the former, not latter, mechanism.

Published
2021
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13. Cardiac‐Specific Deletion of Orai3 Leads to Severe Dilated Cardiomyopathy and Heart Failure in Mice

Author

Jesse Gammons, Mohamed Trebak, and Salvatore Mancarella

Subjects
calcium signaling, dilated cardiomyopathy, ion channels, Orai3, Diseases of the circulatory (Cardiovascular) system, RC666-701
Abstract

Background Orai3 is a mammalian‐specific member of the Orai family (Orai1‒3) and a component of the store‐operated Ca2+ entry channels. There is little understanding of the role of Orai channels in cardiomyocytes, and its role in cardiac function remains unexplored. Thus, we developed mice lacking Orai1 and Orai3 to address their role in cardiac homeostasis. Methods and Results We generated constitutive and inducible cardiomyocyte‐specific Orai3 knockout (Orai3cKO) mice. Constitutive Orai3‐loss led to ventricular dysfunction progressing to dilated cardiomyopathy and heart failure. Orai3cKO mice subjected to pressure overload developed a fulminant dilated cardiomyopathy with rapid heart failure onset, characterized by interstitial fibrosis and apoptosis. Ultrastructural analysis of Orai3‐deficient cardiomyocytes showed abnormal M‐ and Z‐line morphology. The greater density of condensed mitochondria in Orai3‐deficient cardiomyocytes was associated with the upregulation of DRP1 (dynamin‐related protein 1). Cardiomyocytes isolated from Orai3cKO mice exhibited profoundly altered myocardial Ca2+ cycling and changes in the expression of critical proteins involved in the Ca2+ clearance mechanisms. Upregulation of TRPC6 (transient receptor potential canonical type 6) channels was associated with upregulation of the RCAN1 (regulator of calcineurin 1), indicating the activation of the calcineurin signaling pathway in Orai3cKO mice. A more dramatic cardiac phenotype emerged when Orai3 was removed in adult mice using a tamoxifen‐inducible Orai3cKO mouse. The removal of Orai1 from adult cardiomyocytes did not change the phenotype of tamoxifen‐inducible Orai3cKO mice. Conclusions Our results identify a critical role for Orai3 in the heart. We provide evidence that Orai3‐mediated Ca2+ signaling is required for maintaining sarcomere integrity and proper mitochondrial function in adult mammalian cardiomyocytes.

Published
2021
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14. Dichotomous role of the human mitochondrial Na+/Ca2+/Li+ exchanger NCLX in colorectal cancer growth and metastasis

Author

Trayambak Pathak, Maxime Gueguinou, Vonn Walter, Celine Delierneux, Martin T Johnson, Xuexin Zhang, Ping Xin, Ryan E Yoast, Scott M Emrich, Gregory S Yochum, Israel Sekler, Walter A Koltun, Donald L Gill, Nadine Hempel, and Mohamed Trebak

Subjects
calcium signaling, mitochondrial calcium, metastasis, Colorectal cancer, HIF1a, Medicine, Science, Biology (General), QH301-705.5
Abstract

Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer.

Published
2020
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15. The remote allosteric control of Orai channel gating.

Author

Yandong Zhou, Robert M Nwokonko, James H Baraniak, Mohamed Trebak, Kenneth P K Lee, and Donald L Gill

Subjects
Biology (General), QH301-705.5
Abstract

Calcium signals drive an endless array of cellular responses including secretion, contraction, transcription, cell division, and growth. The ubiquitously expressed Orai family of plasma membrane (PM) ion channels mediate Ca2+ entry signals triggered by the Ca2+ sensor Stromal Interaction Molecule (STIM) proteins of the endoplasmic reticulum (ER). The 2 proteins interact within curiously obscure ER-PM junctions, driving an allosteric gating mechanism for the Orai channel. Although key to Ca2+ signal generation, molecular understanding of this activation process remain obscure. Crystallographic structural analyses reveal much about the exquisite hexameric core structure of Orai channels. But how STIM proteins bind to the channel periphery and remotely control opening of the central pore, has eluded such analysis. Recent studies apply both crystallography and single-particle cryogenic electron microscopy (cryo-EM) analyses to probe the structure of Orai mutants that mimic activation by STIM. The results provide new understanding on the open state of the channel and how STIM proteins may exert remote allosteric control of channel gating.

Published
2019
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16. The STIM1-binding site nexus remotely controls Orai1 channel gating

Author

Yandong Zhou, Xiangyu Cai, Natalia A. Loktionova, Xianming Wang, Robert M. Nwokonko, Xizhuo Wang, Youjun Wang, Brad S. Rothberg, Mohamed Trebak, and Donald L. Gill

Subjects
Science
Abstract

How plasma membrane Orai Ca2+ channels are activated by STIM proteins to activate Ca2+signals is still not fully known. Here the authors show that a nexus region located at the Orai1 C-terminus allows channel gating without a direct interaction of STIM1 with the channel pore.

Published
2016
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17. Mitochondrial Calcium Regulation of Redox Signaling in Cancer

Author

Céline Delierneux, Sana Kouba, Santhanam Shanmughapriya, Marie Potier-Cartereau, Mohamed Trebak, and Nadine Hempel

Subjects
mitochondrial ca2+ transport, ros, redox signaling, mitochondrial ros, cancer, Cytology, QH573-671
Abstract

Calcium (Ca2+) uptake into the mitochondria shapes cellular Ca2+ signals and acts as a key effector for ATP generation. In addition, mitochondria-derived reactive oxygen species (mROS), produced as a consequence of ATP synthesis at the electron transport chain (ETC), modulate cellular signaling pathways that contribute to many cellular processes. Cancer cells modulate mitochondrial Ca2+ ([Ca2+]m) homeostasis by altering the expression and function of mitochondrial Ca2+ channels and transporters required for the uptake and extrusion of mitochondrial Ca2+. Regulated elevations in [Ca2+]m are required for the activity of several mitochondrial enzymes, and this in turn regulates metabolic flux, mitochondrial ETC function and mROS generation. Alterations in both [Ca2+]m and mROS are hallmarks of many tumors, and elevated mROS is a known driver of pro-tumorigenic redox signaling, resulting in the activation of pathways implicated in cellular proliferation, metabolic alterations and stress-adaptations. In this review, we highlight recent studies that demonstrate the interplay between [Ca2+]m and mROS signaling in cancer.

Published
2020
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19. Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer

Author

Xiuchao Wang, Yunzhan Li, Zekun Li, Shengchen Lin, Hongwei Wang, Jianwei Sun, Chungen Lan, Liangliang Wu, Dongxiao Sun, Chongbiao Huang, Pankaj K. Singh, Nadine Hempel, Mohamed Trebak, Gina M. DeNicola, Jihui Hao, and Shengyu Yang

Subjects
Cancer Research, Kelch-Like ECH-Associated Protein 1, endocrine system diseases, NF-E2-Related Factor 2, Article, digestive system diseases, Pancreatic Neoplasms, Oncology, Cell Line, Tumor, Cystine, Humans, Calcium, Calcium Channels, Carcinoma, Pancreatic Ductal
Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with few effective treatments. Here we show that the mitochondrial calcium uniporter (MCU) promotes PDAC cell migration, invasion, metastasis, and metabolic stress resistance by activating the Keap1-Nrf2 antioxidant program. The cystine transporter SLC7A11 was identified as a druggable target downstream of the MCU-Nrf2 axis. Paradoxically, despite the increased ability to uptake cystine, MCU-overexpressing PDAC demonstrated characteristics typical of cystine-deprived cells and were hypersensitive to cystine deprivation-induced ferroptosis. Pharmacologic inhibitors of SLC7A11 effectively induced tumor regression and abrogated MCU-driven metastasis in PDAC. In patient-derived organoid models in vitro and patient-derived xenograft models in vivo, MCU-high PDAC demonstrated increased sensitivity to SLC7A11 inhibition compared with MCU-low tumors. These data suggest that MCU is able to promote resistance to metabolic stress and to drive PDAC metastasis in a cystine-dependent manner. MCU-mediated cystine addiction could be exploited as a therapeutic vulnerability to inhibit PDAC tumor growth and to prevent metastasis. Significance: Elevated mitochondrial calcium uptake in PDAC promotes metastasis but exposes cystine addiction and ferroptosis sensitivity that could be targeted to improve pancreatic cancer treatment.

