The [Ca2+]i was measured using the ratiometric fluoroprobe FURA-2 AM. We recorded two groups of epithelial cells with distinct responses to caspofungin exposure, which demonstrated either a rapid transient rise in [Ca2+]i or a sustained elevation of [Ca2+]i. Both patterns of Ca2+ kinetics were still observed when an influx of transmembraneous Ca2+ ions was pharmacologically inhibited. Furthermore, in extracellular buffer solutions without Ca2+ ions, caspofungin exposure still evoked this characteristic rise in [Ca2+]i. To shed light on the origin of the Ca2+ ions responsible for the elevation in [Ca2+]i we investigated the possible intracellular storage of Ca2+ ions. The depletion of mitochondrial Ca2+ stores using 25 µM 2,4-dinitrophenol (DNP) did not prevent the caspofungin-induced rise in [Ca2+]i, which was rapid and transient. However, the application of caffeine (30 mM) to discharge Ca2+ ions that were presumably stored in the endoplasmic reticulum (ER) prior to caspofungin exposure completely inhibited the caspofungin-induced changes in [Ca2+]i levels. When the ER-bound IP3 receptors were blocked by 2-APB (40 µM), we observed a delayed transient rise in [Ca2+]i as a response to the caspofungin. Inhibition of the ryanodine receptors (RyR) using 40 µM ryanodine completely prevented the caspofungin-induced elevation of [Ca2+]i. In summary, caspofungin has been shown to trigger an increase in [Ca2+]i independent from extracellular Ca2+ ions by liberating the Ca2+ ions stored in the ER, mainly via a RyR pathway.The antimycotic drug caspofungin is known to alter the cell function of cardiomyocytes and the cilia-bearing cells of the tracheal epithelium. The objective of this study was to investigate the homeostasis of intracellular Ca Under basal conditions, cilia beat continuously without external stimulation but can beat faster when necessary. This elevated beating frequency depends on several interdepending signal transduction cascades, including changes in intracellular concentrations of Ca2+ ions ([Ca2+]i)6. However, most signal cascades in these cells depend on at least a transient [Ca2+]i to be activated in order to allow the cilia to beat faster for prolonged periods8. Thus, the regulation of [Ca2+]i is a cornerstone for many cellular signal cascades and cell functions. In epithelial cells in the airway, different canonical Ca2+ stores or Ca2+ influx pathways are involved in the regulation of [Ca2+]i. The known intracellular stores are the endoplasmic reticulum (ER) and the mitochondria. The ER can reduce [Ca2+]i via activation of the sarcoplasmic ATPase (SERCA)-carrying Ca2+ ions into the ER. The release of Ca2+ ions from the ER is mainly orchestrated by activating the ryanodine receptors (RyR) or IP3 receptors to enhance [Ca2+]i. There may also be alternative pathways that lead to Ca2+ leakage from the ER; however, these are still unknown. Mitochondria can buffer Ca2+ ions when [Ca2+]i exceeds a threshold of 500 nM and they slowly release Ca2+ into the cytosol when [Ca2+]i falls below the aforementioned threshold9. It is still not known whether further intracellular Ca2+ stores such as lysosomes contribute to the regulation of [Ca2+]i.The mucociliary clearance of lower airways contributes to the removal of debris and pathogens that are trapped in the upper mucus layer from the lower airways. This complex mechanism is driven by cilia-bearing cells of the bronchiolar and tracheal epithelium. The kinocilia of these cells bear over 200 motor proteins as a propelling apparatus, including dynein, the ATP hydrolyzing enzyme2+ channels are known as Ca2+ influx pathways, which include transient receptor potential (TRP) channels, store-operated calcium (SOC) channels or voltage-dependent calcium channels that can all enhance [Ca2+]i. Consequently, any disturbance or rise in [Ca2+]i in epithelial airway cells can be achieved by the activation of different Ca2+ pathways or by liberating them from internal stores. Elevated [Ca2+]i directly contribute to altered cell function, e.g., mucociliary clearance or mucus secretion, which can either lead to colonization of the lower airways by pathogens or can lead to improved clearance rates. When treating mycotic infections in critically ill patients, echinocandines represent the established first-line therapy of antimycotic substances including caspofungin. Caspofungin is used under clinical conditions, with a high capability to treat systemic or local Candida spp. infections. In healthy men, a mean maximum serum concentration Cmax = 9.1–11 µM was achieved after a loading dose of 70 mg caspofungin10.Different plasma membrane-bound Ca12. This distribution pattern depends on the physiology of specific organs13. In critically ill patients and in perfused isolated rat hearts, we have previously reported that caspofungin has severe effects on cardio-circulatory function16. Caused by impaired contractility of cardiomyocytes as a result of changes in [Ca2+]i levels17. It is therefore of interest to determine whether caspofungin may also interact with the homeostasis of [Ca2+]i in airway epithelial cells. [Ca2+]i is part of many cellular signal cascades that are a precondition to the altering of cell functions such as the beat rate of cilia. In order to find out, we investigated whether caspofungin also changes the [Ca2+]i in isolated human tracheal epithelial cells (HTEpC) and tried to elucidate the underlying regulatory mechanism.In order to successfully treat mycotic infections in different organs, caspofungin has to reach therapeutic concentrations in many tissues or regions including the liver and the lower airways of the lungs that exceed plasma concentrations2+]i using the fluoroprobe FURA-2 AM. To determine the amount of Ca2+ ions in the ER, we used Mag-Fluo-4 AM. Pharmacological approaches were used to identify signal pathways that are involved in the regulation of [Ca2+]i. The data gathered by this study should expand our knowledge of the interactions of this antifungal drug in terms of the regulation of [Ca2+]i in airway epithelial cells, which can be considered to be part of the immune system.We measured ER) within the ER, the cells were loaded with 5 µM of the low-affinity calcium indicator Mag-Fluo-4 AM  in darkness at 37 °C for 60 min. Afterward, the cells were rinsed in HEPES buffer for 20 min and were then transferred into the recording chamber of the fluorescence microscope equipped with 20 × immersion lenses , which also contains 2 ml HEPES. Experiments were performed in both Ca2+-containing and Ca2+-free HEPES buffer (for contents see below). As single wavelength indicators are difficult to calibrate, we used normalized Mag-Fluo-4 signals (F/Fo) to express changes in the luminal [Ca2+]ER. Pharmacological agents were applied with a blunt pipette into the recording chamber. The Mag-Fluo-4 was then excited at 490 nm (± 10 nm) and the emitted fluorescence was directed through a dichromatic shortpass filter of 560 nm to a bandpass filter of 510 nm.For calcium imaging in the ER, HTE cells were seeded on laminin-coated coverslips. To assess the intraluminal Ca2O2) in collected cell culture supernatants by using the fluorometric Hydrogen Peroxide Assay Kit . HTE cells were seeded on laminin-coated coverslips and were incubated with 60, 90 or 120 µM caspofungin (CAS) in 1 ml HEPES solution for 30, 60 and 90 min. Additionally, we incubated cells without adding CAS as a control. The cell supernatant (250 µl) was collected and centrifuged at 1,200 G for 15 min to remove all cell particles. To remove the proteins, the sample was then filtered through a 10 kDa MW spin filter . Samples were subsequently prepared in duplicate as follows. 50 µl of the samples were mixed with 50 µl Reaction Mix  in a 96-well plate  and were incubated for 10 min. A H2O2 standard was also established in duplicate with 0, 0.1, 0.2, 0.3, 0.4, or 0.5 nm/well. Fluorescence was measured (Ex/Em = 535/587 nm) in a microplate reader . To calculate the H2O2 quantity, a H2O2 standard curve was plotted after background subtraction using the equation:As an indicator of reactive oxygen species (ROS), we measured hydrogen peroxide , NiCl2 . Caffeine  was sufficient to completely deplete ER Ca2+ stores in these cells. 2,4-dinitrophenol  was used to deplete mitochondrial Ca2+ stores by depolarization of mitochondrial membrane potential, 2-Aminoethoxydiphenylborane ; a high ryanodine concentration  was used to inhibit Ca2+ liberation from ER Ca2+ stores. Ryanodine, 2-APB, and DNP were diluted in Dimethylsulfoxide (DMSO), and end concentrations were achieved by applying the stock solution directly to the buffer solution in the recording chamber. The DMSO did not exceed volumes of 20 µl in 2 ml HEPES in order to prevent the induction of unspecific disturbances of the cilia-bearing cells.The following drugs were administered: caspofungin . A vitality test was performed at the end of each recording with KCl solutions (200 mM). Solutions containing 200 mM KCl also contained 2.5 mM CaCl2.All preparations and experiments were carried out in HEPES solution consisting of 20 mM HEPES, 4.5 mM KCl, 2.5 mM CaClA Mann–Whitney U test was used to compare equivalent measuring points from different experiments, and a Wilcoxon rank-sum test was used to compare dependent variables. Statistical data evaluation and testing were performed using the GraphPad PRISM (Version 5.04) software .2+]i was expressed as changes in the FURA-2-fluorescence ratio. For the controls, isolated tracheal epithelial cells were rested in Ca2+-containing or in Ca2+-free buffer solutions for more than 800 s. In the cells resting in Ca2+-containing buffer, the FURA-2-fluorescence ratio did not change significantly during this period . At the end of all the experiments, we applied a brief pulse of KCl (200 mM), and only the cells that responded to this vitality test by showing a significant increase in the FURA-2-fluorescence ratio were included for further statistical evaluation . The first rapid increasing Ca2+ transient returned to the baseline, while the second one showed a rapid increase in [Ca2+]i which did not return to the baseline during the remaining exposure period  in Ca2+]i, we applied caspofungin to tracheal epithelial cells in Ca2+-free solutions. Exposure to caspofungin in the Ca2+-free solution evoked a rise in [Ca2+]i in all of the investigated cells (n = 30) which was biphasic, and which remained above the baseline level until the end of the experimental observation period  than the peak transients observed in the Ca2+-containing buffer solutions  was used in Ca2+-free buffer solutions to deplete the mitochondrial Ca2+ stores. This uncoupling of the respiratory chain induces a complete depolarization of the mitochondrial membrane potential eliciting an efflux of Ca2+ ions from these organelles.In order to determine the source of the Ca2+ efflux from the mitochondria was visible by brief Ca2+ transients (increase in FURA-2 ratio) that almost immediately returned to baseline levels , a reversible inhibitor of SERCA. In a Ca2+-free buffer solution, CPA induced a prolonged increase in [Ca2+]i that slowly returned to baseline levels  led to a rapid but transient increase in [Ca2+]i. The subsequent application of CPA (10 µM) had no further effect, demonstrating that caffeine had already depleted the ER Ca2+ stores, which are identical to CPA-sensitive stores  had no effect on any tracheal epithelial cells investigated.In further experiments, exposing the cells to caffeine led to a brief Ca2+]i in the presence of caspofungin . However, Ca2+ transients were significantly reduced vs. Ca2+ transients induced by caspofungin in Ca2+-free buffer solutions  was applied to Ca2+-free extracellular solutions in order to inhibit inositol-1,4,5-triphosphate (IP3) receptors prior to caspofungin application. 