Background: The scaffolding protein caveolin-1 coordinates membrane signaling clusters, but how this affects Ca2+ oscillations is unknown.Results: Caveolin-1 accelerates the rundown of Ca2+ oscillations to the agonist leukotriene C4, which is prevented by modest inhibition of protein kinase C.Conclusion: Caveolin-1 increases receptor desensitization through Ca2+-dependent stimulation of protein kinase C.Significance: The findings reveal how caveolin-1 regulates receptor-dependent Ca2+ signaling. 2+ oscillations constitute a widespread signaling mode and are often generated in response to stimulation of G protein-coupled receptors that activate phospholipase C. In mast cells, repetitive Ca2+ oscillations can be evoked by modest activation of cysteinyl leukotriene type I receptors by the physiological trigger, leukotriene C4. The Ca2+ oscillations arise from regenerative Ca2+ release from inositol 1,4,5-trisphosphate-sensitive stores followed by Ca2+ entry through store-operated Ca2+ channels, and the latter selectively activate the Ca2+-dependent transcription factor NFAT. The cysteinyl leukotriene type I receptors desensitize through negative feedback by protein kinase C, which terminates the oscillatory Ca2+ response. Here, we show that the scaffolding protein caveolin-1 has a profound effect on receptor-driven Ca2+ signals and downstream gene expression. Overexpression of caveolin-1 increased receptor-phospholipase C coupling, resulting in initially larger Ca2+ release transients of longer duration but which then ran down quickly. NFAT-activated gene expression, triggered in response to the Ca2+ signal, was also reduced by caveolin-1. Mutagenesis studies revealed that these effects required a functional scaffolding domain within caveolin-1. Mechanistically, the increase in Ca2+ release in the presence of caveolin-1 activated protein kinase C, which accelerated homologous desensitization of the leukotriene receptor and thereby terminated the oscillatory Ca2+ response. Our results reveal that caveolin-1 is a bimodal regulator of receptor-dependent Ca2+ signaling, which fine-tunes the spatial and temporal profile of the Ca2+ rise and thereby its ability to activate the NFAT pathway.Cytoplasmic Ca Receptor desensitization is a universal and conserved mechanism that attenuates responses evoked by prolonged stimulation. The kinetics of receptor desensitization vary over orders of magnitude. Kainate receptors desensitize within milliseconds , whereas2+ oscillations  evokes cytoplasmic Ca2+ oscillations. The CysLT1 receptor shows homologous desensitization through which protein kinase C, including the Ca2+-dependent α isoform  isoform , phospho isoform . Acute itization , likely 2+ signals to occur, thereby bypassing the deleterious consequences of a sustained Ca2+ rise that include excitotoxicity and Ca2+-dependent inhibition of signaling molecules. Mechanisms that control the rate and extent of receptor desensitization will therefore have a profound influence on the spatiotemporal pattern of agonist-evoked Ca2+ signals and the subsequent activation of downstream targets. Here we report that the scaffolding protein caveolin-1 enhances desensitization of CysLT1 receptors. The amplitude of Ca2+ oscillations is initially increased by caveolin-1, because of enhanced coupling between the receptor and phospholipase C. However, the increased Ca2+ mobilization stimulates Ca2+-dependent protein kinase C, which then terminates the oscillatory response by accelerating receptor desensitization. Our work identifies caveolin-1 as a bimodal regulator of intracellular Ca2+ signals.Reversible receptor desensitization enables phasic Ca2 as described (The rat mast cell line RBL-1 was purchased from ATCC (via United Kingdom supplier LCG Standards). For regular maintenance, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% COescribed . For expWild type caveolin-1 tagged with EGFP was kindly provided by Dr. Suetsugu  , and theGCCACTGTGGCAAAATATTGGTTTTACCG-3′ and antisense primer 5′-CGGTAAAACCAATATTTTGCCACAGTGGCGGTGGTGAAGCTGGCCTT-3′; for human caveolin-1 (tagged with