Published
2022
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20. Physiological Functions of CRAC Channels

Author

Mohamed Trebak, Scott M. Emrich, and Ryan E. Yoast

Subjects
Genetically modified mouse, Physiology, Chemistry, Endoplasmic reticulum, Membrane Proteins, CRAC Channels, Calcium Release Activated Calcium Channels, Cell biology, Mice, Knockout mouse, Animals, Humans, Calcium, Calcium Channels, Calcium Signaling, Stromal Interaction Molecule 1, Signal transduction, Receptor
Abstract

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol-1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Published
2022
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21. Supplementary Data from Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer

Author

Shengyu Yang, Jihui Hao, Gina M. DeNicola, Mohamed Trebak, Nadine Hempel, Pankaj K. Singh, Chongbiao Huang, Dongxiao Sun, Liangliang Wu, Chungen Lan, Jianwei Sun, Hongwei Wang, Shengchen Lin, Zekun Li, Yunzhan Li, and Xiuchao Wang

Abstract

Supplementary Data from Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer

Published
2023
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22. Data from Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer

Author

Shengyu Yang, Jihui Hao, Gina M. DeNicola, Mohamed Trebak, Nadine Hempel, Pankaj K. Singh, Chongbiao Huang, Dongxiao Sun, Liangliang Wu, Chungen Lan, Jianwei Sun, Hongwei Wang, Shengchen Lin, Zekun Li, Yunzhan Li, and Xiuchao Wang

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with few effective treatments. Here we show that the mitochondrial calcium uniporter (MCU) promotes PDAC cell migration, invasion, metastasis, and metabolic stress resistance by activating the Keap1-Nrf2 antioxidant program. The cystine transporter SLC7A11 was identified as a druggable target downstream of the MCU-Nrf2 axis. Paradoxically, despite the increased ability to uptake cystine, MCU-overexpressing PDAC demonstrated characteristics typical of cystine-deprived cells and were hypersensitive to cystine deprivation-induced ferroptosis. Pharmacologic inhibitors of SLC7A11 effectively induced tumor regression and abrogated MCU-driven metastasis in PDAC. In patient-derived organoid models in vitro and patient-derived xenograft models in vivo, MCU-high PDAC demonstrated increased sensitivity to SLC7A11 inhibition compared with MCU-low tumors. These data suggest that MCU is able to promote resistance to metabolic stress and to drive PDAC metastasis in a cystine-dependent manner. MCU-mediated cystine addiction could be exploited as a therapeutic vulnerability to inhibit PDAC tumor growth and to prevent metastasis.Significance:Elevated mitochondrial calcium uptake in PDAC promotes metastasis but exposes cystine addiction and ferroptosis sensitivity that could be targeted to improve pancreatic cancer treatment.

Published
2023
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23. Supplementary Table from Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer

Author

Shengyu Yang, Jihui Hao, Gina M. DeNicola, Mohamed Trebak, Nadine Hempel, Pankaj K. Singh, Chongbiao Huang, Dongxiao Sun, Liangliang Wu, Chungen Lan, Jianwei Sun, Hongwei Wang, Shengchen Lin, Zekun Li, Yunzhan Li, and Xiuchao Wang

Abstract

Supplementary Table from Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer

Published
2023
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25. Calcium signaling: A therapeutic target to overcome resistance to therapies in cancer

Author

Olivier Romito, Maxime Guéguinou, William Raoul, Ophélie Champion, Alison Robert, Mohamed Trebak, Caroline Goupille, Marie Potier-Cartereau, Nutrition, croissance et cancer (U 1069) (N2C), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cancéropôle Grand-Ouest [Bretagne-Centre-Pays de Loire], University of Pittsburgh (PITT), Pennsylvania Commonwealth System of Higher Education (PCSHE), Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), This study was supported by grants from INSERM, University of Tours, the 'Région center-Val de Loire', the 'Cancéropôle Grand Ouest', the Association 'CANCEN',Tours’ Hospital oncology association ACORT' and the committees of Ligue Contre le Cancer Grand-Ouest (comities 85, 37, 49, 72, 17)., and Raoul, William

Subjects
Physiology, Carcinogenesis, [SDV]Life Sciences [q-bio], chemoresistance, Cell Biology, potassium channels, [SDV] Life Sciences [q-bio], Neoplasms, cancer, Humans, Calcium, Calcium Signaling, Calcium Channels, Molecular Biology
Abstract

International audience; Innate and acquired resistances to therapeutic agents are responsible for the failure of cancer treatments. Due to the multifactorial nature of resistance, the identification of new therapeutic targets is required to improve cancer treatment. Calcium is a universal second messenger that regulates many cellular functions such as proliferation, migration, and survival. Calcium channels, pumps and exchangers tightly regulate the duration, location and magnitude of calcium signals. Many studies have implicated dysregulation of calcium signaling in several pathologies, including cancer. Abnormal calcium fluxes due to altered channel expression or activation contribute to carcinogenesis and promote tumor development. However, there is limited information on the role of calcium signaling in cancer resistance to therapeutic drugs. This review discusses the role of calcium signaling as a mediator of cancer resistance, and assesses the potential value of combining anticancer therapy with calcium signaling modulators to improve the effectiveness of current treatments.

Published
2022
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28. Curcumin and NCLX inhibitors share anti-tumoral mechanisms in microsatellite-instability-driven colorectal cancer

Author

Maxime Guéguinou, Sajida Ibrahim, Jérôme Bourgeais, Alison Robert, Trayambak Pathak, Xuexin Zhang, David Crottès, Jacques Dupuy, David Ternant, Valérie Monbet, Roseline Guibon, Hector Flores-Romero, Antoine Lefèvre, Stéphanie Lerondel, Alain Le Pape, Jean-François Dumas, Philippe G. Frank, Alban Girault, Romain Chautard, Françoise Guéraud, Ana J. García-Sáez, Mehdi Ouaissi, Patrick Emond, Olivier Sire, Olivier Hérault, Gaëlle Fromont-Hankard, Christophe Vandier, David Tougeron, Mohamed Trebak, William Raoul, Thierry Lecomte, Nutrition, croissance et cancer (U 1069) (N2C), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Groupe innovation et ciblage cellulaire (GICC), EA 7501 [2018-...] (GICC EA 7501), Université de Tours (UT), ERL 7001 LNOx (Leukemic Niche & redOx metabolism / Niche leucémique et métabolisme redOx) (LNOx), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Centre National de la Recherche Scientifique (CNRS)-Microenvironnement des niches tumorales (CNRS GDR 3697 Micronit ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Groupe innovation et ciblage cellulaire (GICC), EA 7501 [2018-...] (GICC EA 7501), Université de Tours (UT)-Université de Tours (UT), Pennsylvania State University (Penn State), Penn State System, Prévention et promotion de la cancérogénèse par les aliments (ToxAlim-PPCA), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Ecole d'Ingénieurs de Purpan (INP - PURPAN), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), EA4245 - Transplantation, Immunologie, Inflammation [Tours] (T2i), Institut de Recherche Mathématique de Rennes (IRMAR), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École normale supérieure - Rennes (ENS Rennes)-Université de Rennes 2 (UR2)-Centre National de la Recherche Scientifique (CNRS)-INSTITUT AGRO Agrocampus Ouest, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), University of Cologne, Eberhard Karls Universität Tübingen = Eberhard Karls University of Tuebingen, Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), Transgenèse et archivage d'animaux modèles (TAAM), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie Cellulaire et Moléculaire - UR UPJV 4667 (LPCM), Université de Picardie Jules Verne (UPJV), Institut de Recherche Dupuy de Lôme (IRDL), École Nationale d'Ingénieurs de Brest (ENIB)-Université de Bretagne Sud (UBS)-Université de Brest (UBO)-École Nationale Supérieure de Techniques Avancées Bretagne (ENSTA Bretagne)-Centre National de la Recherche Scientifique (CNRS), Centre hospitalier universitaire de Poitiers (CHU Poitiers), Université de Poitiers - Faculté de Médecine et de Pharmacie, Université de Poitiers, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), This project was partly supported by grants on behalf of the following french department committees of Ligue Contre le Cancer Grand-Ouest: 16 (Charente), 37 (Indre-et-Loire), 49 (Maine-et-Loire), 72 (Sarthe) and 85 (Vendée), ANR-11-LABX-0020,LEBESGUE,Centre de Mathématiques Henri Lebesgue : fondements, interactions, applications et Formation(2011), LESUR, Hélène, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Pharmacology, Curcumin, [SDV]Life Sciences [q-bio], Calcium signaling, Cell Biology, Colorectal cancer, Sodium-Calcium Exchanger, digestive system diseases, Mitochondria, Mitochondrial Proteins, [SDV] Life Sciences [q-bio], Mice, Cellular and Molecular Neuroscience, Animals, Humans, Molecular Medicine, Calcium, Microsatellite Instability, Colorectal Neoplasms, NCLX, Molecular Biology, Microsatellite Repeats
Abstract

Colorectal cancer (CRC) is associated with high mortality worldwide and new targets are needed to overcome treatment resistance. Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of CRC. In this context, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new potential inhibitor of NCLX.In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis through mitochondrial permeability transition pore opening concomitant with G2/M cell cycle arrest. NCLX inhibition with either CGP37157 (a benzodiazepine derivative), small interfering RNA-mediated knock-down or knockout approaches reproduced the effects of curcumin. Altered mitochondrial respiration, cellular aerobic glycolysis and endoplasmic reticulum–mitochondria membrane perturbations participated in these mechanisms. In a xenograft mouse model, NCLX inhibitors decreased CRC tumor growth. Both transcriptomic analysis of The Cancer Genome Atlas dataset and immunohistochemical analysis of tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC demonstrated that higher NCLX expression was associated with MSI status and for the first time NCLX expression was significantly associated with recurrence-free survival in MSI CRC patients.Our findings provide strong evidence that blocking NCLX inhibits CRC in vitro and in vivo. We highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic treatment of patients with MSI CRC.