2-APB had no effect on the FURA-2 fluorescence ratio indicating transients of [Ca2+]i  compared to Ca2+ transients induced by caspofungin alone . At the end of each experiment, Ca2+ transients were still elicited by applying KCl, demonstrating the vitality of the cells . The stores were replenished after subsequent exposure to a buffer solution containing depolarizing KCl (200 mM) and Ca2+ (2.5 mM), inducing a maximum rise in Mag-Fluo-4 fluorescence  using a fluorescence dye. The results showed no significant ROS generation in the control experiment or when the tracheae were exposed to caspofungin for 30 min  is poor23. Even when the meninges are inflamed, the concentrations of caspofungin in the CNS are far below serum concentrations24. In contrast, caspofungin concentrations are highest in the liver, lungs, kidney, and spleen, where caspofungin can deploy its antifungal properties13. In the lungs, the concentration of caspofungin leads to favorable response rates of up to 62% in neutropenic patients suffering from pulmonary invasive fungal disease25.Caspofungin inhibits the synthesis of 1,3-β-D-glucan, which is an essential component of the fungal cell wallCandida species. However, caspofungin, a membrane-anchored cyclic lipopeptide, is also able to penetrate mammalian cells and can reach high concentrations within the tracheal epithelium allowing it to act upon cilia-bearing cells. We can also conclude that caspofungin concentrations are also high in the epithelial lining fluid, and it may act from the luminal side onto epithelial cells.In mammals, caspofungin mainly acts extracellularly against most Candida spp., it is able to diffuse via the epithelium into the airway lumen so that it can reach these therapeutic concentrations. We found evidence that caspofungin can reach intracellular concentrations in isolated lower airway cells that are high enough to liberate Ca2+ ions from cell organelles. So far, the effects of caspofungin on intracellular organelles have only been described for a few cell types. In stimulated mammalian macrophages, caspofungin has been shown to reach concentrations that will have an effect on trapped Candida glabrata26. Recently, we described a change in the contractility of isolated cardiomyocytes via the liberation of Ca2+ ions from internal Ca2+ stores17. These data support our present findings and lead us to postulate that the diffusion of caspofungin into mammalian cells is not restricted to particular cell types; it seems rather a general characteristic of this antimycotic substance.Since caspofungin is used to treat pulmonary infections caused by 2+ ions from Ca2+ stores in the ER, mainly via a caffeine/ryanodine-sensitive pathway. We found no evidence for the liberation of Ca2+ ions stored in mitochondria, although these organelles do store Ca2+ ions under resting conditions  that refills the ER’s Ca2+ stores. Therefore, the interrupted mitochondrial ATP synthesis after exposure to DNP reduces SERCA activity, which then leads to a slow Ca2+ leakage from the ER stores via different pathways30. Eventually, this results in a slightly reduced Ca2+ response during subsequent caspofungin application, which was what was observed here. Our observations provide evidence that the mitochondria in tracheal epithelial cells store Ca2+ ions under resting conditions but are not affected by caspofungin.When the mitochondrial Casee Fig. . This sm2+ ions into the cytosol from the ER stores is balanced on the one hand by RyR, IP3, or alternative leaks that drive the efflux from the ER, and on the other hand by SERCA, which regulates Ca2+ influx into the ER resulting in a zero net flux of Ca2+ ions into the cytosol32. Ca2+ sparks are transient elevations of the [Ca2+]i that are caused by a net efflux from the ER. This efflux is caused by an increased open probability of the RyR, which cannot immediately be offset by the SERCA-driven influx. Using caffeine to open the RyR pathway leads to a rapid and transient Ca2+ efflux that depletes the ER’s stores. The following balance of efflux and influx of Ca2+ ions to pre-caffeine levels while still in the presence of caffeine is caused by clearing the elevated [Ca2+]i by pumping Ca2+ ions out of the cells or into alternative stores while the RyR is still activated and open33. The refilling of the ER stores cannot be achieved under these conditions, which is the reason why we observed no further Ca2+ transients when caspofungin was applied after the ER stores were depleted by the addition of caffeine. When caspofungin is applied to cells that have not had their ER stores depleted by caffeine, we observed Ca2+ transients in most of the cells.Under steady-state conditions, the influx of Ca2+ contents, we observed a rapid decline in Mag-Fluo-4-fluorescence followed by a partial recovery, which eventually led to a prolonged decline in Mag-Fluo-4-fluorescence. The partial recovery is most probably a response to the SERCA balancing the efflux and influx of Ca2+ ions from the ER, as is generally assumed for many cell types34. A similar mechanism of Ca2+ depletion from the ER has been described previously for smooth muscle cells derived from different organs in rats36. Here, the prolonged decline of Mag-Fluo-4-fluorescence was caused by the continuous presence of caspofungin activating RyR.In our experiments using Mag-Fluo-4 as an indicator for ER luminal Ca2+ ions from the ER stores. Most probably, this net efflux is provoked by caspofungin triggering the increase in open state probability of RyR, since the kinetics of Ca2+ transients are similar. The clearance of cytosolic Ca2+ transients is caused by alternative clearance pathways, e.g., the transmembrane efflux, buffering, or by an uptake in alternative Ca2+ stores like mitochondria. We interpret these findings such that caspofungin specifically activates ER-bound RyR and depletes these Ca2+ stores. Caspofungin now provokes an imbalance in favor of a net Ca2+ efflux that results in an immediate increase in [Ca2+]i, which is either transient or sustained. The rapid amplitude in [Ca2+]i under caspofungin exposure is caused by the steep function of Ca2+ content in the ER as has previously been described in cardiomyocytes38. This Ca2+ content contributes to the regulation of ciliary beat activity by the activation of different signal cascades8.Under these conditions, SERCA was unable to balance the influx and efflux of Ca2+ ions from cytosolic buffers as an alternative source for increasing [Ca2+]i. In many cell types, cytosolic Ca2+ is strongly buffered, and for every free cytosolic Ca2+ ion approximately 100 to 200 Ca2+ ions are bound to buffers40. In cardiomyocytes, the major buffers are troponin and SERCA, whereas in airway epithelial cells the Ca2+ is supposed to be buffered by calmodulin34. However, in this case, the kinetics of increased [Ca2+]i would be different.Alternatively, caspofungin may promote the dissociation of Ca2+ transients or sustained elevations of [Ca2+]i. This is because Ca2+ pumps on the plasma membrane, the ER and mitochondrial Ca2+ uptake would still be fully active and would be sufficient to immediately reduce elevated [Ca2+]i content.We have not observed biphasic Ca2+ ions to SERCA contributes significantly to Ca2+ buffering, it may be that caspofungin contributes to the dissocitaion of Ca2+ ions from these binding sites41. Nevertheless, in this case, we would observe Ca2+ transients while RyR was inhibited by ryanodine, or when the ER stores were depleted by caffeine. However, we did not observe these Ca2+ kinetics. These data argue against the assumption that caspofungin drives Ca2+ ions out of the buffering sites of the SERCA. Furthermore, the inhibition of SERCA by caspofungin would lead to a slow, sustained increase in [Ca2+]i which then returns to the baseline, as we observed for CPA  could not occur. Further experiments are necessary to explore whether the IP3 receptor pathway is affected by using a more specific IP3 receptor blocker such as Xestospongin C or to examine SOCE pathways, which is more complicated due to diverse interactions and a finetuned orchestration of multiple targets. Regardless, we conclude that RyR is the principal target of caspofungin since we measured the main effects of caspofungin via this Ca2+ efflux pathway.In our experiments, we noted a reduced response to caspofungin while using 2-APB, which binds to the IP2+ ions under caspofungin exposure may be due to several reasons, as a similar effect has been described when these cells are exposed to histamine or acetylcholine45. Most probably, this prolonged kinetic is caused by an imbalance between the influx and efflux of Ca2+ ions from the cytosol due to a slow clearance rate when reaching baseline levels of [Ca2+]i during the observation period. These findings imply that caspofungin has effects on other cell organelles. It may also interfere with mitochondrial ATP synthesis, as has been described for mammalian mitochondria28. Under these circumstances, the remaining ATP supply from mitochondria, glycolysis or phosphocreatine stores is too low to fully activate Ca2+ ATPase in the plasma membrane. This mechanism prevents a rapid return of [Ca2+]i to baseline levels. Beyond plasma membrane-bound, sodium-calcium exchangers are also activated when [Ca2+]i is high and may blunt the peak of observed the Ca2+ transients46.The observed sustained elevation of Ca2+ storage in these organelles. This is supported by our findings that the number of Ca2+ transients was only slightly altered when mitochondria were depolarized using DNP. Therefore, in many tracheal epithelial cells, caspofungin may have additional effects on other cellular structures that cause a prolonged elevation of [Ca2+]i, with the slow return to the baseline level due to an energetic restriction.We also found no evidence that caspofungin depolarizes mitochondrial membrane potential, which is the driving force behind Ca2+ influx pathways, e.g. store operated calcium entry (SOCE) or IP3 receptors in the intact tracheal epithelium, should also be considered.HTE cells, which are isolated cells from the human tracheal epithelium, are suitable for investigating the different effects and signal pathways under stable conditions, whereas freshly isolated human tracheal cells should only be used in further studies to underline these observations. Additionally, a contribution of alternative Ca2+ ions from internal ER stores and enhances [Ca2+]i either transiently or in a sustained kinetic. This mechanism likely occurs due to the activation of a RyR pathway. A sustained elevation in [Ca2+]i levels may be supported via energetic imbalances during caspofungin application. Further studies should focus on RyR activation by caspofungin, detecting its binding site to these receptors, and determining how cellular ATP synthesis is compromised by caspofungin.Caspofungin liberates Ca