RFP), sense primer 5′- AAGGCCAGCTTCACCACCGCCACTGTGGCGAAATACTGGTTTTACC-3′ and antisense primer 5′- GGTAAAACCAGTATTTCGCCACAGTGGCGGTGGTGAAGCTGGCCTT-3′.The scaffolding domain mutant caveolin-1  was generated by using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies) with primers list as follows: for mouse caveolin-1 (tagged with EGFP), sense primer 5′-AAGGCCAGCTTCACCACCm NaCl, 2.8 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mmd-glucose, and 10 mm HEPES, pH 7.4, with NaOH as described  for 1 h and then incubated with specific primary antibody against FLAG tag (Sigma-Aldrich). Secondary antibody against rabbit IgG was conjugated with Alexa-488 purchased from Invitrogen. Images were obtained by using an Olympus confocal microscope. Relative fluorescence intensity was analyzed using ImageJ software. For cells transfected with GFP-PHD, immunofluorescence images were obtained with a Leica microscope, and the fluorescence intensity was analyzed by ImageJ software. For colocalization studies, confocal images were taken with an FV-1000 confocal microscope , and the colocalization coefficient between two different channels was assessed by the Olympus Fluoview FV1000 system. At least five representative images in each group were used for analysis and 10 different areas on the cell membrane and in the cytosol were selected to obtain Pearson's correlation coefficient.4 (see text for specific times). The percentage of GFP-positive cells was measured as describe previously . Statistical significance was set at p < 0.05, with one, two, and three asterisks denoting p < 0.05, 0.01, and 0.001, respectively.All results were expressed as mean ± S.E. A two-tailed Student's 2+ oscillations. In non-transfected (wild type) cells, stimulation with LTC4 evoked a series of cytoplasmic Ca2+ oscillations (A), which decreased slightly over time due to receptor desensitization (B) . The amplitudes of the initial Ca2+ oscillations evoked by LTC4 were now considerably larger than in non-transfected cells (A and C), but the oscillations ran down more quickly and so were fewer in number over a 600 to 700-s recording period . Analysis of the various oscillatory parameters revealed that the total Ca2+ rise associated with each oscillation (area under the spike) was significantly larger in cells expressing caveolin-1-GFP ; this reflected both an increase in the amplitude of each Ca2+ oscillation  as well as an increase in duration . Cytoplasmic Ca2+ during each oscillation was therefore elevated for a longer time in the presence of caveolin-1-GFP.Endogenous levels of caveolin-1 were virtually undetectable in Western blots from RBL-1 cells (data not shown), so we overexpressed the GFP-tagged protein to study its impact on Callations A, which tization B 6). Ex. Ex2+ osB and C). However, caveolin-1-RFP replicated the marked effects of caveolin-1-GFP on the pattern of Ca2+ oscillations (data not shown).The effects of caveolin-1-GFP were not mimicked by expression of GFP alone , B and C2+ reflect both InsP3-dependent Ca2+ release and Ca2+ influx through CRAC channels, the latter being required to replenish the stores with Ca2+ in readiness for the next oscillatory cycle. To see which of these processes was affected by caveolin-1, we separated Ca2+ release from Ca2+ entry by stimulating cells with LTC4 in the absence of external Ca2+ and then readmitting external Ca2+ once the oscillations had run down. Because of the lack of Ca2+ influx, Ca2+ oscillations decreased in size over time and were lost typically within 400 s after stimulation . Readmission of external Ca2+ after 600 s resulted in Ca2+ entry through CRAC channels . Expression of caveolin-1-GFP increased the amplitude of the Ca2+ oscillations in Ca2+-free solution considerably , but these oscillations ran down more quickly than the corresponding control recordings . Readmission of external Ca2+ led to a significantly larger rate of rise of Ca2+ (F and I), indicating increased store-operated Ca2+ influx. Unlike the case of caveolin-1-GFP, expression of GFP alone had no effect on the number of Ca2+ oscillations , the size of the