Published
2022
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29. Orai3 and Orai1 are essential for CRAC channel function and metabolic reprogramming in B cells

Author

Scott M. Emrich, Ryan E. Yoast, Xuexin Zhang, Adam J. Fike, Yin-Hu Wang, Kristen N. Bricker, Anthony Tao, Ping Xin, Vonn Walter, Martin T. Johnson, Trayambak Pathak, Adam C. Straub, Stefan Feske, Ziaur S.M. Rahman, and Mohamed Trebak

Abstract

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in expression of Orai isoforms in response to B cell activation. We show that Orai3 and Orai1 are essential components of native CRAC channels in B cells and are critical for primary B cell proliferation and survival. The combined loss of Orai1 and Orai3 strongly impairs SOCE, nuclear factor for activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of B cells in response to antigenic stimulation. Our results clarify the molecular composition and cellular functions of SOCE in B lymphocytes.

Published
2022
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30. The anatomy of native CRAC channel(s)

Author

Mohamed Trebak, Scott M. Emrich, and Ryan E. Yoast

Subjects
0301 basic medicine, Gene isoform, Physiology, Chemistry, Orai1 protein, CRAC Channels, Store-operated calcium entry, Cell biology, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Cell culture, Physiology (medical), Knockout mouse, 030217 neurology & neurosurgery, Calcium signaling
Abstract

The ubiquitous store-operated Ca2+ entry pathway mediated by plasma membrane Ca2+ release-activated Ca2+ (CRAC) channels regulates a wide variety of physiological functions. While it is clearly established that the ORAI1 protein is essential for native mammalian CRAC channels, the contribution of ORAI2 and ORAI3 have remained nebulous. The crystal structure of the sole Orai isoform in drosophila (dOrai) revealed a hexameric assembly, suggesting that mammalian CRAC channels are hexamers of ORAI. Nevertheless, the relative contribution of each isoform of the mammalian ORAI trio to the stoichiometry of native CRAC channels remains elusive. The recent generation of ORAI isoform single, double and triple knockout cell lines and tissue-specific knockout mice has shed light on how native ORAI isoform heteromerization fine tunes CRAC-mediated Ca2+ signaling.

Published
2020
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31. L-type Ca 2+ channel blockers promote vascular remodeling through activation of STIM proteins

Author

Aparna Gudlur, Robert M. Nwokonko, Ryan E. Yoast, Mohamed Trebak, Nadine Hempel, Donald L. Gill, Martin Johnson, Scott M. Emrich, Ping Xin, Xuexin Zhang, Patrick G. Hogan, Raphael Jean Courjaret, Khaled Machaca, and Wei Li

Subjects
Multidisciplinary, Vascular smooth muscle, biology, Chemistry, Endoplasmic reticulum, Cell, STIM1, medicine.disease, Cav1.2, Cell biology, medicine.anatomical_structure, Heart failure, medicine, biology.protein, Ca2 channels, Calcium signaling
Abstract

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

Published
2020
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32. The native ORAI channel trio underlies the diversity of Ca2+ signaling events

Author

Nadine Hempel, David I. Yule, Ping Xin, James Sneyd, Ryan E. Yoast, Adam J. Fike, Mohamed Trebak, Scott M. Emrich, Vonn Walter, Martin Johnson, Xuexin Zhang, and Donald L. Gill

Subjects
0301 basic medicine, Gene isoform, Multidisciplinary, ORAI1, Chemistry, Extramural, Science, HEK 293 cells, General Physics and Astronomy, General Chemistry, General Biochemistry, Genetics and Molecular Biology, Cell biology, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Transcriptional regulation, lcsh:Q, lcsh:Science, Receptor activation, 030217 neurology & neurosurgery
Abstract

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.

Published
2020

33. A New Selective Pharmacological Enhancer of the Orai1 Ca2+ Channel Reveals Roles for Orai1 in Smooth and Skeletal Muscle Functions

Author

Jack U. Flanagan, Ryan E. Yoast, Gregory R. Monteith, Bradley S. Launikonis, Irina Vetter, Iman Azimi, Ping Xin, Mohamed Trebak, Jeevak Sopanrao Kapure, Martin Johnson, Mark R. Ashton, Aldo Meizoso-Huesca, Benjamin P. Ross, Xuexin Zhang, Silke B. Chalmers, Ralph J. Stevenson, and William A. Denny

Subjects
Pharmacology, 0303 health sciences, ORAI1, Chemistry, Endoplasmic reticulum, Growth factor, medicine.medical_treatment, Skeletal muscle, Translation (biology), Cell migration, Cell biology, 03 medical and health sciences, 0302 clinical medicine, medicine.anatomical_structure, medicine, Pharmacology (medical), Enhancer, 030217 neurology & neurosurgery, Intracellular, 030304 developmental biology
Abstract

Store-operated calcium (Ca2+) entry is an important homeostatic mechanism in cells, whereby the release of Ca2+ from intracellular endoplasmic reticulum stores triggers the activation of a Ca2+ influx pathway. Mediated by Orai1, this Ca2+ influx has specific and essential roles in biological processes as diverse as lactation to immunity. Although pharmacological inhibitors of this Ca2+ influx mechanism have helped to define the role of store-operated Ca2+ entry in many cellular events, the lack of isoform specific modulators and activators of Orai1 has limited our full understanding of these processes. Here we report the identification and synthesis of an Orai1 activity enhancer that concurrently potentiated Orai1 Ca2+-dependent inactivation (CDI). This unique enhancer of Orai1 had only a modest effect on Orai3 with weak inhibitory effects at high concentrations in intact MCF-7 breast cancer cells. The Orai1 enhancer heightened vascular smooth muscle cell migration induced by platelet-derived growth factor and the unique store-operated Ca2+ entry pathway present in skeletal muscle cells. These studies show that IA65 is an exemplar for the translation and development of Orai isoform selective agents. The ability of IA65 to activate CDI demonstrates that agents can be developed that can enhance Orai1-mediated Ca2+ influx but avoid the cytotoxicity associated with sustained Orai1 activation. IA65 and/or future analogues with similar Orai1- and CDI-activating properties could function to fine-tune physiological processes important in specific disease states, such as cellular migration and immune cell function.

Published
2020
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34. The airway smooth muscle sodium/calcium exchanger NCLX is critical for airway remodeling and hyperresponsiveness in asthma

Author

Martin T. Johnson, J. Cory Benson, Trayambak Pathak, Ping Xin, Abagail S. McKernan, Scott M. Emrich, Ryan E. Yoast, Vonn Walter, Adam C. Straub, and Mohamed Trebak

Subjects
Mice, Sodium, Airway Remodeling, Animals, Calcium, Muscle, Smooth, Cell Biology, Molecular Biology, Biochemistry, Asthma, Sodium-Calcium Exchanger
Abstract

The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca

Published
2022

35. Functional communication between IP 3 R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE

Author

Moaz Ahmad, Hwei Ling Ong, Hassan Saadi, Ga-Yeon Son, Zahra Shokatian, Lara E. Terry, Mohamed Trebak, David I. Yule, and Indu Ambudkar

Subjects
Multidisciplinary, STIM2, Physiology, endogenous protein, Cell Membrane, ER–PM junction, Biological Sciences, Endoplasmic Reticulum, Neoplasm Proteins, HEK293 Cells, Cluster Analysis, Humans, Inositol 1,4,5-Trisphosphate Receptors, Calcium, Calcium Signaling, Stromal Interaction Molecule 1, IP3R, Stromal Interaction Molecule 2, clustering
Abstract

Significance STIM proteins sense decreases in [Ca2+]ER and cluster in endoplasmic reticulum (ER)–plasma membrane (PM) junctions where they recruit and activate Orai1. While STIM1 clustering requires substantial [Ca2+]ER decrease, STIM2 displays preclustering under resting conditions and regulates basal Ca2+ entry. The mechanism(s) underlying constitutive clustering of STIM2 is not known. We show herein that endogenous STIM2 assembles as mobile and immobile clusters and that Orai1 is recruited to the latter. Anchoring of STIM2 clusters is triggered by decreases in local [Ca2+]ER that are mediated by ambient activity of IP3R and sensed by the STIM2 N terminus. This functional link between IP3R and STIM2 governs constitutive STIM2 clustering and ensures coupling of [Ca2+]ER decrease at subthreshold stimuli with activation of Ca2+ entry., Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER–plasma membrane (ER–PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER–PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER–PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER–PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

Published
2022
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37. STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma

Author

Martin T. Johnson, Ping Xin, J. Cory Benson, Trayambak Pathak, Vonn Walter, Scott M. Emrich, Ryan E. Yoast, Xuexin Zhang, Gaoyuan Cao, Reynold A. Panettieri, and Mohamed Trebak

Subjects
inorganic chemicals, Transcription, Genetic, Physiology, calcium signaling, Mice, Cell Movement, Respiratory Hypersensitivity, Animals, metabolic reprogramming, Stromal Interaction Molecule 1, Cell Proliferation, Mice, Knockout, Multidisciplinary, Ion Transport, Muscle, Smooth, respiratory system, Biological Sciences, asthma, Cellular Reprogramming, respiratory tract diseases, Mitochondria, smooth muscle remodeling, Chronic Disease, Airway Remodeling, Calcium, CRAC channels
Abstract