In these studies, only micromolar [Ca2+]i were believed to affect gating—concentrations reachable only in cell death, which would discard Ca2+i as a fine modulator of cell coupling. More recently, however, numerous researchers, including us, have reported the effectiveness of nanomolar [Ca2+]i. Since connexins do not have high-affinity calcium sites, the effectiveness of nanomolar [Ca2+]i suggests the role of Ca-modulated proteins, with calmodulin (CaM) being most obvious. Indeed, in 1981 we first reported that a CaM-inhibitor prevents chemical gating. Since then, the CaM role in gating has been confirmed by studies that tested it with a variety of approaches such as treatments with CaM-inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent co-localization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as CaM targets. Our gating model envisions Ca2+-CaM to directly gate the channels by acting as a plug (“Cork” gating model), and probably also by affecting connexin conformation.Evidence that neighboring cells uncouple from each other as one dies surfaced in the late 19th century, but it took almost a century for scientists to start understanding the uncoupling mechanism . The role of cytosolic free calcium (Ca The neighboring cells of most tissues freely exchange small cytosolic molecules via cell-to-cell channels clustered at gap junctions. This form of direct cell–cell communication (cell coupling) provides a fundamental mechanism for coordinating and regulating a host of cellular activities in mature and developing organs ,4,5,6,7.2+]i i ,10.herzmuskelzellen leben zusamme und sterben einzeln ” i rise [2+i role in gating was proven by evidence that cell-to-cell uncoupling coincides with an increase in [Ca2+]i, monitored at the cell–cell contacts by aequorin luminescence i rise . The Ca2nescence . The Ca2tebrates ,15,16,172+]i in the high µM range causes cell-to-cell uncoupling [2+]i, in the range of ~100 nM to low µM, are effective for channel gating. The effectiveness of low [Ca2+]i was first published in studies on Chironomus salivary gland cells i as low as 251 nM in cardiac cell-pairs [2+]i = ~685 nM or greater [2+]i uncoupled cells subjected to ischemia followed by reperfusion [2+]i were also effective in crayfish axons i and pHi in crayfish septate axons uncoupled by intracellular acidification caused by superfusion with Na+-acetate (pH 6.3) [2+]i rise of approximately one order of magnitude from basal values of 100–300 nM greatly increased Rj [2+]i and Rj time-courses coincided, while those of pHi and Rj did not  at pHi = 7.2 or 6.1. Gating was activated by [Ca2+]i ranging from 500 nM to 1 µM, regardless of pHi [2+]i in the nM range and are insensitive to pHi as low as 6.1, as long as [Ca2+]i is kept at resting level with BAPTA in the patch pipettes i monitored with fura-2 increased with AA to 0.7–1.5 µM in cells with normal extracellular Ca2+ concentration ([Ca2+]) i increased from ~110 to ~400 nM and cell–cell transfer of Lucifer Yellow was drastically reduced i dropped to ~260 nM and dye transfer resumed [2+]i drastically decreased Gj in pancreatic β-cells, in which [Ca2+]i was increased by lowering the temperature from 37 to 30 °C and increasing [Ca2+] from 2.56 to 7.56 mM i in the nM range was also effective in astrocytes co-injected with Lucifer Yellow and Ca2+, as the dye-transfer blockage was linearly related to [Ca2+]i ranging from 150 to 600 nM [2+]i. Similarly, dye coupling was blocked in ionomycin-treated astrocytes by [Ca2+]i = 500 nM [2+ influx and reduced Gj by 95% [2+]i increased from ~80 to ~250 nM i ~250 nM .2+i is a fine modulator of cell communication via gap junction channels. Furthermore, evidence for the effectiveness of nanomolar [Ca2+]i suggests the role of a Ca2+-modulated protein, calmodulin (CaM), as being the most likely candidate (see the following).Based on these data, it is reasonable to believe that CaXenopus laevis embryonic cells i rise i rise .i-sensitivity varies among cell types, and in most cases is related to the type of connexin expressed. In Xenopus oocyte pairs, for example, the rat liver connexin (Cx32) is much less sensitive than Cx38, the native oocyte connexin i almost one order of magnitude lower (pKa = 6.5 versus 7.3) i relationship i was kept to resting levels with BAPTA in the patch pipettes  and by cytosolic dialysis with a strong acid (HCl) i effect, in one study on hemichannels it appears that direct protonation may affect gating i–Rj and [Ca2+]i–Rj relationships were first studied in crayfish axons uncoupled by acetate superfusion [+]i markedly differ in shape, and [H+]i maxima preceded Rj maxima by 40–90s  by monitoring Gj, with a double voltage clamp, as well as pHi and pCai i, as they greatly inhibited the effect of acidification on Gj [2+-uniporter [2+]i by releasing it, at least partially, from the mitochondria. A low-pHi-induced Ca2+-release from the endoplasmic reticulum (ER) is also likely to be involved, as indicated by our study on crayfish axons uncoupled by a low pHi in the presence of caffeine or ryanodine i maxima resulted from the addition of caffeine to acetate solutions, whereas a substantial drop in Rj and [Ca2+]i maxima was seen when the acetate-caffeine treatment was preceded by caffeine pretreatment, suggesting that the caffeine pretreatment depleted the Ca2+-stores. In contrast, ryanodine always caused a significant decrease in Rj and [Ca2+]i maxima [2+ entry was excluded because Ca2+-channel blockers were ineffective [2+] as high 40 mM were also ineffective [2+ entry does not play a role in acetate-induced [Ca2+]i increase.To understand the mechanism by which low pH2+ entry . A largei maxima . Acetateffective . Acetateffective , furtheri on gating is mediated by Ca2+i [i = 6.1 had no effect on gating as long as [Ca2+]i was carefully buffered to resting levels by BAPTA in the patch pipettes ,772-induon V2/V1 A. The TFon V2/V1 A. Xenopus embryonic cells ..Xenopus The CaM blocker W7 also inhibited uncoupling of crayfish axons superfused with acetate-containing salines (pH = 6.3) . W7 100C, while 2+ increased from pCa 5.7 to pCa 7 when the cells were perfused with 10 µM CaM. Significantly, W7 (but not W5) prevented uncoupling o increased [Ca2+]i from ~110 to ~620 nM and blocked the cell-to-cell diffusion of AlexaFluor594. The block was prevented by pre-incubation with 10 µM CDZ. Recently, CDZ was also effective in murine Neuro-2a cells (N2a) expressing Cx43 [2+]i from ~80 to 250 nM and reduced Gj by ~95% in 15 min; the Gj drop was prevented by bathing the cells in Ca2+-free solutions or pretreating them with 2 µM CDZ . . 2 sensily 17.3% B [106].2-terminal EF-hand pair (res. 9–76) of CaM is replaced by a duplication of the COOH-terminal pair (res. 82–148). Since the Ca2+ affinity constant of the COOH-terminal EF-hand pair is almost one order of magnitude greater than that of the NH2-terminal pair [2+]i, monitored with Calcium Green-1, was lowered by 180 µM BAPTA superfusion—note the reversible drop in F/F0  in CaM’s EF–hand domains—these mutations greatly decreased the Ca2+ affinity of the Ca2+-binding EF–hand loops i results from the activation of Protein kinase C (PKC) or CaM-kinase II was tested in lens epithelial cells by raising [Ca2+]i via Ca2+-ionophore or ATP treatment [2+]i and was prevented by CDZ treatment before ionomycin addition, but not by inhibitors of PKC or CaM-kinase II. Significantly, in HeLa cells transfected with a CT-truncated Cx43 mutant (Cx43-D257), the drop in coupling caused by a [Ca2+]i rise to ~300 nM was similarly prevented by CDZ i i .In 2000, we tested the direct CaM–connexin interaction by immunofluorescence microscopy ,108. In The direct CaM–Cx32 interaction was also demonstrated by confocal fluorescence microscopy in HeLa cells, expressing Cx32 bound to the green fluorescent protein (Cx32-GFP) and CaM bound to the red fluorescent protein (CaM-RFP) ,10,108. 2+-dependent and blocked by W7. The role of CL2’s CaM-binding site  was confirmed by its high-affinity interaction (Kd = ~5 nM) with a peptide mimicking the CL2 domain, monitored by fluorescence-labeled CaM i (~50 nM) . Csitivity  [116]; tsitivity ,116. Theven more ,135. The2 sensitivity of 5R/N and 5R/E mutants seems to indicate that the greater the reduction in CT1’s CaM-binding affinity, the greater the CO2 channel gating sensitivity. Indeed, presently we doubt that the CT1’s CaM binding is relevant to chemical gating, because CT-deleted Cx32 mutants (Cx32–D219) generated channels indistinguishable from Cx32 wild-type in CO2 gating sensitivity i [CaM binding to Cx32’s CT1 was confirmed by testing the interaction with Isothermal Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) . However2+]i .CaM also binds to the CT1 domain of mouse Cx34.7 ,143. The1/2’s for Ca2+ range from 3 to 5 µM i rise [2+]i and caused uncoupling at pHi > 7.8 i rise . In factHi > 7.8 .2+-dependent gating, confirming that the CL2 domain (res. 136–158) contains the CaM-binding site relevant to Cx43’s Ca2+ gating i and caused Gj to drop by 95%; in contrast, ionomycin did not significantly affect Gj in N2a cells expressing human–Cx40 channels. The chemical gating incompetence of the human–Cx40 i has recently been confirmed by in vitro experiments testing CaM-binding to peptides matching the CL2’s CaM site of Cx32, Cx35, Cx45 and Cx57, in the presence and absence of Ca2+  . Hemicha2+] . We furtolutions . In contcontrols .2+ clearly plays a major role in keeping hemichannels closed [2+]i actually causes hemichannel opening [2+]i rise to ~500 nM, caused by treatment with 2 μM A23187 (a Ca2+ ionophore), triggered ATP release and dye uptake that was blocked by a Cx32 mimetic peptide [2+]i rise to ~500 nM opened hemichannels, this phenomenon vanished with a greater [Ca2+]i rise. Note, however, that the hemichannel closure at high [Ca2+]i is likely to be CaM-independent i; significantly, Ca2+-sensitivity is restored by application of the peptide “32gap 24”. These Authors suggested that the interaction of “32gap 24” with the Cx32–D220 hemichannel stabilizes CL fluctuations, and proposed that CL fluctuations may prevent the exposure of CL residues to a target domain relevant to gating [2+]i by a plugging molecule likely to be a CaM lobe [2+]i rise, hence the kinetics of the hemichannel’s “cork” unplugging i i ,112. Perinus end . The CaMinus end .2+, even though Ca2+ still binds . T. T135]. The slow change in Gj was interpreted as a gating phenomenon based on the activity of the chemical/slow gate, clearly distinct from that of the fast Vj-gate. There are several reasons for making this distinction , one beiThis is significant because the fast Vj-gates of Cx26 and Cx32 are sensitive to opposite voltage polarities—Cx32 is a “negative gater” while Cx26 is a “positive gater” . This obOne may question why most diverse mutations unmask a similar slow gating behavior. We feel that, while without uncouplers the chemical/slow gate of most connexins, perhaps except Cx45 channels ,149, is 2 exposure , the chemical/slow gate is inactive in the absence of uncouplers or Cx-mutations, our 2007 data indicate that this gate can also be activated by applying a series of large Vj gradients  115]. W. W115]. These data clearly confirm the idea that the gate responsible for this phenomenon is the chemical/slow gate, rather than the fast Vj-gate. The manifestation of the activity of the chemical/slow gate in wild-type Cx32 indicates that this negatively charged gate, likely to be the CaM’s N-lobe, can be rendered operational even without chemical uncouplers and/or connexin mutations. Indeed, our data  confirm 2 sensitivity of Cx45 channels; indeed, with CO2, Gj reversibly decreased by only ~17%, while, in controls, it rapidly dropped to zero  by a CaM lobe A 10,16],1610,16]l gating ,10,16,80l gating ,135.2+]i in the nM range i;Peptides mimicking the CaM-binding sites of various connexins bind CaM with high affinity ,128,133.ent ways ,112, sugCaM and connexins co-localize at gap junctions and intracellular spots ,126,127;negatively charged CaM’s lobes is ~25 × 35 Å in size [positively charged channel’s mouth  prevent uncoupling by acidification and/or increased [Ca2+]i ,98,122;2+-affinity, greatly increases the chemical gating sensitivity of Cx32 channels , c, c161], 2+]i i . In cont2+]i ;2+]i opens Cx32 and Cx43 hemichannels i rise and in most connexins, except in Cx45 i rise above resting levels (> ~50 nM) activates CaM and enables a CaM lobe (probably the N-lobe) to plug the channel’s mouth  at the CL2 site  i increases above resting levels. This model agrees with evidence for the separate functions of CaM’s N- and C-lobes in interacting with Cx32 i (~50 nM) CaM is anchored to each connexin by its C-lobe to the CL2 site  by a Ca2+]i rise, potential scenarios may be as follows:trans-domain interaction) and changes connexin conformation (trans-subunit interaction) and interacting electrostatically and hydrophobically with the positively charged channel’s mouth  and plugs the pore (cork gating) by interacting with the positively charged channel’s mouth electrostatically and hydrophobically  of the opposite connexin  by plugging the channel’s mouth and preventing other N-lobes from accessing the channel’s mouth.In both cases, one N-lobe would win the competition  rather than the fast Vj-gate because the channel is opened by positive Vj at the mutant side both with “negative fast-Vj-gaters” like Cx32 and “positive fast-Vj-gaters” like Cx26  9,16]. ,16. 