oscillations , or store-operated Ca2+ entry . A significant fraction of caveolin-1-RFP was also found in the cytoplasm, likely reflecting its contribution to vesicle sorting . CysLT1 receptor distribution showed two clear peaks, corresponding to plasma membrane at the two edges of the cell . Although caveolin-1-RFP was present within the cytoplasm, two peaks at the cell periphery were also resolvable, indicating a plasma membrane location. We quantified the extent of overlap of the two proteins using Pearson's correlation coefficient . Under both basal and stimulated conditions (LTC4 exposure for 10 min), there was a much better correlation between FLAG-tagged CysLT1 receptor and caveolin-1-RFP in the membrane than in the cytoplasm, and stimulation did not change the correlation coefficient .Caveolin-1 increases the interaction between the heterotrimeric GTP-binding protein Glipase C , a mechamembrane A. A sign sorting . To test3 levels should increase more following stimulation in the presence of caveolin-1 than in wild type cells, and second, less Ca2+ should remain within the InsP3-sensitive store after the Ca2+ oscillations have run down in cells expressing caveolin-1. Using the GFP-PHD construct as a means for monitoring InsP3 levels in individual cells , and this was slightly more pronounced when caveolin-1-RFP was expressed . To test the second prediction, we stimulated cells with LTC4 in the absence of external Ca2+, and then once the oscillations had stopped, we applied thapsigargin in Ca2+-free solution to estimate how much Ca2+ remained within the store (C). The thapsigargin-mobilizable Ca2+ pool was significantly reduced in cells expressing caveolin-1-GFP .If caveolin-1 increases receptor-phospholipase C coupling, two predictions are that, first, InsPal cells –17, we fhe store C. The th92FTVT95, is critical for association with G proteins  and threonine (Thr-95) had been mutated to alanines, several Ca2+ oscillations were seen in Ca2+-free solution (A); these were similar in size to those obtained in wild type cells (B). The number of oscillations in Ca2+-free solution (data not shown) and the rate of rise of the Ca2+ signal due to store-operated entry were also not significantly different from control cells (C). Cytoplasmic Ca2+ oscillations in response to LTC4 showed only modest rundown when transfected with the mutated caveolin-1 (D), which was not different from wild type cells (E). The size of these oscillations was also similar to that in wild type cells (F).The scaffolding domain of caveolin-1, which involves amino acids between residues 82 and 101, is required for interaction with receptors, G proteins, and other signaling molecules , 19. A cproteins . To detesolution A; these pe cells B. The nuol cells C. Cytoplveolin-1 D, which pe cells E. The sipe cells F.2+ oscillations. We therefore compared GFP fluorescence in cells transfected with either caveolin-1-GFP or F92A,T95A caveolin-1-GFP. There was no difference in either the profile of GFP expression between the two groups (G) or the averaged GFP fluorescence between the groups (H). In Fig. 4I, Ca2+ signals evoked by LTC4 are compared between a cell expressing caveolin-1-GFP and one expressing F92A,T95A caveolin-1-GFP. The cells had almost identical levels of GFP expression . However, only the presence of caveolin-1-GFP altered the pattern of the Ca2+ oscillations. Confocal images showed that both caveolin-1-RFP and F92A,T95A caveolin-1-RFP were expressed at the plasma membrane with FLAG-tagged CysLT1 receptors . Pearson's correlation coefficient between mutant caveolin-1-RFP and CysLT1 receptors was similar to that seen for caveolin-1-RFP and the receptors (C). Collectively, these results show that the scaffolding domain of caveolin-1 is important for the modulation of agonist-evoked Ca2+ oscillations.We considered the possibility that expression of F92A,T95A caveolin-1-GFP was considerably lower than caveolin-1-GFP, thereby explaining the lack of effect of mutant caveolin-1 on Cao groups G or the e groups H. In Figeceptors C. CollecJ) and mimicked the effects of caveolin-1-GFP expression on agonist-induced Ca2+ oscillations. The initial Ca2+ transients were larger , and fewer oscillations were obtained . Internalization of caveolin-1 through phosphorylation of tyrosine 14 therefore does not contribute to the effects of caveolin-1 on LTC4-driven Ca2+ signals.Phosphorylation of caveolin-1 on tyrosine 14 by Src family kinases potentiates growth factor signaling and is required for internalization of caveolae . ExpressFig. 