Significance Stromal-interacting molecule 1 (STIM1) proteins are essential for the function of store-operated Ca2+ entry (SOCE). Using transcriptomics, metabolomics, imaging, and inducible smooth muscle–specific STIM1 knockout mice expressing genetically encoded Ca2+ sensors, we reveal a crucial function of STIM1 in airway remodeling and airway hyperresponsiveness in asthma. STIM1-mediated Ca2+ oscillations in airway smooth muscle (ASM) cells are critical for ASM remodeling through metabolic and transcriptional reprogramming and cytokine secretion, including IL-6. These effects are driven by Ca2+-dependent activation of the transcription factor isoform NFAT4 specifically in ASM. Our data provide evidence that ASM STIM1 and SOCE are central triggers of asthma manifestations and advocate for the future use of STIM1 as a molecular target in asthma therapy., Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

Published
2021

39. PKC-ε regulates vesicle delivery and focal exocytosis for efficient IgG-mediated phagocytosis

Author

Mohamed Trebak, Ananya Murali, Michelle R. Lennartz, Anna E. D’Amico, Cheryl M. Zajd, Xuexin Zhang, and Alexander C. Wong

Subjects
Phagocytosis, Vesicle, Protein Kinase C-epsilon, Cell Biology, Golgi apparatus, Biology, Exocytosis, Cell biology, Vesicular transport protein, Mice, symbols.namesake, Nocodazole, chemistry.chemical_compound, chemistry, Immunoglobulin G, Phagosomes, symbols, Animals, Protein kinase C, Research Article, Phagosome
Abstract

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis – its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.This article has an associated First Person interview with the first author of the paper.

Published
2021
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41. The Mitochondrial Ca2+ uniporter is a central regulator of interorganellar Ca2+ transfer and NFAT activation

Author

Ahmed Emam Abdelnaby, Ryan E. Yoast, Jung Min Han, Natalia Lakomski, Vikas Arige, Scott M. Emrich, David I. Yule, James Sneyd, Geneviève Dupont, J. Cory Benson, Mohamed Trebak, Martin Johnson, Xuexin Zhang, Trayambak Pathak, Ping Xin, and Nadine Hempel

Subjects
T-Lymphocytes, Mitochondrion, Endoplasmic Reticulum, Lymphocyte Activation, IP3R, IP3 receptor, Biochemistry, TIRF, total internal reflection fluorescence, Gene Knockout Techniques, Jurkat Cells, chemistry.chemical_compound, Cytosol, NCLX, mitochondrial Na+/Ca2+ exchanger, BCR, B cell receptor, calcium oscillations, MCU, mitochondrial Ca2+ uniporter, Calcium signaling, NFAT, nuclear factor for activated T cells, RBL, rat basophilic leukemia, NFAT, Editors' Pick, FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone, IM, ICRAC microdomain, Mitochondria, Cell biology, PM, plasma membrane, CRAC, Ca2+ release–activated Ca2+, SOCE, store-operated Ca2+ entry, LPS, lipopolysaccharide, CRAC channels, Research Article, SOCE, Thapsigargin, SERCA, Biochimie, 4-OHT, 4-hydroxytamoxifen, SERCA, sarcoplasmic/ER Ca2+ ATPase, CCh, Carbachol, ER, endoplasmic reticulum, Humans, Calcium Signaling, Uniporter, Molecular Biology, NFATC Transcription Factors, Endoplasmic reticulum, Biologie moléculaire, Cell Biology, Inositol trisphosphate receptor, HCT116 Cells, IP3, inositol-1,4,5-trisphosphate, MCU, HEK293 Cells, chemistry, CDI, Ca2+-dependent inactivation, Tg, thapsigargin, STIM, stromal interaction molecule, Calcium, Biologie cellulaire, MAM, mitochondria-associated membrane, Calcium Channels, CRISPR-Cas Systems
Abstract

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C–coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release–activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol–mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function., SCOPUS: ar.j, info:eu-repo/semantics/published

Published
2021

42. The mitochondrial sodium/calcium exchanger NCLX (Slc8b1) in B lymphocytes

Author

Scott M. Emrich, Ryan E. Yoast, Adam J. Fike, Kristen N. Bricker, Ping Xin, Xuexin Zhang, Ziaur S.M. Rahman, and Mohamed Trebak

Subjects
Mice, Knockout, Mice, B-Lymphocytes, Physiology, Sodium, Animals, Calcium, Calcium Signaling, Cell Biology, Molecular Biology, Sodium-Calcium Exchanger, Mitochondria
Abstract

Antigen receptor stimulation triggers cytosolic Ca

Published
2022
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44. Functional Communication between IP3R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

Author

Hwei Ling Ong, Lara E. Terry, Hassan Saadi, Mohamed Trebak, Zahra Shokatian, Ga-Yeon Son, Indu S. Ambudkar, Moaz Ahmad, and David I. Yule

Subjects
chemistry.chemical_compound, chemistry, ORAI1, Endoplasmic reticulum, Biophysics, Inositol, STIM1, Stimulation, STIM2, Receptor, Synaptotagmin 1
Abstract

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters which co- ordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2-N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.SignificanceSTIM proteins sense decreases in [Ca2+]ER and cluster in endoplasmic reticulum (ER)-plasma membrane (PM) junctions where they recruit and activate Orai1. While STIM1 clustering requires substantial [Ca2+]ER decrease, STIM2 displays pre-clustering under resting conditions and regulates basal Ca2+ entry. The mechanism(s) underlying constitutive clustering of STIM2 is not known. We show herein that endogenous STIM2 assembles as mobile and immobile clusters and that Orai1 is recruited to the latter. Anchoring of STIM2 clusters is triggered by decreases in local [Ca2+]ER that is mediated by ambient activity of IP3R and sensed by the STIM2 N-terminus. This functional link between IP3R and STIM2 governs constitutive STIM2 clustering and ensures coupling of [Ca2+]ER decrease at sub-threshold stimuli with activation of Ca2+ entry.

Published
2021
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45. Crth2 receptor signaling down‐regulates lipopolysaccharide‐induced NF‐αB activation in murine macrophages via changes in intracellular calcium

Author

Mohamed Trebak, Randall M. Rossi, K. Sandeep Prabhu, Robert F. Paulson, Ian Huffnagle, Tai Jung Lee, Ryan E. Yoast, Fenghua Qian, Svanjita Berry, Shaneice K. Nettleford, and Bastihalli T. Diwakar

Subjects
0301 basic medicine, Cyclopentenone, Lipopolysaccharide, Prostanoid, Biochemistry, Calcium in biology, Cell biology, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, 0302 clinical medicine, chemistry, Genetics, Prostaglandin D2, Protein kinase A, Molecular Biology, 030217 neurology & neurosurgery, Cyclopentenone prostaglandins, Biotechnology, G protein-coupled receptor
Abstract

Prostaglandin D2 and its cyclopentenone metabolites [cyclopentenone prostaglandins (CyPGs)], Δ12prostaglandin J2 and 15-deoxy-Δ12,14-prostaglandin J2, act through 2 GPCRs, d-type prostanoid 1 and t...

Published
2019
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46. MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

Author

Julia S. Jacobs, Young-Rae Kim, Diana Castañeda, Kaikobad Irani, Karima Ait-Aissa, Mohanad Gabani, Jing Liu, Isabella M. Grumbach, Mohamed Trebak, Olha M. Koval, Modar Kassan, and Ajit Vikram

Subjects
0301 basic medicine, medicine.medical_specialty, Contraction (grammar), Vascular smooth muscle, Physiology, Thromboxane, chemistry.chemical_element, Cell Biology, Calcium, Contractility, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Endocrinology, Blood pressure, chemistry, Internal medicine, cardiovascular system, medicine, medicine.symptom, Molecular Biology, Phenylephrine, 030217 neurology & neurosurgery, Vasoconstriction, medicine.drug
Abstract

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca2+) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204-/- mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204-/- mice compared to WT mice. Aortas and MRA of miR-204-/- mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP3R1) as a target of miR-204. Aortas and MRA of miR-204-/- mice had higher expression of IP3R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204-/- and WT mice was abolished with pharmacologic inhibition of IP3R1. Furthermore, Ang II-induced aortic IP3R1 was greater in miR-204-/- mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204-/- and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP3R1, and boosted SR Ca2+ release in response to PE, while overexpression of miR-204 downregulated IP3R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP3R1 interaction rescued miR-204-induced downregulation of IP3R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP3R1-dependent regulation of SR calcium release.