9,16Cytosolic connexin domains have a high ratio of basic versus acidic residues. For example, in Cx32, if we neglect most of CT, whose deletion by over 80% does not affect chemical gating sensitivity , and assOne may question why the channel’s mouth (vestibule) would be freely accessible to the gating CaM’s N-lobe in Cx32 mutants only. Possibly, in wild-type connexins the inaccessibility of the channel’s mouth may result from the postulated CL1–CT1 electrostatic/hydrophobic interaction . The abs2+-activation. Therefore, the hemichannel’s mouth (vestibule) may not be totally inaccessible, so that the N-lobe could be forced to access it by large negative Vj-gradients (100 mV)   115]. . 2+-actiIn several wild-type connexin channels, large Vj-gradients may not be needed to force the gating element (CaM’s N-lobe) into the channel’s mouth. Indeed, in cells expressing Cx45, a number of channels are closed by the chemical/slow gate, even at Vj = 0 and without uncouplers ,149. Pos2+-dependent gating of Cx43 channels in N2a cell pairs exposed to 1 µM ionomycin and 1.8 mM Ca2+; competitive peptides or CDZ prevented the Gj drop, confirming the CaM role in gating i was increased by exposure to uncouplers i, all of the CaM molecules but the gating one, might become detached from connexins. This would allow the channels to tightly pack into crystalline  arrays  arrays B, inset,2+]i? In 2004, Black and coworkers developed a genetically encoded fluorescent biosensor to determine the intracellular concentration of both Ca2+-free (apo) and Ca2+-activated (holo) CaM [i at rest is 8.8 ± 2.2 µM. A [Ca2+]i rise induced by agonist, store-operated Ca2+-entry, or ionophores caused a Ca2+-dependent consumption of available CaM, resulting in a [CaM]i drop to ≤200 nM. This is caused by the drastic buffering of free CaM due to excess availability of CaM binding sites. The same results were obtained in cardiac myocytes of adult rabbits. In fact, in both permeabilized and intact myocytes, with a [Ca2+]i rise, CaM reversibly moved into the nucleus i rise, the five non-gating CaM molecules become detached from connexins and bind to other CaM binding sites with a higher affinity to Ca2+–CaM, which would successfully compete against connexins for CaM binding.The same phenomenon might apply to gap junctions. A potential scenario, therefore, could be that, with a great/prolonged [Ca2+–CaM at nanomolar [Ca2+]i. This is important not only because this finding brings together the vast knowledge of two major fields of cell biology, but also because it proves that the function of chemical gating is much greater than just protecting healthy cells from damaged or dead neighboring cells .While much still needs to be investigated to understand the gating mechanism of gap junction channels in detail, most relevant, thus far, is evidence that direct cell-to-cell communication is finely modulated by the direct action of CaFurther knowledge of the CaM-role in modulating the permeability of channels and hemichannels made of connexins/innexins may also pave the way for understanding the behavior of intracellular connexin/innexin channels. Indeed, evidence accumulated over several decades indicates that these proteins form hexameric connexons/innexons in non-junctional plasma membranes, the Golgi apparatus, mitochondria and, in some cases, also in the endoplasmic reticulum . Many qu


The modulation of cytoplasmic Ca2+ concentration regulates a plethora of cellular processes, such as: synaptic plasticity, neuronal survival, chemotaxis of immune cells, platelet aggregation, vasodilation, and cardiac excitation–contraction coupling. Rap1 GTPases are ubiquitously expressed binary switches that alternate between active and inactive states and are regulated by diverse families of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Active Rap1 couples extracellular stimulation with intracellular signaling through secondary messengers—cyclic adenosine monophosphate (cAMP), Ca2+, and diacylglycerol (DAG). Much evidence indicates that Rap1 signaling intersects with Ca2+ signaling pathways to control the important cellular functions of platelet activation or neuronal plasticity. Rap1 acts as an effector of Ca2+ signaling when activated by mechanisms involving Ca2+ and DAG-activated  GEFs. Conversely, activated by other GEFs, such as cAMP-dependent GEF Epac, Rap1 controls cytoplasmic Ca2+ levels. It does so by regulating the activity of Ca2+ signaling proteins such as sarcoendoplasmic reticulum Ca2+-ATPase (SERCA). In this review, we focus on the physiological significance of the links between Rap1 and Ca2+ signaling and emphasize the molecular interactions that may offer new targets for the therapy of Alzheimer’s disease, hypertension, and atherosclerosis, among other diseases.Ca K-Ras (one of the mutated Ras genes) . ,12. 9,12Rap1 is a soluble cytosolic protein that undergoes isoprenylation (geranylgeranylation), a covalent binding of geranylgeraniol to the –SH group of cysteine in the C-terminal CAAX motif . This posttranslational modification, combined with CAAX motif cystine carboxymethylation, enhances Rap1 hydrophobicity and facilitates its membrane localization ,14,15. RSignaling events that interfere with the prenylation of Rap1 may decrease its membrane localization and, thus, interfere with cell–cell adhesion. For instance, adenosine A2B receptor-mediated signaling induces Rap1b (ser179 and ser180) phosphorylation and leads to decreased binding to chaperone protein small G-protein dissociation stimulator (SmgGDS). This signaling inhibits Rap1 prenylation and membrane localization and results in cell scattering ,20.2+ influx .,60.2+ an GTPases ,46. CalD GTPases ,45,60. T studies .αs-coupled GPCRs stimulation, induces the activation of Rap1 GEFs and Epacs (exchange proteins directly activated by cAMP) [Rap1 is an important mediator of cellular cAMP signaling . Elevatiby cAMP) ,65,66. Tby cAMP) . Epac1 iby cAMP) .Epac1 and Epac2 share a similar structural organization, with the C-terminal catalytic GEF region and N-terminal regulatory region. The catalytic regions of Epacs possess a Ras exchange motif (REM domain), a Cdc25-homology catalytic domain that mediates the GEF activity for Rap GTPases and a RAS-association domain (RA domain), which translocates Epac2 to the plasma membrane. The regulatory region of Epac1 consists of a DEP domain  that is responsible for membrane anchoring, and a conserved cAMP-binding domain ,68 Tabl. In the 2+ homeostasis and Epac and Ca2+ signaling pathways crosstalk at multiple levels, converging on effectors like IP3 receptor and ryanodine receptor (RyR), mediating Ca2+ release, or sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), mediating Ca2+ clearance, effectively forming a signaling network in non-excitable cells [2+-induced Ca2+ release to mobilize intracellular Ca2+ levels, as found in the regulation of exocytosis in human pancreatic β-cells and INS-1 insulin-secreting cells [2+ can also modulate the Epac signaling pathway by activating the adenylyl cyclase to increase the production of cAMP levels [2+ signaling intersect to regulate important functions in several tissues. Some of the mechanisms uncovered are described below.Epac plays an important role in the regulation of Cale cells ,72,73. Tng cells ,75. InteP levels . It is iP levels ,64, the 2+-dependent processes, such as neuronal excitability, synaptic plasticity, long-term potentiation and gene transcription [2+ influx leading to CalDAG-GEFI activation and the subsequent formation of CalDAG-GEFI/Rap1/B-Raf cassette to stimulate the ERK pathway [2+-dependent Rap1 activation vary in different neuronal cells depending on the source and magnitude of Ca2+ signal. In PC12 and hippocampal neurons, Rap1-ERK signaling is mediated by PKA activation upon depolarization-induced Ca2+ influx through L-type Ca2+ [2+ release activates the Rap1/B-Raf/ERK pathway to regulate cAMP-response element binding protein (CREB)-phosphorylation and gene expression [2+ release, but is not further induced by direct or indirect PKA- or protein kinase C-dependent phosphorylation [2+ and Rap1 signaling can intersect at multiple signaling modules.Via the activation of the ERK signaling pathway, Rap1 is involved in a number of Cacription ,78,79,80 pathway   in hippocampal CA1 pyramidal neurons [2+ influx stimulates the nuclear pool of Rap1-ERK signaling, which phosphorylates nuclear targets involved in the expression of hippocampal long term potentiation [2+-signaling.Furthermore, the magnitude of intracellular Caignaling , which l neurons . Conversntiation  and CREBntiation . Thus, i2+-mediated ERK signaling, Epac-mediated Rap1 activation controls Ca2+-dependent signaling events, such as resting membrane potential, glutamate release, and cortico-amygdala plasticity in neurons. In mouse cerebellar granule cells, the Epac-induced activation of Rap1 and p38 mitogen-activated protein kinase (MAPK) mobilizes intracellular Ca2+ release, facilitating the opening of large conductance Ca2+-activated K+ channels to modulate resting membrane potential and after-hyperpolarization [2+ channels act as the downstream Rap1 effectors to mediate the suppression of glutamate release required for cortico-amygdala plasticity and fear learning. Rap1 deletion in these cells leads to increased axonal Ca2+ influx, ERK inhibition, and increased plasma membrane expression of L-type voltage-gated Ca2+ channels (Cav1.2 or Cav1.3), enabling the Ca2+-regulated glutamate release [In addition to acting as an important mediator of Carization . In prim release .2+ aberrations and neuronal activity. The inhibition of Rap1 interaction with PDE6δ restrains disease-associated abnormal Ca2+ influx and neuronal hyperactivity and confers neuroprotection in models of Alzheimer’s disease [2+ aberrations, such as Alzheimer’s disease.Interestingly, the modulation of prenylated Rap1 appears to play a role in controlling disease-associated Ca disease . Rap1 ma2+ is required and sufficient for Rap1 activation in platelets [2+-dependent