42+ oscillations seen in the presence of caveolin-1. To see whether this was dependent on Ca2+ release or Ca2+ entry, we stimulated cells in the absence of external Ca2+ but with the plasma membrane Ca2+ pump blocked with La3+. Under these conditions, Ca2+ release can no longer be exported out of the cell and instead is sequestrated back into the stores. Ca2+ oscillations therefore continue for several minutes, reflecting regenerative Ca2+ release in the absence of Ca2+ influx (4 in wild type cells evoked a series of repetitive Ca2+ oscillations that decreased slightly in number over time (A and B). By contrast, in cells expressing caveolin-1-GFP, larger Ca2+ spikes were obtained initially, which then ran down quickly (A and B). As with the responses in the presence of external Ca2+, the amplitude of the first oscillation (C), as well as the duration of the oscillations (D), was significantly increased in the presence of caveolin-1-GFP. Rundown of Ca2+ oscillations in the presence of caveolin-1 therefore arises from Ca2+ release.We designed experiments to identify the mechanism responsible for the accelerated rundown of Ca+ influx , 22. Stiver time , A and B quickly , A and Billation C, as welllations D, was si2+ release from the stores in caveolin-1-expressing cells contributes to the rundown of the oscillations is shown in 2+ content of the stores in order to reduce the size of each Ca2+ oscillation upon stimulation. We therefore incubated control (non-transfected) cells in Ca2+-free solution for 10 min and found that this was sufficient to reduce the extent of Ca2+ release by thapsigargin by ∼ 30% when compared with control cells pre-exposed to Ca2+-free solution for just a few seconds prior to stimulation with thapsigargin (A and C). We then stimulated cells in Ca2+-free solution containing 1 mm La3+ to eliminate the increased Ca2+ influx due to the reduced store Ca2+ content from affecting the oscillatory pattern. Oscillatory Ca2+ responses to LTC4 were sustained both in cells pretreated with Ca2+-free solution acutely (D and I) and in those following 10 min of pretreatment (E and I), although the size of the oscillations was smaller in the latter case (H), reflecting the reduced store Ca2+ content. In cells expressing caveolin-1-GFP and incubated in Ca2+-free solution for 10 min, the extent of Ca2+ release induced by thapsigargin was similar to control cells treated in the same way (B and C). Whereas only a few Ca2+ oscillations were seen in response to LTC4 challenge in caveolin-1-GFP-expressing cells exposed to Ca2+-free solution for a few seconds prior to stimulation (F and I), preincubation for 10 min with Ca2+-free external solution resulted in more prolonged oscillatory Ca2+ signals following agonist stimulation (G and I). The amplitude of the first Ca2+ oscillation was reduced following the 10-min preincubation in Ca2+-free solution prior to stimulation (H). Hence, lowering the Ca2+ content of the stores results in prolonged oscillatory Ca2+ signals in the presence of caveolin-1-GFP. These results are consistent with the view that the enhanced Ca2+ release normally seen in caveolin-1-expressing cells is responsible for the accelerated rundown of the oscillations.Further evidence that Casigargin , A and C acutely , D and Ireatment , E and Iter case H, reflecsame way , B and Cmulation , F and Imulation , G and Imulation H. Hence,2+ release can increase the rundown of Ca2+ oscillations is through Ca2+-dependent inactivation of InsP3 receptors. However, the Ca2+ release transient following phospholipase C-coupled P2Y receptor activation after CysLT1 receptors had been desensitized was slightly