Published
2019
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47. The lysosomal TRPML1 channel regulates triple negative breast cancer development by promoting mTORC1 and purinergic signaling pathways

Author

Bing Shen, Yiming Yang, Peng Huang, Mohamed Trebak, Yassine El Hiani, Shashi Gujar, Andra M. Sterea, Alia Kazim Rizvi Syeda, Jun Wang, Clements Richard Derek, Mengnan Xu, Chi Yan, Xian-Ping Dong, Shekoufeh Almasi, and Wei-Xing Zong

Subjects
0301 basic medicine, Cell Survival, Physiology, Triple Negative Breast Neoplasms, Mice, SCID, mTORC1, Mechanistic Target of Rapamycin Complex 1, Biology, Article, Mice, 03 medical and health sciences, Transient Receptor Potential Channels, 0302 clinical medicine, Downregulation and upregulation, Cell Movement, Mice, Inbred NOD, Animals, Humans, Molecular Biology, Cells, Cultured, Triple-negative breast cancer, Cell Proliferation, Tumor microenvironment, Purinergic receptor, Cell Biology, Purinergic signalling, 030104 developmental biology, Targeted drug delivery, Cancer research, Calcium, Female, Signal transduction, Lysosomes, 030217 neurology & neurosurgery, Signal Transduction
Abstract

The triple-negative breast cancer (TNBC) that comprises approximately 10%–20% of breast cancers is an aggressive subtype lacking effective therapeutics. Among various signaling pathways, mTORC1 and purinergic signals have emerged as potentially fruitful targets for clinical therapy of TNBC. Unfortunately, drugs targeting these signaling pathways do not successfully inhibit the progression of TNBC, partially due to the fact that these signaling pathways are essential for the function of all types of cells. In this study, we report that TRPML1 is specifically upregulated in TNBCs and that its genetic downregulation and pharmacological inhibition suppress the growth of TNBC. Mechanistically, we demonstrate that TRPML1 regulates TNBC development, at least partially, through controlling mTORC1 activity and the release of lysosomal ATP. Because TRPML1 is specifically activated by cellular stresses found in tumor microenvironments, antagonists of TRPML1 could represent anticancer drugs with enhanced specificity and potency. Our findings are expected to have a major impact on drug targeting of TNBCs.

Published
2019
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48. Calcium signalling in T cells

Author

Jean-Pierre Kinet and Mohamed Trebak

Subjects
0301 basic medicine, History, Calcium Channels, L-Type, T-Lymphocytes, T cell, Endoplasmic Reticulum, Article, Education, Calcium Channels, T-Type, 03 medical and health sciences, Transient Receptor Potential Channels, 0302 clinical medicine, Immune system, medicine, Humans, Calcium Signaling, Calcium signaling, Voltage-dependent calcium channel, Chemistry, Endoplasmic reticulum, Cell Membrane, Cytoplasmic Vesicles, Mitochondria, Computer Science Applications, Cell biology, Cytosol, Crosstalk (biology), 030104 developmental biology, medicine.anatomical_structure, Cytokine secretion, Calcium Channels, Receptors, Purinergic P2X2, 030215 immunology
Abstract

Calcium (Ca(2+)) signalling is of paramount importance to immunity. Regulated increases in cytosolic and organellar Ca(2+) concentrations in lymphocytes control complex and crucial effector functions such as metabolism, proliferation, differentiation, antibody and cytokine secretion and cytotoxicity. Altered Ca(2+) regulation in lymphocytes leads to various autoimmune, inflammatory and immunodeficiency syndromes. Several types of plasma membrane and organellar Ca(2+)-permeable channels are functional in T cells. They contribute highly localized spatial and temporal Ca(2+) microdomains that are required for achieving functional specificity. While the mechanistic details of these Ca(2+) microdomains are only beginning to emerge, it is evident that through crosstalk, synergy and feedback mechanisms, they fine tune T cell signalling to match complex immune responses. In this article, we review the expression and function of various Ca(2+)-permeable channels in the plasma membrane, endoplasmic reticulum, mitochondria and endolysosomes of T cells, and their role in shaping immunity and the pathogenesis of immune-mediated diseases.

Published
2019
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49. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Author