mechanism of CalDAG-GEFI, and downstream, Rap1 activation. In resting platelets, where cytosolic Ca2+ levels are low, CalDAG-GEFI remains in an auto-inhibited state. In response to agonist stimulation, elevated cytosolic Ca2+ binds to the EF hands and induces structural rearrangements that free the catalytic surface of CalDAG-GEFI to activate Rap1b [3 integrin subunit and contributes to integrin activation [IIbβ3 integrin activation has not been fully validated in vivo [2+-dependent rapid activation of Rap1 and a marked defect in platelet aggregation [2+ homeostasis [2+ signaling with integrin αIIbβ3 activation, thromboxane A2 formation, and granule release in platelets [Early studies demonstrated that an increase in cytosolic Calatelets . Much relatelets  and recete Rap1b . Downstrtivation ,87. Whil in vivo ,89, evidregation , similarregation . Human peostasis . Importalatelets ,92,93  [2+ pump that transports Ca2+ into ER, reducing cytosolic Ca2+ concentration, thereby controlling IP3-sensitive intracellular Ca2+ pools in platelets [In addition to acting as an effector of Ca (SERCA) ,95. SERClatelets . Studyinlatelets . Later, 2+ pool to induce platelet inhibition [2+ pool, thus decreasing IP3-sensitive Ca2+ release to promote platelet activation [The dynamic regulation of the interaction between Rap1b and SERCA 3b by cAMP-dependent phosphorylation of Rap1 may act to regulate a transition between platelet inhibition and activation ,98. Incrhibition   and enables Ca2+ release from lysosomal acidic organelles to stimulate long-lasting Ca2+ entry through transient receptor potential melastatin 2 (TRPM2) channels required for cell migration [Once activated by CalDAG-GEFI or Epac, Rap1 controls chemotaxis and the trafficking of immune cells via additional, distinct mechanisms. In neutrophils, CalDAG-GEFI-activated Rap1 controls chemotaxis in an integrin-independent manner through a mechanism that involves actin cytoskeleton and cellular polarization . In lympigration   signaling in immune cells [2+ signal has been linked with differential activation signaling by Ras and Rap1 as well as a differential effect on ERK activation and cytokine production [2+ influx mediated by stromal interaction molecule 1 (STIM1) favors Rap1 inhibition and ERK activation, while high-intensity TLR stimuli trigger more intense Ca2+ influx, leading to Ras activation and cytokine production [2+-dependent, key aspects of the immune response.In addition to the regulation of migration, Rap1 signaling is an important modulator of Cane cells ,116. Theoduction . Inducedoduction . These f2+ signaling to regulate cardiac excitation–contraction coupling, with phosplipase Cε (PLCε) and CamKII as Epac effectors [2+-induced Ca2+ release (CICR) in adult ventricular cardiomyocytes. In response to β-adrenergic receptor (βAR) stimulation, Epac–Rap2b induce both Rap GEF activity and hydrolytic activity of PLCε. This leads to sustained Rap activation and the induction of PKCε and CamKII phosphorylation downstream from PIP2 breakdown. These signaling events lead to enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release (CICR) [2+ homeostasis through the regulation of CICR in cardiomyocytes. Although these studies identified Rap2b as the main isoform involved in the cardiac Ca2+ signaling, the role of Rap1 in this context is not known. Therefore, Rap1 isoform-specific knockout animal models will be useful to determine the in vivo role of Rap1 in cardiac Ca2+ signaling.Epac proteins play an important role in the regulation of cardiac physiology, with some of its functions being mediated by Ras family members other than Rap1 ,118,119.ffectors ,71. PLCεffectors . Importae (CICR) ,122  [2+ signaling network [2+ homeostasis and heart function is indisputable, the identity and function of its specific effectors, including Rap GTPases, in normal and pathologic conditions, remain to be fully elucidated.In addition to its role in cardiac physiology, Epac is also implicated in cardiac pathology. Epac stimulation induces cardiac hypertrophy via activation of IPs (NFAT) ,124. In s (NFAT) —macromol network . While t2+ and Epac-dependent Rap1 activation in vascular smooth muscle cells to control smooth muscle relaxation. The activation of Epac modulates Ca2+ sensitivity of the contractile proteins by a Rap1-dependent reduction in RhoA GTPase activity in several types of smooth muscle cell from airway, gut, and vascular tissues [20) phosphorylation and the disinhibition of myosin light chain phosphatase (MLCP) activity, which leads to Ca2+-desensitization and relaxation of force in smooth muscle [2+, medicated by decreased RLC20 phosphorylation [Rap1 plays an important role in the regulation of vascular tone. At least two distinct mechanisms connect Ca tissues ,127. In h muscle ,127 , inducing smooth muscle hyperpolarization. This, subsequently, leads to a decrease in the activity of voltage-gated Ca2+ channels, reducing Ca2+ influx and promoting vasorelaxation [2+ sensitivity of vascular smooth muscle cells, plays an important role in maintaining normal vascular contractile state and contributes to blood pressure regulation.In addition to triggering signaling that modulates Calaxation   release is where Rap1 and Ca2+ signaling intersect and this is one of the most physiologically impactful functions of Rap1 [Some of the best-described functions of Rap1 in the endothelium involve the dynamic regulation of endothelial junctions and the control of the endothelial and vascular barrier ,130. Acteability . Howevereability ,140. Whing edema , it is n of Rap1 .2+-dependent eNOS activation, as endothelium-specific deletion of Rap1 leads to a significant impairment of acetylcholine induced-Ca2+-dependent vasodilation and is sufficient to induce hypertension in vivo [2+, eNOS activity, and subsequent NO production contributing to endothelium-dependent vasorelaxation [2+ signaling in EC homeostasis.The endothelial cell-specific deletion of Rap1 during development leads to an impaired vascular barrier and is not compatible with vascular maturation . However in vivo ,143. Conlaxation  (Figure 2+ signaling—the dynamic regulation of endothelial barrier and release of NO. While the identity of some of the Rap1 effectors and GEFs regulating these processes is known, more research is needed to understand the exact underlying mechanisms. Such analysis may reveal novel therapeutic targets for treating diseases associated with endothelial dysfunction such as atherosclerosis, myocardial infarction and hypertension.In summary, endothelial Rap1 signaling plays an important role in the fundamental endothelial processes regulated by Ca2+ signaling to control important functions in the multiple tissues. The significance of Rap1/Ca2+ cross-talk is particularly evident in the CNS, where increased Ca2+ from external Ca2+ influx or internal Ca2+ release activates Rap1 to stimulate B-Raf/ERK signaling. This, in turn, controls neuronal function and gene expression [2+ signals, with different frequencies and amplitudes, activate Rap1 to induce ERK activation. Better understanding of the kinetics of Rap1 activity and identity of Rap1 activating GEFs involved in Ca2+ signaling is needed. To this end, novel tools [In two different signaling modalities, as an upstream activator and downstream effector, active Rap1 intersects with Capression ,79. Howeel tools  and modeel tools  might he2+ signaling elicited downstream from several GPCR-agonists [2+ entry is required for sustained eNOS activation in endothelial cells [2+ signaling in smooth muscle cells, promoting their relaxation. Thus, in both tissues, Rap1 controls vascular tone [Studies of Rap1 in the endothelium, heart and platelets have revealed its novel effectors and functions important for tissue and organ homeostasis ,129,143.agonists ,148,149.al cells . Intereslar tone . CalDAG-lar tone . Howeverlar tone .2+ signals. A thorough characterization of Rap1 regulators, effectors, and the mechanisms connecting them in time and space may provide novel therapeutic strategies for several cardiovascular and neurological diseases.It is becoming evident that Rap1 signaling is essential for the function of multiple organs, and that some key Rap1 functions rely on cross-talk with Ca


NAADP and thereby promotes the opening of Ca2+‐permeable two‐pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo‐lysosomal Ca2+ nanodomains rather than global cytoplasmic or ER Ca2+ signals. Motile endolysosomes contact nascent phagosomes to promote phagocytosis, whereas endo‐lysosome immobilization prevents it. We show that TPC‐released Ca2+ rapidly activates calcineurin, which in turn dephosphorylates and activates the GTPase dynamin‐2. Finally, we find that different endo‐lysosomal Ca2+ channels play diverse roles, with TPCs providing a universal phagocytic signal for a wide range of particles and TRPML1 being only required for phagocytosis of large targets.Macrophages clear pathogens by phagocytosis and lysosomes that fuse with phagosomes are traditionally regarded as to a source of membranes and luminal degradative enzymes. Here, we reveal that endo‐lysosomes act as platforms for a new phagocytic signalling pathway in which FcγR activation recruits the second messenger  2+ nanodomains formed by two‐pore channels (TPCs) on endolysosomes, rather than global cytoplasmic or ER calcium signals, promote phagocytosis in murine macrophages.Local Ca Phagocytosis is a multi‐step process that involves signal transduction pathways, rearrangements of the actin cytoskeleton and membrane trafficking  . A major pathway for endo‐lysosomal Ca2+ release is controlled by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which activates Ca2+‐permeable two‐pore channels (TPCs) located on acidic organelles , but how the FcγR generates Ca2+ signals remains incompletely understood and the role of Ca2+ in phagocytosis has proven controversial  upon FcγR stimulation either during “frustrated phagocytosis” or by phagocytosis of IgG‐opsonized beads.Pathogens opsonized by IgG are recognized by the Fc‐gamma receptors (FcγR) of macrophages and undergo phagocytosis, involving a rearrangement of the actin cytoskeleton and internalization of the microorganism : extracellular Ca2+ (Ca2+o) was chelated with EGTA whereas cytosolic Ca2+ was buffered with EGTA (or BAPTA) pre‐loaded as their AM‐ester precursors.Frustrated phagocytosis can be induced by dropping BMDMs onto IgG‐coated coverslips, engaging Fcγ receptors as they come into contact though they are unable to engulf the target. We measured single‐cell  Glu to Gln in each). The triple mTagBFP2 construct (FKBP12‐(mTagBFP2)3‐HA) was generated so that a similarly sized protein could also be repositioned in the cell, to account for any steric hindrances. The rapalog‐FRB*  was targeted to the cytosolic face of lysosomes by fusion with human LAMP1 to produce LAMP1‐mCherry‐FRB*. The dynein‐binding protein for rapalog‐induced organelle repositioning to MTOC (mTagBFP2‐BicD2‐FKBP12) was constructed in‐house using pBa‐tdTomato‐flag‐BicD2 594‐FKBP   supplemented with essential amino acids, for 45–60 min at room temperature, followed by a 15 min de‐esterification. Alternatively, cells were transfected with plasmids to express cytosolic GECIs or TPC‐tethered GECIs. For experiments conducted in Ca2+‐free medium (−Ca2+o), cells were washed once with Ca2+‐free ECM supplemented with 1 mM EGTA, followed by two washes in Ca2+‐free ECM plus 100 μM EGTA and experiments conducted in this same medium. Unless otherwise stated, cytosolic Ca2+ was