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Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca2+ signals of low amplitude. Subsequently, also global Ca2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic. Upon stimulation of receptors, NAADP is formed as calcium-mobilizing second messenger in many cells. Here, the authors develop MASTER-NAADP, a membrane-permeant precursor of NAADP, and characterize its biological activity. [ABSTRACT FROM AUTHOR]

CD8+ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca2+ microdomains in CD4+ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8+ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca2+ microdomains, global Ca2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8+ T cells lacking P2X4 and P2X7 channels, global Ca2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca2+ concentration ([Ca2+]i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8+ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8+ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca2+ events during CD8+ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

Ca2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatiotemporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community. [ABSTRACT FROM AUTHOR]

An origin story for NAADP in T cells: Early steps in T cell activation are mediated by the synthesis of Ca2+-mobilizing second messenger NAADP, which is produced through oxidation of NAADPH by a previously unknown enzyme. Gu et al. identified NAADPH-oxidizing enzymes that were critical for the early phases of T cell activation. Stimulation of T cell receptors initially results in rapid production of NAADP that induces the formation of localized Ca2+ microdomains that eventually lead to more global and sustained intracellular Ca2+ signaling. In cultured rat T cells, knockout of DUOX2 reduced local Ca2+ microdomain formation, whereas functional knockout of both DUOX1 and DUOX2 in murine T cells suppressed global intracellular Ca2+ signaling. The findings identify a critical pair of enzymes in T cell activation. The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction—NAADP to NAADPH—is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate. [ABSTRACT FROM AUTHOR]

Connecting Ca2+ channels with NAADP: Although generation of the second messenger NAADP stimulates the release of Ca2+ from intracellular stores, binding sites for NAADP have not been characterized on NAADP-sensitive ion channels. Two papers independently identified an NAADP-binding protein called HN1L, which is also known as JPT2, that interacts with ryanodine receptors in the endoplasmic reticulum in T cells and two-pore channels (TPCs) in endosomes and lysosomes. Roggenkamp et al. found that HN1L deletion suppressed the formation of Ca2+ microdomains in stimulated Jurkat and primary rat T cells, one of the earliest responses to T cell receptor activation, thereby reducing global Ca2+ signaling. Gunaratne et al. found that knockdown of JPT2 attenuated NAADP-evoked Ca2+ signals from endosomes and lysosomes and the ability of a SARS-CoV-2 pseudocoronavirus to infect cells, a process that depends on TPC activity. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through ryanodine receptors and from endosomes and lysosomes through TPCs. NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1–like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3–dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR. [ABSTRACT FROM AUTHOR]

Local, sub-second Ca 2+ signals, termed Ca 2+ microdomains, are highly dynamic and short-lived Ca 2+ signals, which result in a global [Ca 2+ ]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca 2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca 2+ ]i. To capture these fast and dynamically occurring Ca 2+ signals, we developed a high-resolution imaging technique using a combination of two Ca 2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca 2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca 2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

CD8 + T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca 2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca 2+ microdomains in CD4 + T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8 + T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca 2+ microdomains, global Ca 2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca 2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8 + T cells lacking P2X4 and P2X7 channels, global Ca 2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca 2+ concentration ([Ca 2+ ] i ). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8 + T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8 + T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca 2+ events during CD8 + T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Brock, Lory, Möckl, Birus, Stähler, Woelk, Jaeckstein, Heeren, Koch-Nolte, Rissiek, Mittrücker, Guse, Werner and Diercks.)

Ca 2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca 2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca 2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca 2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca 2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatio-temporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca 2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community. Code available at: 10.5281/zenodo.10459243., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GD declared a past co-authorship with one of the authors B-PD, AG to the handling editor., (Copyright © 2024 Woelk, Kovacevic, Husseini, Förster, Gerlach, Möckl, Altfeld, Guse, Diercks and Werner.)