Lara Gibellini, Sussan Nourshargh, Susanna Cardell, Wlodzimierz Maslinski, Mar Felipo-Benavent, Florian Mair, Hans-Martin Jäck, Lilly Lopez, Klaus Warnatz, John Trowsdale, Diana Ordonez, Marcus Eich, William Hwang, Anne Cooke, Dirk Mielenz, Alberto Orfao, Winfried F. Pickl, Vladimir Benes, Alice Yue, T. Vincent Shankey, Maria Tsoumakidou, Virginia Litwin, Gelo Victoriano Dela Cruz, Andrea Cavani, Sara De Biasi, Larissa Nogueira Almeida, Jonathan J M Landry, Claudia Haftmann, Charlotte Esser, Ana Cumano, Anneke Wilharm, Francesco Dieli, Rudi Beyaert, Alessio Mazzoni, Burkhard Ludewig, Carlo Pucillo, Dirk H. Busch, Joe Trotter, Stipan Jonjić, Marc Veldhoen, Josef Spidlen, Aja M. Rieger, Dieter Adam, Srijit Khan, Todd A. Fehniger, Giuseppe Matarese, Maximilien Evrard, Christian Maueröder, Steffen Schmitt, Kristin A. Hogquist, Barry Moran, Raghavendra Palankar, Markus Feuerer, S Schmid, Susann Rahmig, Amy E. Lovett-Racke, James V. Watson, Megan K. Levings, Susanne Melzer, Dinko Pavlinic, Christopher M. Harpur, Christina Stehle, A. Graham Pockley, Toshinori Nakayama, Attila Tárnok, Juhao Yang, Michael Lohoff, Paulo Vieira, Francisco Sala-de-Oyanguren, Christian Kurts, Anastasia Gangaev, Alfonso Blanco, Hans Scherer, Regine J. Dress, Bruno Silva-Santos, Kiyoshi Takeda, Bimba F. Hoyer, Ilenia Cammarata, Daryl Grummitt, Isabel Panse, Günnur Deniz, Bianka Baying, Friederike Ebner, Esther Schimisky, Leo Hansmann, Thomas Kamradt, Edwin van der Pol, Daniel Scott-Algara, Anna Iannone, Giorgia Alvisi, Sebastian R. Schulz, Francesco Liotta, Irmgard Förster, Beatriz Jávega, Hans-Peter Rahn, Caetano Reis e Sousa, Livius Penter, Xuetao Cao, David P. Sester, Keisuke Goda, Peter Wurst, Iain B. McInnes, Ricardo T. Gazzinelli, Federica Piancone, Gerald Willimsky, Yotam Raz, Pärt Peterson, Wolfgang Fritzsche, Yvonne Samstag, Martin Büscher, Thomas Schüler, Susanne Hartmann, Robert J. Wilkinson, Anna E. S. Brooks, Steven L. C. Ketelaars, Catherine Sautès-Fridman, Anna Rubartelli, Petra Bacher, Katja Kobow, Marco A. Cassatella, Andrea Hauser, Henrik E. Mei, Kilian Schober, Silvia Della Bella, Graham Anderson, Michael D. Ward, Garth Cameron, Sebastian Lunemann, Katharina Kriegsmann, Katarzyna M. Sitnik, Brice Gaudilliere, Chantip Dang-Heine, Marcello Pinti, Paul Klenerman, Frank A. Schildberg, Joana Barros-Martins, Laura G. Rico, Hanlin Zhang, Christian Münz, Thomas Dörner, Jakob Zimmermann, Andrea M. Cooper, Jonni S. Moore, Andreas Diefenbach, Yanling Liu, Wolfgang Bauer, Tobit Steinmetz, Katharina Pracht, Leonard Tan, Peter K. Jani, Alan M. Stall, Petra Hoffmann, Christine S. Falk, Jasmin Knopf, Simon Fillatreau, Hans-Dieter Volk, Luis E. Muñoz, David L. Haviland, William W. Agace, Jonathan Rebhahn, Ljiljana Cvetkovic, Mohamed Trebak, Jordi Petriz, Mario Clerici, Diether J. Recktenwald, Anders Ståhlberg, Tristan Holland, Helen M. McGuire, Sa A. Wang, Christian Kukat, Thomas Kroneis, Laura Cook, Wan Ting Kong, Xin M. Wang, Britta Engelhardt, Pierre Coulie, Genny Del Zotto, Sally A. Quataert, Kata Filkor, Gabriele Multhoff, Bartek Rajwa, Federica Calzetti, Hans Minderman, Cosima T. Baldari, Jens Geginat, Hervé Luche, Gert Van Isterdael, Linda Schadt, Sophia Urbanczyk, Giovanna Borsellino, Liping Yu, Dale I. Godfrey, Achille Anselmo, Rachael C. Walker, Andreas Grützkau, David W. Hedley, Birgit Sawitzki, Silvia Piconese, Maria Yazdanbakhsh, Burkhard Becher, Ramon Bellmas Sanz, Michael Delacher, Hyun-Dong Chang, Immanuel Andrä, Hans-Gustaf Ljunggren, José-Enrique O'Connor, Ahad Khalilnezhad, Sharon Sanderson, Federico Colombo, Götz R. A. Ehrhardt, Inga Sandrock, Enrico Lugli, Christian Bogdan, James B. Wing, Susann Müller, Tomohiro Kurosaki, Derek Davies, Ester B. M. Remmerswaal, Kylie M. Quinn, Christopher A. Hunter, Andreas Radbruch, Timothy P. Bushnell, Anna Erdei, Sabine Adam-Klages, Pascale Eede, Van Duc Dang, Rieke Winkelmann, Thomas Korn, Gemma A. Foulds, Dirk Baumjohann, Matthias Schiemann, Manfred Kopf, Jan Kisielow, Lisa Richter, Jochen Huehn, Gloria Martrus, Alexander Scheffold, Jessica G. Borger, Sidonia B G Eckle, John Bellamy Foster, Anna Katharina Simon, Alicia Wong, Mübeccel Akdis, Gisa Tiegs, Toralf Kaiser, James McCluskey, Anna Vittoria Mattioli, Aaron J. Marshall, Hui-Fern Koay, Eva Orlowski-Oliver, Anja E. Hauser, J. Paul Robinson, Jay K. Kolls, Luca Battistini, Mairi McGrath, Jane L. Grogan, Natalio Garbi, Timothy Tree, Kingston H. G. Mills, Stefan H. E. Kaufmann, Wolfgang Schuh, Ryan R. Brinkman, Tim R. Mosmann, Vincenzo Barnaba, Andreas Dolf, Lorenzo Cosmi, Bo Huang, Andreia C. Lino, Baerbel Keller, René A. W. van Lier, Alexandra J. Corbett, Paul S. Frenette, Pleun Hombrink, Helena Radbruch, Sofie Van Gassen, Olivier Lantz, Lorenzo Moretta, Désirée Kunkel, Kirsten A. Ward-Hartstonge, Armin Saalmüller, Leslie Y. T. Leung, Salvador Vento-Asturias, Paola Lanuti, Alicia Martínez-Romero, Sarah Warth, Zhiyong Poon, Diana Dudziak, Andrea Cossarizza, Kovit Pattanapanyasat, Konrad von Volkmann, Jessica P. Houston, Agnès Lehuen, Andrew Filby, Pratip K. Chattopadhyay, Stefano Casola, Annika Wiedemann, Hannes Stockinger, Jürgen Ruland, Arturo Zychlinsky, Claudia Waskow, Katrin Neumann, Ari Waisman, Lucienne Chatenoud, Sudipto Bari, Kamran Ghoreschi, David W. Galbraith, Yvan Saeys, Hamida Hammad, Andrea Gori, Miguel López-Botet, Gabriel Núñez, Sabine Ivison, Michael Hundemer, Dorothea Reimer, Mark C. Dessing, Günter J. Hämmerling, Rudolf A. Manz, Tomas Kalina, Jonas Hahn, Holden T. Maecker, Hendy Kristyanto, Martin S. Davey, Henning Ulrich, Michael L. Dustin, Takashi Saito, Yousuke Takahama, Milena Nasi, Johanna Huber, Jürgen Wienands, Paolo Dellabona, Andreas Schlitzer, Michael D. Leipold, Kerstin H. Mair, Christian Peth, Immo Prinz, Chiara Romagnani, José M. González-Navajas, Josephine Schlosser, Marina Saresella, Matthias Edinger, Dirk Brenner, Nicole Baumgarth, Rikard Holmdahl, Fang-Ping Huang, Guadalupe Herrera, Malte Paulsen, Gergely Toldi, Luka Cicin-Sain, Reiner Schulte, Christina E. Zielinski, Thomas Winkler, Christoph Goettlinger, Philip E. Boulais, Jennie H M Yang, Antonio Celada, Heike Kunze-Schumacher, Julia Tornack, Florian Ingelfinger, Jenny Mjösberg, Andy Riddell, Leonie Wegener, Thomas Höfer, Christoph Hess, James P. Di Santo, Anna E. Oja, J. Kühne, Willem van de Veen, Mary Bebawy, Alberto Mantovani, Bart Everts, Giovanna Lombardi, Laura Maggi, Anouk von Borstel, Pia Kvistborg, Elisabetta Traggiai, A Ochel, Nima Aghaeepour, Charles-Antoine Dutertre, Matthieu Allez, Thomas Höllt, Wenjun Ouyang, Regina Stark, Maries van den Broek, Shimon Sakaguchi, Paul K. Wallace, Silvano Sozzani, Francesca LaRosa, Annette Oxenius, Malgorzata J. Podolska, Ivana Marventano, Wilhelm Gerner, Oliver F. Wirz, Britta Frehse, Gevitha Ravichandran, Martin Herrmann, Carl S. Goodyear, Gary Warnes, Helen Ferry, Stefan Frischbutter, Tim R. Radstake, Salomé LeibundGut-Landmann, Yi Zhao, Axel Schulz, Angela Santoni, Pablo Engel, Daniela C. Hernández, Andreas Acs, Cristiano Scottà, Francesco Annunziato, Thomas Weisenburger, Wolfgang Beisker, Sue Chow, Fritz Melchers, Daniel E. Speiser, Immanuel Kwok, Florent Ginhoux, Dominic A. Boardman, Natalie Stanley, Carsten Watzl, Marie Follo, Erik Lubberts, Andreas Krueger, Susanne Ziegler, Göran K. Hansson, David Voehringer, Antonia Niedobitek, Eleni Christakou, Lai Guan Ng, Sabine Baumgart, Nicholas A Gherardin, Antonio Cosma, Orla Maguire, Jolene Bradford, Daniel Schraivogel, Linda Quatrini, Stephen D. Miller, Rheumatology, Università degli Studi di Modena e Reggio Emilia (UNIMORE), Deutsches Rheuma-ForschungsZentrum (DRFZ), Deutsches Rheuma-ForschungsZentrum, Swiss Institute of Allergy and Asthma Research (SIAF), Universität Zürich [Zürich] = University of Zurich (UZH), Institut de Recherche Saint-Louis - Hématologie Immunologie Oncologie (Département de recherche de l’UFR de médecine, ex- Institut Universitaire Hématologie-IUH) (IRSL), Université de Paris (UP), Ecotaxie, microenvironnement et développement lymphocytaire (EMily (UMR_S_1160 / U1160)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Department of Internal Medicine, Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI)-DENOTHE Center, Institute of Clinical Molecular Biology, Kiel University, Department of Life Sciences [Siena, Italy], Università degli Studi di Siena = University of Siena (UNISI), Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP), Dulbecco Telethon Institute/Department of Biology, Caprotec Bioanalytics GmbH, International Occultation Timing Association European Section (IOTA ES), International Occultation Timing Association European Section, European Molecular Biology Laboratory [Heidelberg] (EMBL), VIB-UGent Center for Inflammation Research [Gand, Belgique] (IRC), VIB [Belgium], Fondazione Santa Lucia (IRCCS), Department of Immunology, Chinese Academy of Medical Sciences, FIRC Institute of Molecular Oncology Foundation, IFOM, Istituto FIRC di Oncologia Molecolare (IFOM), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Physiopatology and Transplantation, University of Milan (DEPT), University of Milan, Monash University [Clayton], Institut des Maladies Emergentes et des Thérapies Innovantes (IMETI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institute of Cellular Pathology, Université Catholique de Louvain = Catholic University of Louvain (UCL), Lymphopoïèse (Lymphopoïèse (UMR_1223 / U1223 / U-Pasteur_4)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Experimental Immunology Unit, Dept. of Oncology, DIBIT San Raffaele Scientific Institute, Immunité Innée - Innate Immunity, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Department of Biopharmacy [Bruxelles, Belgium] (Institute for Medical Immunology IMI), Université libre de Bruxelles (ULB), Charité Hospital, Humboldt-Universität zu Berlin, Agency for science, technology and research [Singapore] (A*STAR), Laboratory of Molecular Immunology and the Howard Hughes Institute, Rockefeller University [New York], Kennedy Institute of Rheumatology [Oxford, UK], Imperial College London, Theodor Kocher Institute, University of Bern, Leibniz Research Institute for Environmental Medicine [Düsseldorf, Germany] ( IUF), Université Lumière - Lyon 2 (UL2), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), University of Edinburgh, Integrative Biology Program [Milano], Istituto Nazionale Genetica Molecolare [Milano] (INGM), Singapore Immunology Network (SIgN), Biomedical Sciences Institute (BMSI), Universitat de Barcelona (UB), Rheumatologie, Cell Biology, Department of medicine [Stockholm], Karolinska Institutet [Stockholm]-Karolinska University Hospital [Stockholm], Department for Internal Medicine 3, Institute for Clinical Immunology, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Delft University of Technology (TU Delft), Medical Inflammation Research, Karolinska Institutet [Stockholm], Department of Photonics Engineering [Lyngby], Technical University of Denmark [Lyngby] (DTU), Dpt of Experimental Immunology [Braunschweig], Helmholtz Centre for Infection Research (HZI), Department of Internal Medicine V, Universität Heidelberg [Heidelberg], Department of Histology and Embryology, University of Rijeka, Freiburg University Medical Center, Nuffield Dept of Clinical Medicine, University of Oxford [Oxford]-NIHR Biomedical Research Centre, Institute of Integrative Biology, Molecular Biomedicine, Berlin Institute of Health (BIH), Laboratory for Lymphocyte Differentiation, RIKEN Research Center, Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Immunité et cancer (U932), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Department of Surgery [Vancouver, BC, Canada] (Child and Family Research Institute), University of British Columbia (UBC)-Child and Family Research Institute [Vancouver, BC, Canada], College of Food Science and Technology [Shangai], Shanghai Ocean University, Institute for Medical Microbiology and Hygiene, University of Marburg, King‘s College London, Erasmus University Medical Center [Rotterdam] (Erasmus MC), Centre d'Immunophénomique (CIPHE), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Brustzentrum Kantonsspital St. Gallen, Immunotechnology Section, Vaccine Research Center, National Institutes of Health [Bethesda] (NIH)-National Institute of Allergy and Infectious Diseases, Heinrich Pette Institute [Hamburg], Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI), Department of Immunology and Cell Biology, Mario Negri Institute, Laboratory of Molecular Medicine and Biotechnology, Don C. Gnocchi ONLUS Foundation, Institute of Translational Medicine, Klinik für Dermatologie, Venerologie und Allergologie, School of Biochemistry and Immunology, Department of Medicine Huddinge, Karolinska Institutet [Stockholm]-Karolinska University Hospital [Stockholm]-Lipid Laboratory, Università di Genova, Dipartimento di Medicina Sperimentale, Department of Environmental Microbiology, Helmholtz Zentrum für Umweltforschung = Helmholtz Centre for Environmental Research (UFZ), Department of Radiation Oncology [Munich], Ludwig-Maximilians-Universität München (LMU), Centre de Recherche Publique- Santé, Université du Luxembourg (Uni.lu), William Harvey Research Institute, Barts and the London Medical School, University of Michigan [Ann Arbor], University of Michigan System, Centro de Investigacion del Cancer (CSIC), Universitario de Salamanca, Molecular Pathology [Tartu, Estonia], University of Tartu, Hannover Medical School [Hannover] (MHH), Centre d'Immunologie de Marseille - Luminy (CIML), Monash Biomedicine Discovery Institute, Cytometry Laboratories and School of Veterinary Medicine, Purdue University [West Lafayette], Data Mining and Modelling for Biomedicine [Ghent, Belgium], VIB Center for Inflammation Research [Ghent, Belgium], Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, RIKEN Research Center for Allergy and Immunology, Osaka University [Osaka], Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Institute of Medical Immunology [Berlin, Germany], FACS and Array Core Facility, Johannes Gutenberg - Universität Mainz (JGU), Otto-von-Guericke University [Magdeburg] (OVGU), SUPA School of Physics and Astronomy [University of St Andrews], University of St Andrews [Scotland]-Scottish Universities Physics Alliance (SUPA), Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), General Pathology and Immunology (GPI), University of Brescia, Université de Lausanne (UNIL), Terry Fox Laboratory, BC Cancer Agency (BCCRC)-British Columbia Cancer Agency Research Centre, Department of Molecular Immunology, Medizinische Universität Wien = Medical University of Vienna, Dept. Pediatric Cardiology, Universität Leipzig [Leipzig], Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (UKE), Center for Cardiovascular Sciences, Albany Medical College, Dept Pathol, Div Immunol, University of Cambridge [UK] (CAM), Department of Information Technology [Gent], Universiteit Gent, Department of Plant Systems Biology, Department of Plant Biotechnology and Genetics, Universiteit Gent = Ghent University [Belgium] (UGENT), Division of Molecular Immunology, Institute for Immunology, Department of Geological Sciences, University of Oregon [Eugene], Centers for Disease Control and Prevention [Atlanta] (CDC), Centers for Disease Control and Prevention, University of Colorado [Colorado Springs] (UCCS), FACS laboratory, Cancer Research, London, Cancer Research UK, Regeneration in Hematopoiesis and Animal Models of Hematopoiesis, Faculty of Medicine, Dresden University of Technology, Barbara Davis Center for Childhood Diabetes (BDC), University of Colorado Anschutz [Aurora], School of Computer and Electronic Information [Guangxi University], Guangxi University [Nanning], School of Materials Science and Engineering, Nanyang Technological University [Singapour], Max Planck Institute for Infection Biology (MPIIB), Max-Planck-Gesellschaft, Work in the laboratory of Dieter Adam is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 125440785 – SFB 877, Project B2.Petra Hoffmann, Andrea Hauser, and Matthias Edinger thank BD Biosciences®, San José, CA, USA, and SKAN AG, Bale, Switzerland for fruitful cooperation during the development, construction, and installation of the GMP‐compliant cell sorting equipment and the Bavarian Immune Therapy Network (BayImmuNet) for financial support.Edwin van der Pol and Paola Lanuti acknowledge Aleksandra Gąsecka M.D. for excellent experimental support and Dr. Rienk Nieuwland for textual suggestions. This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO‐TTW), research program VENI 15924.Jessica G Borger, Kylie M Quinn, Mairi McGrath, and Regina Stark thank Francesco Siracusa and Patrick Maschmeyer for providing data.Larissa Nogueira Almeida was supported by DFG research grant MA 2273/14‐1. Rudolf A. Manz was supported by the Excellence Cluster 'Inflammation at Interfaces' (EXC 306/2).Susanne Hartmann and Friederike Ebner were supported by the German Research Foundation (GRK 2046).Hans Minderman was supported by NIH R50CA211108.This work was funded by the Deutsche Forschungsgemeinschaft through the grant TRR130 (project P11 and C03) to Thomas H. Winkler.Ramon Bellmàs Sanz, Jenny Kühne, and Christine S. Falk thank Jana Keil and Kerstin Daemen for excellent technical support. The work was funded by the Germany Research Foundation CRC738/B3 (CSF).The work by the Mei laboratory was supported by German Research Foundation Grant ME 3644/5‐1 and TRR130 TP24, the German Rheumatism Research Centre Berlin, European Union Innovative Medicines Initiative ‐ Joint Undertaking ‐ RTCure Grant Agreement 777357, the Else Kröner‐Fresenius‐Foundation, German Federal Ministry of Education and Research e:Med sysINFLAME Program Grant 01ZX1306B and KMU‐innovativ 'InnoCyt', and the Leibniz Science Campus for Chronic Inflammation (http://www.chronische-entzuendung.org).Axel Ronald Schulz, Antonio Cosma, Sabine Baumgart, Brice Gaudilliere, Helen M. McGuire, and Henrik E. Mei thank Michael D. Leipold for critically reading the manuscript.Christian Kukat acknowledges support from the ISAC SRL Emerging Leaders program.John Trowsdale received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant Agreement 695551)., European Project: 7728036(1978), Università degli Studi di Modena e Reggio Emilia = University of Modena and Reggio Emilia (UNIMORE), Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Università degli Studi di Firenze = University of Florence (UniFI)-DENOTHE Center, Università degli Studi di Milano = University of Milan (UNIMI), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Humboldt University Of Berlin, Leibniz Research Institute for Environmental Medicine [Düsseldorf, Germany] (IUF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Danmarks Tekniske Universitet = Technical University of Denmark (DTU), Universität Heidelberg [Heidelberg] = Heidelberg University, Universitäts Klinikum Freiburg = University Medical Center Freiburg (Uniklinik), University of Oxford-NIHR Biomedical Research Centre, Universität Bonn = University of Bonn, Università degli Studi di Firenze = University of Florence (UniFI), Università degli studi di Genova = University of Genoa (UniGe), Universidad de Salamanca, Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Otto-von-Guericke-Universität Magdeburg = Otto-von-Guericke University [Magdeburg] (OVGU), Université de Lausanne = University of Lausanne (UNIL), Universität Leipzig, Universiteit Gent = Ghent University (UGENT), HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany., Cossarizza, A., Chang, H. -D., Radbruch, A., Acs, A., Adam, D., Adam-Klages, S., Agace, W. W., Aghaeepour, N., Akdis, M., Allez, M., Almeida, L. N., Alvisi, G., Anderson, G., Andra, I., Annunziato, F., Anselmo, A., Bacher, P., Baldari, C. T., Bari, S., Barnaba, V., Barros-Martins, J., Battistini, L., Bauer, W., Baumgart, S., Baumgarth, N., Baumjohann, D., Baying, B., Bebawy, M., Becher, B., Beisker, W., Benes, V., Beyaert, R., Blanco, A., Boardman, D. A., Bogdan, C., Borger, J. G., Borsellino, G., Boulais, P. E., Bradford, J. A., Brenner, D., Brinkman, R. R., Brooks, A. E. S., Busch, D. H., Buscher, M., Bushnell, T. P., Calzetti, F., Cameron, G., Cammarata, I., Cao, X., Cardell, S. L., Casola, S., Cassatella, M. A., Cavani, A., Celada, A., Chatenoud, L., Chattopadhyay, P. K., Chow, S., Christakou, E., Cicin-Sain, L., Clerici, M., Colombo, F. S., Cook, L., Cooke, A., Cooper, A. M., Corbett, A. J., Cosma, A., Cosmi, L., Coulie, P. G., Cumano, A., Cvetkovic, L., Dang, V. D., Dang-Heine, C., Davey, M. S., Davies, D., De Biasi, S., Del Zotto, G., Dela Cruz, G. V., Delacher, M., Della Bella, S., Dellabona, P., Deniz, G., Dessing, M., Di Santo, J. P., Diefenbach, A., Dieli, F., Dolf, A., Dorner, T., Dress, R. J., Dudziak, D., Dustin, M., Dutertre, C. -A., Ebner, F., Eckle, S. B. G., Edinger, M., Eede, P., Ehrhardt, G. R. A., Eich, M., Engel, P., Engelhardt, B., Erdei, A., Esser, C., Everts, B., Evrard, M., Falk, C. S., Fehniger, T. A., Felipo-Benavent, M., Ferry, H., Feuerer, M., Filby, A., Filkor, K., Fillatreau, S., Follo, M., Forster, I., Foster, J., Foulds, G. A., Frehse, B., Frenette, P. S., Frischbutter, S., Fritzsche, W., Galbraith, D. W., Gangaev, A., Garbi, N., Gaudilliere, B., Gazzinelli, R. T., Geginat, J., Gerner, W., Gherardin, N. A., Ghoreschi, K., Gibellini, L., Ginhoux, F., Goda, K., Godfrey, D. I., Goettlinger, C., Gonzalez-Navajas, J. M., Goodyear, C. S., Gori, A., Grogan, J. L., Grummitt, D., Grutzkau, A., Haftmann, C., Hahn, J., Hammad, H., Hammerling, G., Hansmann, L., Hansson, G., Harpur, C. M., Hartmann, S., Hauser, A., Hauser, A. E., Haviland, D. L., Hedley, D., Hernandez, D. C., Herrera, G., Herrmann, M., Hess, C., Hofer, T., Hoffmann, P., Hogquist, K., Holland, T., Hollt, T., Holmdahl, R., Hombrink, P., Houston, J. P., Hoyer, B. F., Huang, B., Huang, F. -P., Huber, J. E., Huehn, J., Hundemer, M., Hunter, C. A., Hwang, W. Y. K., Iannone, A., Ingelfinger, F., Ivison, S. M., Jack, H. -M., Jani, P. K., Javega, B., Jonjic, S., Kaiser, T., Kalina, T., Kamradt, T., Kaufmann, S. H. E., Keller, B., Ketelaars, S. L. C., Khalilnezhad, A., Khan, S., Kisielow, J., Klenerman, P., Knopf, J., Koay, H. -F., Kobow, K., Kolls, J. K., Kong, W. T., Kopf, M., Korn, T., Kriegsmann, K., Kristyanto, H., Kroneis, T., Krueger, A., Kuhne, J., Kukat, C., Kunkel, D., Kunze-Schumacher, H., Kurosaki, T., Kurts, C., Kvistborg, P., Kwok, I., Landry, J., Lantz, O., Lanuti, P., Larosa, F., Lehuen, A., LeibundGut-Landmann, S., Leipold, M. D., Leung, L. Y. T., Levings, M. K., Lino, A. C., Liotta, F., Litwin, V., Liu, Y., Ljunggren, H. -G., Lohoff, M., Lombardi, G., Lopez, L., Lopez-Botet, M., Lovett-Racke, A. E., Lubberts, E., Luche, H., Ludewig, B., Lugli, E., Lunemann, S., Maecker, H. T., Maggi, L., Maguire, O., Mair, F., Mair, K. H., Mantovani, A., Manz, R. A., Marshall, A. J., Martinez-Romero, A., Martrus, G., Marventano, I., Maslinski, W., Matarese, G., Mattioli, A. V., Maueroder, C., Mazzoni, A., Mccluskey, J., Mcgrath, M., Mcguire, H. M., Mcinnes, I. B., Mei, H. E., Melchers, F., Melzer, S., Mielenz, D., Miller, S. D., Mills, K. H. G., Minderman, H., Mjosberg, J., Moore, J., Moran, B., Moretta, L., Mosmann, T. R., Muller, S., Multhoff, G., Munoz, L. E., Munz, C., Nakayama, T., Nasi, M., Neumann, K., Ng, L. G., Niedobitek, A., Nourshargh, S., Nunez, G., O'Connor, J. -E., Ochel, A., Oja, A., Ordonez, D., Orfao, A., Orlowski-Oliver, E., Ouyang, W., Oxenius, A., Palankar, R., Panse, I., Pattanapanyasat, K., Paulsen, M., Pavlinic, D., Penter, L., Peterson, P., Peth, C., Petriz, J., Piancone, F., Pickl, W. F., Piconese, S., Pinti, M., Pockley, A. G., Podolska, M. J., Poon, Z., Pracht, K., Prinz, I., Pucillo, C. E. M., Quataert, S. A., Quatrini, L., Quinn, K. M., Radbruch, H., Radstake, T. R. D. J., Rahmig, S., Rahn, H. -P., Rajwa, B., Ravichandran, G., Raz, Y., Rebhahn, J. A., Recktenwald, D., Reimer, D., Reis e Sousa, C., Remmerswaal, E. B. M., Richter, L., Rico, L. G., Riddell, A., Rieger, A. M., Robinson, J. P., Romagnani, C., Rubartelli, A., Ruland, J., Saalmuller, A., Saeys, Y., Saito, T., Sakaguchi, S., Sala-de-Oyanguren, F., Samstag, Y., Sanderson, S., Sandrock, I., Santoni, A., Sanz, R. B., Saresella, M., Sautes-Fridman, C., Sawitzki, B., Schadt, L., Scheffold, A., Scherer, H. U., Schiemann, M., Schildberg, F. A., Schimisky, E., Schlitzer, A., Schlosser, J., Schmid, S., Schmitt, S., Schober, K., Schraivogel, D., Schuh, W., Schuler, T., Schulte, R., Schulz, A. R., Schulz, S. R., Scotta, C., Scott-Algara, D., Sester, D. P., Shankey, T. V., Silva-Santos, B., Simon, A. K., Sitnik, K. M., Sozzani, S., Speiser, D. E., Spidlen, J., Stahlberg, A., Stall, A. M., Stanley, N., Stark, R., Stehle, C., Steinmetz, T., Stockinger, H., Takahama, Y., Takeda, K., Tan, L., Tarnok, A., Tiegs, G., Toldi, G., Tornack, J., Traggiai, E., Trebak, M., Tree, T. I. M., Trotter, J., Trowsdale, J., Tsoumakidou, M., Ulrich, H., Urbanczyk, S., van de Veen, W., van den Broek, M., van der Pol, E., Van Gassen, S., Van Isterdael, G., van Lier, R. A. W., Veldhoen, M., Vento-Asturias, S., Vieira, P., Voehringer, D., Volk, H. -D., von Borstel, A., von Volkmann, K., Waisman, A., Walker, R. V., Wallace, P. K., Wang, S. A., Wang, X. M., Ward, M. D., Ward-Hartstonge, K. A., Warnatz, K., Warnes, G., Warth, S., Waskow, C., Watson, J. V., Watzl, C., Wegener, L., Weisenburger, T., Wiedemann, A., Wienands, J., Wilharm, A., Wilkinson, R. J., Willimsky, G., Wing, J. B., Winkelmann, R., Winkler, T. H., Wirz, O. F., Wong, A., Wurst, P., Yang, J. H. M., Yang, J., Yazdanbakhsh, M., Yu, L., Yue, A., Zhang, H., Zhao, Y., Ziegler, S. M., Zielinski, C., Zimmermann, J., Zychlinsky, A., UCL - SSS/DDUV - Institut de Duve, UCL - SSS/DDUV/GECE - Génétique cellulaire, Netherlands Organization for Scientific Research, German Research Foundation, European Commission, European Research Council, Repositório da Universidade de Lisboa, CCA - Imaging and biomarkers, Experimental Immunology, AII - Infectious diseases, AII - Inflammatory diseases, Biomedical Engineering and Physics, ACS - Atherosclerosis & ischemic syndromes, and Landsteiner Laboratory