chelated by loading BMDMs with 25 μM EGTA/AM or 25 μM BAPTA/AM for 45 min at room temperature in the presence of 0.03% w/v Pluronic F‐127 in ECM, followed by a 15 min de‐esterification.Cells were loaded with 2 μM Fura‐2/AM, 2 μM Calbryte 520/AM or 2 μM Calbryte 590/AM in the presence of 0.03% w/v Pluronic F‐127 in extracellular medium  on a single‐cell basis.Kd for Ca2+ of G‐GECO1.2 proteins was determined in situ in permeabilized Cos‐7 cells equilibrated with intracellular‐like media (ICM) buffered to different free [Ca2+]. The basic ICM was (mM): 10 NaCl, 140 KCl, 20 HEPES, pH 7.2 at room temperature. To this ICM, 5 mM of a Ca2+ chelator was added (“Free”); to half of this chelator solution, 5 mM CaCl2 was added  and all solutions were re‐adjusted to pH 7.2 with KOH. Different free [Ca2+] were generated by mixing the “Free” and “Total” solutions in different ratios, as calculated using Winmax chelator . To generate a broad range of free [Ca2+], it was necessary to use different Ca2+ chelators with different Ca2+ affinities . Where possible, we overlapped the common free [Ca2+] ranges of different chelators to minimize potential errors from inter‐chelator variation.The 2+], and the final ICM addition was supplemented with 30 μM sulforhodamine B as a permeabilization marker (MW 559). Cells were imaged with standard green and red channels. The plasma membrane was discretely permeabilized with 60 μg/ml β‐escin for ~ 5 min, breaching indicated by the entry of small MW marker, sulforhodamine B, while the large MW protein G‐GECO1.2 was retained (even the cytosolic form). As extracellular Ca2+ equilibrated, the GECI fluorescence reached a plateau and this value was normalized to the basal fluorescence prior to permeabilization (F0). A plot of the free [Ca2+] versus F/F0 values was fitted to a sigmoidal dose–response (GraphPad Prism 5) to determine the in situ Kd of G‐GECO1.2 either cytosolic or fused to TPC2. The calculated Ca2+Kd was as follows: 0.98 μM  for cyto‐G‐GECO1.2 and 0.81 μM  for human TPC2‐G‐GECO1.2.Briefly, Cos‐7 cells transiently expressing cytosolic G‐GECO1.2 or human TPC2‐G‐GECO1.2 were washed 3× with an ICM at a fixed free  sheep polyclonal . Anti‐dynamin [pSer778] is a phospho‐specific antibody to phospho‐Ser 778 in dynamin‐1  and scanned using an Odyssey infrared scanner in the 700 and 800 nm channels.BMDM were seeded into a 96‐well U‐bottom, suspension culture plate (Greiner Bio‐one). FcγR‐mediated phagocytosis of rabbit IgG‐3 μm beads proceeded at 37°C for various times. Phagocytosis was arrested by placing the plate on ice and the cells washed 3× with ice‐cold PBS. Cells were fixed and permeabilized with ice‐cold methanol for 10 min at RT. Following block in Odyssey2 (0.75 mM) were added to the lysates and incubated at 30°C for 1 h before Western blotting. Proteins were resolved by SDS–PAGE (50 μg/lane) using 4–12% TruPAGE gels (Sigma) and transferred to PVDF membranes. Membranes were blocked in Odyssey® blocking buffer (LI‐COR) and incubated with dynamin‐2 (G‐4) monoclonal (Santa Cruz), HA (3F10) monoclonal (Roche), dynamin [pSer778]  or actin (JLA20) monoclonal (DSHB). Secondary antibodies were anti‐mouse, anti‐rat and anti‐goat conjugated to IRDyes® (LI‐COR). Immunoreactive bands were detected by fluorescence using an Odyssey infrared scanner in the 700 and 800 nm channels.Total‐protein lysates were prepared by solubilizing cells in RIPA buffer  supplemented with 1× Halt protease and phosphatase inhibitors (Thermo Scientific). For protein phosphatase treatment, a pellet of WT BMDM was resuspended in NEBuffer (New England BioLabs) and sonicated on ice. Lambda protein phosphatase (100–400 U) and MnCl2+‐free ECM supplemented with 1 mM EGTA, followed by two washes in Ca2+‐free ECM with 100 μM EGTA and phagocytosis of Alexa 647‐conjugated 3 μm‐IgG‐beads conducted in this same medium.RAW 264.7 macrophages were transfected with LAMP1‐mCherry‐FRB* and the dynein‐binding protein for CID‐mediated repositioning to the MTOC (mTagBFP2‐BicD2‐FKBP12). Cells were treated with 250 nM AP21967 (Takara) or 0.05% ethanol (control) for 45 min at 37°C in DMEM, washed once with Cat‐test (unpaired) for two conditions, or a one‐way ANOVA for multiple conditions with significance determined as P < 0.05 using GraphPad Prism or Instat programs. Imaging data are analysed as pooled single‐cell values. Normality was tested using a Kolmogorov–Smirnov test (Instat). Normally distributed data were analysed using a Tukey–Kramer post‐test, otherwise a non‐parametric post‐test  was applied. Graphs were annotated using the following conventions: P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).Each experimental condition was performed on at least three preparations on multiple days. Data are presented as mean ± S.E.M. and analysed by either Student's et al, 2+ diffusion coefficient of 233 μm2/s  from a diameter of 0.5–2.0 μm . An 8‐nm diameter TPC  and occupy an area of ~ 100 nm2. Therefore, 500 channels occupy 50,265 nm2 which is 0.4–6.4% of the total vesicular SA.To estimate the relative space occupied by TPCs on a vesicle, we first calculated the surface area (SA) of the vesicle sphere (4πrConceptualization: LCD, AJM, AG; Methodology: LCD, AJM; Investigation: LCD, AJM; Writing: LCD, AJM, AG; Funding acquisition: AG.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.


BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t. Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients. RUNX1-RUNX1T1 (AML1-ETO) . Th. ThBRCC322) Fig. , Table 1lgorithm , the codlgorithm . The BRCBRCC3 mutated and nonmutated patients  treated with intensive chemotherapy  Kasumi-1 and SKNO-1 and in the OCI-AML5 cell line without t. Complete knockout (KO) of BRCC3 was confirmed by western blot  Kasumi-1 and SKNO-1  AML had a similar beneficial effect on cell proliferation in Kasumi-1. To this end, we ectopically over-expressed BRCC3 WT, R81X, or R81G in BRCC3 KO cells. Again, cell proliferation of Kasumi-1 BRCC3 R81X and R81G expressing cells was significantly higher as compared with BRCC3 WT cells suggesting that both mutants are inactivating  AML patients. We therefore analyzed the impact of a subclone of BRCC3 KO on cell proliferation. We found that a fraction of 40% of Kasumi-1 cells harboring a BRCC3 KO was sufficient to significantly increase cell proliferation of all cells in the culture as compared with BRCC3 WT cells .In the next step, we investigated whether loss of Brcc3 inactivation on the self-renewal capacity in mouse hematopoietic progenitor cells we performed colony forming assays. To this end, we infected primary murine stem- and progenitor Lineage- Sca1+ c-Kit+ (LSK) cells isolated from the transgenic Rosa26-Cas9 mouse that constitutively express the Cas9 enzyme [AML1-ETO or a control as well as a lentiviral vector expressing sgRNAs targeting Brcc3 or a non-targeting sgRNA control  and MDS patients, we cloned the two missense mutations R81G and R82H, located within the MPN + domain as well as D135E located at the end of the JAMM motif  background [BRCC3  , 27. Botnd NLRP3 , 27. NLRlls Fig. . When weI44 Fig.  as well ckground . ConsistCC3 Fig. .Fig. 4InBRCC3 inactivation on cytokine release systematically using a cytokine array in unstimulated Kasumi-1 cells. This revealed an upregulation of several cytokines in BRCC3 KO cells with G-CSF (1.75 ± 0.03) being the top hit . For this purpose, either parental or CRISPR-mediated stable BRCC3 KO Kasumi-1 cells  AML. In a large sequencing study of CBF AML we found recurrent inactivating mutations in BRCC3 in t AML with a frequency of 4.7% but not in inv(16)(p13.1q22) AML [BRCC3 mutations were found in 5 patients in a recent study of 331 t AML cases [BRCC3 mutations for t AML. In our cohort, BRCC3 mutations had no impact on preclinical characteristics but were associated with a favorable outcome with the caveat that the case number is small. Seven out of the nine BRCC3 mutations were located within the catalytically active MPN + domain, four of them at the exact position as mutations previously found in MDS, while the other three were located in close proximity [BRCC3 mutations found in AML and MDS are predicted to be deleterious and are hemizygous we hypothesized that loss of BRCC3 function contributes to malignant transformation of hematopoietic cells in a specific genetic context.Here, we investigated the role of q22) AML . ConsistML cases , but in ML cases . These rroximity . Given tBRCC3 mutations with t we found a pro-proliferative effect of BRCC3 inactivation in the t AML cell lines Kasumi-1 and SKNO-1 but not in a cell line without t. CRISPR-mediated BRCC3 inactivation alone did not enhance the self-renewal capacity in mouse hematopoietic progenitor cells (LSK) implying that loss of BRCC3 alone is not sufficient for malignant transformation. In contrast, self-renewal capacity was markedly increased in LSK cells when BRCC3 was inactivated together with expression of AML1-ETO supporting that both genetic alterations cooperate.In accordance with the close association of BRCC3 contributes to malignant transformation. One possible mechanism would be that loss of BRCC3 in the BRCA1-A complex leads to impaired DNA damage repair what may increase the likelihood for acquisition of genomic aberrations that activate oncogenic pathways. However, we considered this possibility for several reasons unlikely. First, in our AML t cohort no increase in secondary cytogenetic aberrations was observed and in MDS, BRCC3 mutations are associated with a normal karyotype [BRCC3 mutations found in AML and also in MDS are subclonal, suggesting that they are a secondary event and not an initiating founder mutation that promotes further genetic alterations. Besides the role in DNA damage repair, BRCC3 is involved in other cellular pathways including cytokine signaling and regulation. BRCC3 as part of the BRISC-complex deubiquitinates its substrate IFNAR1 which affects interferon signaling [BRCC3 mutations abrogate the DUB activity on IFNAR1 resulting in an attenuated interferon response in AML cells. In a study by DeKelver et al. it was shown that AML1-ETO induces strong interferon signaling and that this antagonizes the leukemic potential of AML1-ETO [IFNAR1 has not been found mutated or deleted in t AML, we could show that BRCC3 mutations render t AML cells less sensitive to IFNα-mediated toxicity through impaired deubiquitination and function of IFNAR1. This provides a possible explanation for the close association of BRCC3 mutations with t. We also demonstrate that AML cells with BRCC3 inactivation had an impaired inflammasome activity with decreased IL-1β release after activation. In our models, IL-1β had no negative effect on cell proliferation of AML1-ETO cell lines. However, inflammasome-driven IL-1β has been found to suppress AML proliferation in other models [BRCC3 mutations.We next sought to investigate how inactivation of aryotype . Second,ignaling . We founAML1-ETO . While Ir models . Thus, dBRCC3 inactivation leads to deregulation of release of other cytokines by AML cells. Although not sufficient to induce cytokine-independence in several cell lines, some of the more released cytokines in BRCC3 inactivated AML cells like G-CSF, HGF, and IL-4 have been shown to stimulate proliferation of AML cells [BRCC3 WT, but not to other cell lines with a different cellular background including inv(16)(p13.1q22), which further links BRCC3 mutations to t AML. As loss of BRCC3 on a subclonal level was sufficient to increase cell proliferation, this implies that BRCC3 mutations at least in part act through a paracrine mechanism by release of G-CSF. A paracrine activation of AML cells would also explain how subclonal BRCC3 mutations, as present in some of the AML and MDS patients, stimulate proliferation of nonmutated clones through pro-proliferative cytokines. Remarkably, primary AML cells and cell lines with t have a higher expression of G-CSF receptor molecules (G-CSFR) than other AML subtypes [BRCC3 was associated with an increased K63-ubiquitination and activation of JAK2 [BRCC3 inactivation enhances JAK2 signaling by two mechanisms. Remarkably, JAK2 signaling is a particularly important driver in t AML as indicated by the presence of activating JAK2 mutations specifically in this but not other AML subtypes [In addition to IL-1β and interferon signaling, we found that ML cells –43. We wsubtypes , 44. G-Csubtypes . Of note of JAK2 . Therefosubtypes .BRCC3 mutations would suggest that they are associated with a more aggressive disease. However, the BRCC3 mutated t AML patients in our cohort had an excellent outcome. In addition, in the TCGA dataset of de novo AML lower BRCC3 expression was associated with a better overall survival [BRCC3 inactivation leads to an impaired capability of the BRCA1-A complex to repair DNA damage and subsequently higher sensitivity to DNA damaging chemotherapy. In fact, none of the BRCC3 mutated patients in our cohort that received intensive chemotherapy relapsed. Consistently, downregulation of BRCC3 and its complex members has been previously associated with a higher sensitivity to DNA damage inducing agents in other types of cancer [Enhanced self-renewal and proliferation by survival . Our obsf cancer , 37.BRCC3 mutations lead to altered ubiquitination of its substrates and cytokine release which cooperates with AML1-ETO to induce AML and sensitizes the leukemic cells to cytotoxic chemotherapy. Future studies are needed to investigate the clinical impact of BRCC3 mutations in larger cohorts in the context of different treatments and elucidate whether altered ubiquitination of additional, yet unknown substrates of BRCC3 contributes to the development of AML and MDS.In conclusion, we demonstrate that Supplement


Single solute experiments with Suwannee river NOM (SRNOM) performed with environmentally-relevant concentrations showed MWCNT surface saturation at initial SRNOM concentrations between 10 and 15 mgC/L, for water pH between 5.2 and 8.7. These results suggested that at NOM:virus ratios found in natural waters, the NOM would competitively suppress virus adsorption onto MWCNT, even at low NOM concentrations. We confirmed this expectation with SRNOM-MS2 co-solute experiments, which showed an exponential decrease of the MS2 LRV by MWCNT with an increase in the initial SRNOM concentration. More interestingly, we showed that pre-equilibrating MWCNT with a SRNOM solution at a concentration as low as 0.4 mgC/L resulted in a LRV decrease of 3 for MS2, due to the formation of a negatively charged SRNOM adlayer on the MWCNT surface. Complementary batch experiments with natural NOM-containing waters and competition experiments with SRNOM in the presence of CaCl2 confirmed that the presence of NOM in waters challenges virus removal by MWCNT-filters, irrespective of the concentration and type of NOM and also in the presence of Ca2+. We therefore conclude that MWCNT-filters produced with commercially available pristine MWCNT cannot be considered as a viable technology for drinking water virus removal.A leading challenge in drinking water treatment is to remove small-sized viruses from the water in a simple and efficient manner. Multi-walled carbon nanotubes (MWCNT) are new generation adsorbents with previously demonstrated potential as filter media to improve virus removal. This study therefore aimed to evaluate the field applicability of MWCNT-filters for virus removal in water containing natural organic matter (NOM) as co-solute to viruses, using batch equilibrium experiments. Contrary to previous studies, our results showed with MS2 bacteriophages single-solute systems that the affinity of MWCNT for MS2 was low, since after 3 h of equilibration only 4 log  •LRV of MS2 increase with MWCNT mass, but are little affected by solution pH.•SRNOM adsorption onto MWCNT is pH dependent.•SRNOM competes with MS2 for adsorption to MWCNT, even at low SRNOM concentrations.•NOM suppresses MS2 adsorption, irrespective Ca2+ concentration and NOM composition.•In practice, MWCNT is insufficiently selective for viruses to ensure virus removal. In another study, MS2 removal by a MWCNT filter was between 5 and 8 LRV at low pressure and LRV was higher than what was achieved using a SWCNT filter (Carbon nanotubes (CNT) are broadly considered promising materials for future water treatment applications, including filtration and/or adsorption. The unique features of CNT include high surface areas, hydrophobicity, porosity, rapid adsorption kinetics, simple regeneration techniques and good mechanical and thermal stability. As a result, CNT are promising alternatives to traditional adsorbents . Since tT filter , 2010c. T filter . In addi3+)) at pH > IEP of MS2. Nonetheless, 2+, Mg2+ and Al3+. Higher adsorption of MS2 to sandy loam soil in presence of oxidized metal ions like ferric oxihydroxides was noted by 2 improved MS2 virus removal by MWCNT-filters in presence of NOM. NOM competitive effect mitigation during virus removal by MWCNT-filters in presence of Ca2+ may result from Ca2+ forming cationic bridges between negatively charged carboxylate and phenolate groups in the NOM adlayer and the negatively charged amino acids on the MS2 virion surface. The cation bridges formation would increase MS2 adsorption through the formation of NOM-Ca2+-MS2 complexes, as compared to Ca2+-free solutions, thereby overcoming direct MS2-NOM electrostatic repulsion  that can affect the efficiency of the MWCNT-filters by clogging the filter. Applying a first pre-filtration step, like gravity-driven membrane (GDM) filtration, would eliminate the potential interference from suspended particles . Howeverepulsion . Evidenc2+ on MS2 removal in SRNOM-Ca2+ batch experiments. The results from this work inform on the efficacy of MWCNT to produce virus-filters, a necessary first step toward application in the field for drinking water treatment.The goal of our work was to experimentally assess the competitive co-adsorption between MS2 bacteriophages and NOM to MWCNT. This information is critical to inform the feasibility of using MWCNT as adsorbent to produce filters to remove virus from waters that contain different types and concentrations of NOM and/or ionic compositions. To do so, we studied adsorption of MS2 bacteriophages to MWCNT in batch reactors, both in the absence and presence of NOM as co-adsorbate. MS2 bacteriophages were used as a surrogate for apolar and negatively charged enteric viruses of human health concern, such as Adenovirus . Further22/g. Their length and outer diameter were equal to 10–30 3 (65%) and H2O2. Details about metal impurities are provided in the supplementary information . The MWCNT had a specific surface area of 117 mormation .Prior to use MWCNT in batch adsorption experiments, MWCNT were bath-sonicated in ethanol . The resulting suspension was subsequently vacuum-filtered onto 0.1 2PO4 and 0.58 g/L NaCl, IS = 26 mM, pH = 4.75), which was used as a dilution buffer for all batch experiments, and stirred overnight to ensure complete dissolution of SRNOM. The pH of the SRNOM stock solution was then adjusted to the targeted pH required  by adding small volumes of 5 M NaOH. The dissolved organic carbon (DOC) of the resulting SRNOM solutions was quantified using a TOC-L total carbon analyzer .Suwannee river natural organic matter  served as model NOM and was purchased from the International Humic Substances Society (IHSS). SRNOM stock solution was prepared by adding 0.05 g of SRNOM into 500 mL of buffer solution  and ion chromatography (Metrohm 930 Compact IC Flex), respectively.To demonstrate the transferability of the results obtained with the model NOM to NOM in natural water samples, we also determined MS2 adsorption to MWCNT in water samples that we collected from four different sources: tap water, ground water, river water and wastewater treatment plant secondary effluent. We collected these water samples at Eawag . More specifically, river and ground water were collected from the sampling stations of Chriesbach river and the local aquifer, respectively. The secondary effluent was sampled from Eawag’s pilot-scale wastewater treatment plant outlet. After collection, the samples were filtered through sterile 0.45 2+ concentrations used in our study are based on a literature review we have made from 85 studies that reported water chemistry characteristics of natural water samples from 42 countries. The data from the literature review were used to calculate average, upper and lower values (5th and 95th percentiles), thereby capturing the range of values found in natural waters that can be used as a drinking water source. The references of these studies are available in supplementary information  followed by adding the targeted solution (20 mL), as shown in i) and thus accounted for any potential dilution variability, decay and adsorption to the glass vial during the batch experiment .After 3 h mixing at 40 rpm with a rotating mixer at room temperature, the samples were filtered through sterile 0.45 b) was then subtracted from the DOC value measured after the single solute batch experiment with SRNOM and MWCNT (DOCf), in order to estimate the SRNOM adsorbed mass per mass of MWCNT, as described in Equation e(SRNOM) is the SRNOM adsorbed mass per mass of MWCNT (mgC/g), DOCi is the initial DOC concentration (mgC/L) of the SRNOM solution measured after pH and concentration adjustment and prior to the batch experiment, DOCf is the DOC concentration measured after the single solute batch experiment performed in presence of SRNOM and MWCNT (mgC/L), DOCb is the DOC value from the blank measured to evaluate the DOC released from MWCNT (mgC/L), V is the volume of the batch reactor (L), and mMWCNT is the MWCNT mass used in each batch experiment (g).A reactor with 15 mg MWCNT in buffer solution was also ran as a blank for single solute SRNOM adsorption experiments, in order to quantify the amount of DOC that leached from the MWCNT. This DOC value (DOCE. coli host (DSMZ 5695) were purchased from DSMZ German Collection of Microorganisms and Cell Cultures . MS2 stock solution was prepared at the beginning of the study by amplifying MS2 commercial stock solution in 1 L of E. coli culture. After the amplification step, the culture was filtered over a 0.22 μm PES vacuum filter membrane to remove E. coli cells. Cell removal prevents any proliferation of MS2 during the batch adsorption experiments. Following filtration, we additionally purified the virus stock solution using centrifugal membrane filters  before using the MS2 solution in experiments. It was previously shown that the additional centrifugal membrane filtration step is critical to remove lower molecular weight organics that may heavily interfere with MS2 adsorption to sorbent surfaces  and its associated surfaces .E. coli host in soft agar (0.7% Agar) and poured onto a hard agar plate (1.5% Agar). After overnight incubation at 37 °C, plaques formed from the bacteriophages were counted. MS2 LRV was calculated as follows:i (PFU/mL) is the concentration of MS2 available for adsorption (measured from the blank experiment containing no MWCNT) and Cb (PFU/mL) is the final MS2 concentration after the adsorption batch experiment in the presence of MWCNT. To evaluate MWCNT MS2 adsorption capacity and compare it to the SRNOM adsorption capacity, the adsorbed mass of MS2 per mass of MWCNT , mg/mg) was calculated, considering that the total number of MS2 capsids in solution is higher than the total number of PFU, given that not all virions are infective (The double agar layer assay was used to enumerate infectious bacteriophages (PFU/mL) , combined with the PFU/mL concentration measured with the double layer assay, were used to calculate the number of RNA-virions and RNA-free-virions, referring to individual virions with and without RNA in the assembled capsid, respectively. These values were taken from the table provided in supporting information of The ratios between the different MS2 subpopulations (i.e. Rinfectious-virions) was calculated using Equation infectious-virion/plaque-forming-units is the ratio of infectious virions to plaque forming units (PFU), which we assumed to be equal to 1, and V as the volume used for the batch experiment (mL).Briefly, the number of infectious virions adsorbed onto MWCNT during batch experiment  was calculated using Equation Based on NRNA-free-virions) was calculated using Equations total-virions is the total number of virions present in solution, accounting for both RNA-containing and RNA-free virions  and a single capsid (Mcapsid = 4.18∗10-18 g), the mass of virions adsorbed per mass of MWCNT was calculated using Equation (7)Then, the number of RNA-free-virions (N virions .