Subjects
0301 basic medicine, Consensus, Immunology, Consensu, Cell Separation, Biology, Article, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, Guidelines, Allergy and Immunology, medicine, Cell separation, Immunology and Allergy, Humans, guidelines, flow cytometry, immunology, medicine.diagnostic_test, BIOMEDICINE AND HEALTHCARE. Basic Medical Sciences, Cell sorting, Flow Cytometry, Cell selection, Data science, 3. Good health, 030104 developmental biology, Phenotype, [SDV.IMM]Life Sciences [q-bio]/Immunology, BIOMEDICINA I ZDRAVSTVO. Temeljne medicinske znanosti, 030215 immunology, Human
Abstract

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Published
2019
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50. Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation

Author

Sarah A. Kazzaz, Jazmin M. Stenson, Mohamed Trebak, Youjun Wang, Robert M. Nwokonko, Yandong Zhou, James H. Baraniak, Abigale L. Whitsell, Donald L. Gill, and Michelle R. Jennette

Subjects
Boron Compounds, Models, Molecular, 0301 basic medicine, ORAI1 Protein, Transcription, Genetic, QH301-705.5, Peptide, Endoplasmic Reticulum, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Cytosol, 0302 clinical medicine, Leucine, Transcriptional regulation, Humans, Amino Acid Sequence, Calcium Signaling, Stromal Interaction Molecule 1, Biology (General), chemistry.chemical_classification, Binding Sites, NFATC Transcription Factors, Endoplasmic reticulum, Cell Membrane, STIM1, NFAT, Coupling (electronics), Transmembrane domain, HEK293 Cells, 030104 developmental biology, chemistry, Cytoplasm, Mutation, Biophysics, Calcium Channels, Protein Multimerization, Peptides, Ion Channel Gating, 030217 neurology & neurosurgery, HeLa Cells, Protein Binding
Abstract

Summary: Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals.

Published
2021

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