(6)NRNA− be 0.07 . Based o2 present in water after mixing the sample with phosphoric acid . Then, the sample was exposed to UV-light in the lower part of the Gräntzel thin-film reactor to produce hydroxyl radical (.OH). C-compounds were fully oxidized by hydroxyl radicals to CO2, which was subsequently analyzed with a Siemens IR detector. More detailed information on LC-OCD-OND is available in . Phosphate-buffer  was used as eluent and the flow rate was set at 1 mL/min. After the chromatographic column, the flow was split into a Gräntzel thin-film reactor (C-compounds oxidation) and a special DONOX-reactor . The upper part of the Gräntzel thin-film reactor was shielded from the UV-bulb to allow stripping of the COlable in .The obtained chromatograms are divided in five fractions according to the NOM molecular weight (MW), i.e. biopolymers , humics (MW∼1000 Da), building blocks (MW∼300–500 Da), low molecular weight (LMW) organics (MW < 350 Da) and neutrals (MW < 350 Da), as described in .3p < 2.2∗10-16), with a R2 of 0.955. MS2 LRV was significantly influenced by MWCNT mass , but neither pH 7.7  nor pH 8.7  were statistically significant relative to pH 5.2. This finding strongly suggests that the higher the MWCNT mass, the higher the MS2 LRV, as visible in a = 4–5)  . Nonethea = 4–5)  and theya = 4–5) . Furthera = 4–5) .Fig. 2MSp = 2.8∗10-14), with a R2 of 0.974. MS2 LRV predicted is equal to -0.14 + 0.28 (MWCNT mass) when MWCNT mass is measured in milligrams. At an initial MS2 concentration of 106 PFU/mL, MS2 LRV increased by 0.28 for each mg of MWCNT, over the pH range tested. Given that the multiple linear regression showed that pH had no significant effect on MS2 LRV, a single linear regression was calculated from all data points from the batch experiments conducted at pH 5.2, 7.7 and 8.7 to predict the MS2 LRV based on MWCNT mass . A signi batches , 2016. G3 PFU/L or 101-103 gene copy/L in river water and that these viruses adsorb similarly to MS2 , with a R2 of 0.999. The adsorbed mass of MS2 per mass of MWCNT predicted is equal to 2.1∗10-4+1.6∗10-10  when MS2 initial concentration is measured in PFU/mL. The adsorbed mass of MS2 per mass of MWCNT increased by 1.6∗10-10 mg/g per PFU/mL of MS2. At the maximum initial MS2 concentration tested (i.e. 109 PFU/mL), the adsorbed mass of MS2 per mass of MWCNT was 0.16 ± 0.002 mg/g, corresponding to a total capsid number of 4∗1011 ± 4∗109 adsorbed per 15 mg (corresponding to a MWCNT surface area of 1.75∗104 cm2).A single linear regression was calculated to predict the adsorbed mass of MS2 per mass of MWCNT based on the initial MS2 concentration . A signi9 PFU/mL, the total number of virions adsorbed to the MWCNT covered a surface of 2.6 cm2, corresponding to only 0.015% of the MWCNT total surface. This is approximately 2000 times lower than the hypothetical maximum surface coverage  if one assumes random sequential adsorption of individual MS2 virions up to the surface jamming limit, following 2, while in our study the highest adsorbed MS2 concentration was only 0.13 ng/cm2 (at a MS2 concentration of 109 PFU/mL). The differences in the adsorbed mass solely reflect the different experimental set-ups for adsorption in both studies. In 12 PFU/mL, calculated from 9 PFU/mL, the jamming limit of MWCNT surface was not reached.Based on the MS2 capsid diameter of approximately 28.8 nm, we estimated that at an initial MS2 concentration of 10Natural waters containing viruses also contain NOM as a co-solute that may interfere with virus removal by MWCNT-filters, as already observed by 2 and 19.3 ngC/cm2, respectively) at pH 7.7 and 8.7, respectively. These values are in agreement with ractions . In contractions ). The mo9H9NO6 (i.e. 1.79∗10-19 mgC), that assemblies can be modeled as rigid spheres having a diameter of 1 nm . Even for initial DOC concentrations where MWCNT surface saturation by SRNOM was not reached, MWCNT surface coverage by SRNOM was significantly higher than the surface coverage observed for MS2, irrespectively the initial MS2 concentration. Indeed, at low SRNOM concentration (i.e. 2.5 mgC/L), the calculated MWCNT surface coverage by adsorbed SRNOM was approximately 29%. This drastic difference between SRNOM and MS2 MWCNT surface coverage might be explained by the comparable or higher affinity of SRNOM to MWCNT in comparison to MS2 and/or to higher concentrations of SRNOM assemblies than MS2 capsids at the tested concentrations . The finding of competitive co-adsorption between MS2 and SRNOM at such low SRNOM concentrations is in apparent contrast to the finding in 3+) surface as investigated using QCM-D  would therefore be electrostatically repelled and not retained by the filter. Electrostatic repulsion of viruses from NOM adlayers was already demonstrated by 2+ on MS2 removal.Our results strongly suggest that virus adsorption by MWCNT-based filters is impractical from waters that also contain NOM. To demonstrate that the results obtained with SRNOM are transferrable to natural waters with different types of NOM, we subsequently analyzed competitive co-adsorption of MS2 and NOM in different natural water samples. Furthermore, we assessed the effect of Ca2+ (1 mM CaCl2) to a solution containing MS2 and alginate increased by 1.2 the MS2 LRV by a MWCNT-filter. The authors ascribed this increased removal in the presence of Ca2+ to the formation of cation bridges between negatively charged groups in the NOM adlayer and on the MS2 surface. Evidence for such cation bridges was provided already in earlier studies  and three different CaCl2 concentrations . LRV of MS2 increased with increasing Ca2+ concentration  from natural waters will likely be influenced by factors beyond the concentration of NOM as a co-solute. For example,  studies . Enhancentration A. Specifervation . However2+ alleviated competitive co-adsorption of SRNOM and MS2, we also conducted additional MS2 removal experiments with natural waters containing different types of NOM and a different ionic matrix than the dilution buffer used for the co-solutes batch experiments, to compare them to the previous batch experiments. Tap, ground and river water, as well as wastewater treatment plant secondary effluent, were sampled twice (December 2018 and January 2019) to obtain samples that contain a range of NOM types and sources, as well as Ca2+ concentration. The water sources ranged in Ca2+ concentrations from 1.6 to 2.6 mmolCa2+/L, and in DOC concentrations between 0.91 and 9.77 mgC/L. We characterized differences in size and chemical functions of the NOM in the different samples by LC-OCD-OND (2+ (2+. The difference in LRV value and trend observed between the batch experiments conducted in natural waters, in the DOC range of 0.91 and 3.53, and the SRNOM co-solute experiments conducted without Ca2+ may be due to the presence of divalent and trivalent cations in natural waters that might mitigate the competitive co-adsorption of MS2 and NOM on MWCNT, like Ca2+ that was present at similar concentrations in all natural water samples. Indeed, the MS2 LRV measured for natural waters having an initial DOC concentration between 0.91 and 3.53 mgC/L was in a similar range than the MS2 LRV measured for the batch experiment performed with an initial SRNOM concentration of 4.8 mgC/L and a Ca2+ concentration of 1.9 mmolCa2+/L.Given that Ca-OCD-OND . Specifi-OCD-OND . For DOC-OND (2+ , contrar2+, as described above (2+ concentration. The findings highlight that competitive co-adsorption is independent of natural water types and that the competition increased with increasing NOM:virus ratios and decreasing Ca2+ concentrations (Overall, the natural NOM samples, similar to SRNOM, lowered MS2 LRV in comparison with single solute experiment with MS2 and MWCNT, indicative of competitive co-adsorption. At low initial DOC concentrations, the competition was likely alleviated by the presence of divalent or trivalent cations, especially Caed above . Howevertrations  and 8.2+ concentrations.We therefore conclude that while MWCNT may be a promising material for adsorption of virus in the absence of NOM, its application to virus removal in natural waters is expected to not be suitable due to the presence of NOM, which undergoes competitive co-adsorption with the viruses. Competitive suppression of virus removal by NOM is expected to occur for NOM that vary broadly in chemical composition and also in waters that show varying Ca4•2+, competitively suppresses virus adsorption.This study provides evidence for competitive co-adsorption of MS2 and NOM, thereby highlighting that the occurrence of NOM as a co-solute in natural waters questions the suitability of MWCNT as a virus adsorbent to produce filters for POU systems. Despite the fact that MWCNT show outstanding adsorption properties in controlled systems, we show that the presence of NOM, even at low DOC concentration and in presence of Ca•Adsorption of SRNOM onto MWCNT impairs MS2 adsorption due to the formation of a negatively charged SRNOM adlayer on the MWCNT surface, resulting in electrostatic repulsion of the net negatively charged MS2 from the adlayer. This effect occurs irrespective of which MWCNT mass is used and irrespective of the initial NOM concentration. Indeed, batch experiments with MS2 and SRNOM as co-solutes showed that MS2 LRV decreased exponentially with increasing SRNOM concentration down to very low LRV values at the highest tested SRNOM concentrations.•2+ and natural water samples showed that NOM of different sources and with varying chemical compositions competed with MS2 for adsorption onto the MWCNT. Although dissolved Ca2+ presence might punctually alleviated the NOM competitive effect, we expect that for most natural waters, where virus:NOM ratio is low, MWCNT should not be considered promising adsorbents for the removal of negatively charged human viruses.Batch adsorption experiments with dissolved CaThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.