larger in caveolin-1-expressing cells (B) than in the corresponding controls . Inactivation of the InsP3 receptor therefore plays little role in the rundown of Ca2+ oscillations in the presence of caveolin-1.One way whereby enhanced Cang cells B than in2+ oscillations was a consequence of the accelerated desensitization of the CysLT1 receptor. These receptors are desensitized following Ca2+-dependent protein kinase C-mediated phosphorylation of a series of serine residues on the carboxyl terminus of the receptor, and we had previously found a major role for protein kinase Cα in the desensitization process  to reduce but not abolish kinase activity, as substantial block of the kinase results in non-oscillatory Ca2+ signals (2+ response in wild type cells induced by LTC4 stimulation (A) was only weakly affected by the low concentration of Go6983 (B and J). However, the rapid rundown of Ca2+ oscillations in cells expressing caveolin-1-GFP (C) was largely prevented by the protein kinase C inhibitor (D and J). Identical results were obtained with a structurally different protein kinase C blocker, GF109203X . Many agonists of G protein-coupled receptors elicit responses by occupying only a fraction of the total receptors. We therefore reasoned that increasing the number of available CysLT1 receptors in the plasma membrane in cells expressing caveolin-1-GFP should lead to an increased likelihood for LTC4 to encounter a non-desensitized receptor, which should reduce the rate of rundown of Ca2+ oscillations. We therefore transfected cells with plasmids for caveolin-1-GFP and the CysLT1 receptor. Increased expression of CysLT1 receptors significantly prolonged the oscillatory Ca2+ response compared with cells transfected with caveolin-1-GFP alone (I and J). Despite coupling to phospholipase C via Gq proteins, P2Y receptor-driven Ca2+ release was unaffected by caveolin-1-GFP expression ). This suggests that P2Y and CysLT1 receptors might couple to phospholipase C differently, with the leukotriene receptor more prominent in caveolin-1-rich domains. Lipid rafts can be disrupted by methyl-β-cyclodextrin (MβCD), a compound that removes cholesterol from the plasma membrane. Treatment with MβCD abolished LTC4-dependent Ca2+ responses (A) but had no significant effect on P2Y-evoked Ca2+ signals (B). Different agonists thus differ in their sensitivity to regulation by caveolin-1 and lipid rafts.We considered that rundown of the Ca process . Increasmulation A was onlf Go6983 , B and Jin-1-GFP C was larnhibitor , D and JFP alone , I and Jesponses A but had signals B. Differ2+ signaling by caveolin-1 had functional relevance, we measured Ca2+-dependent gene expression using a GFP construct under a promoter driven by the Ca2+-dependent transcription factor NFAT (A), but it increased ∼4-fold after LTC4 was added to the culture medium. Basal gene expression was also low in caveolin-1-RFP-expressing cells, but stimulation resulted in a relatively weaker rise . Because NFAT activation is tightly linked to local Ca2+ entry through CRAC channels following physiological levels of stimulation in RBL cells (2+ release transients in the presence of caveolin-1 (C and E) increased Ca2+-dependent slow inactivation of CRAC channels . In fact, expression increased somewhat, in accordance with the increase in store-operated Ca2+ entry that arises from the more extensive store depletion . Because Ca2+-dependent slow inactivation requires a rise in bulk Ca2+, it can be prevented by the slow Ca2+ chelator EGTA , but it prevented the reduction in gene expression seen in the presence of caveolin-1-RFP . The reduction in LTC4-driven gene expression in caveolin-1-RFP-expressing cells was not seen when F92A,T95A caveolin-1-RFP was expressed instead . Gene expression was also impaired after lipid raft disruption with MβCD . The reduction in gene expression to LTC4 in cells expressing caveolin-1-RFP was prevented by pretreating cells with 1 nm Go6983 , a concentration that rescued repetitive Ca2+ signaling to agonist (To see whether the altered pattern of Cator NFAT , 23. In tor NFAT A, but itBL cells , 24, we channels , 26 and llations . Readmistor EGTA , 26. We  agonist .q and phospholipase C, thereby generating larger increases in InsP3 (2+-dependent responses following physiological levels of stimulation.Caveolin-1 is a conserved plasma membrane scaffolding protein that facilitates interaction between signaling molecules within subcompartments of the membrane. One such interaction involves enhanced coupling between Gin InsP3 . Our dat4 leads to repetitive Ca2+ oscillations, which reflect regenerative Ca2+ release followed by transient Ca2+ entry through CRAC channels (2+ oscillations can be converted into a more prolonged non-oscillatory Ca2+ rise by interfering with protein kinase C activity (2+ oscillations with larger amplitude and greater duration, as expected from increased Gq-phospholipase C coupling. However, the oscillations ran down more quickly and Ca2+-dependent gene expression was reduced following overexpression of caveolin-1. The rundown was not due to compromised store refilling or inactivation of the InsP3 receptors. Rather, the increased Ca2+ release in the presence of caveolin-1 led to stronger Ca2+-dependent activation of protein kinase C, which resulted in increased leukotriene receptor desensitization. Partial block of protein kinase C reversed the effects of caveolin-1 on oscillation amplitude, duration, rundown, and gene expression. The increase in size and duration of Ca2+ release in the presence of caveolin-1 would lead to enhanced Ca2+-dependent inactivation of CRAC channels (2+ microdomains near these channels activate gene expression, larger or prolonged Ca2+ release impairs transcription by reducing CRAC channel activity.Stimulation of CysLT1 receptors with LTCchannels . The Ca2activity . Proteinactivity . Overexpchannels , 26. BecCysLT1 receptors and caveolin-1 are co-expressed in various tissues, suggesting that the interaction we have described here might occur in other cell types as well. Airway smooth muscle expresses both CysLT1 receptors  and cave2+ release due to increased coupling between the receptor and phospholipase C both activates Ca2+-dependent protein kinase C, which leads to pronounced receptor desensitization, and accelerates Ca2+-dependent slow inactivation of CRAC channels. Activation of this pathway likely involves subcompartments within the membrane, as P2Y receptor-dependent Ca2+ release was unaffected by caveolin-1. By regulating desensitization, caveolin-1 is therefore an important determinant of the duration of receptor stimulation and thus of subsequent Ca2+-dependent downstream signaling.Our results reveal a novel mechanism for cytsteinyl leukotriene receptor desensitization involving caveolin-1. Enhanced Ca


Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca2+ ([Ca2+]c) dynamics and a distinct Ca2+ signature that involves Ca2+ influx from the external medium and internal Ca2+ stores. We investigated the molecular basis of this Ca2+ response by using [Ca2+]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca2+ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca2+ entry does not occur through the hitherto described high- and low-affinity Ca2+ uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca2+ release from internal stores following IP3 formation combines with the extracellular Ca2+ influx.The model organism  The ion binds to a diverse range of proteins, commonly at EF-hand or EF-hand-like domains, and promotes conformational and electrostatic alterations in these proteins. These interactions modulate their activity and contribute to a cascade of signaling events that ultimately defines a Ca2+-mediated cellular response to a stimulus  or in the membranes of organelles that act as intracellular stores. In fungi, Ca2+ uptake from the extracellular milieu can occur by different mechanisms, depending on the stimulus. The high-affinity Ca2+ uptake system (HACS) is the best described fungal Ca2+ uptake system. It comprises the channel Cch1 and the regulatory proteins Mid1 and Ecm7 (2+ system (LACS) is more active in nutrient-rich medium  is important for polarized growth. Hyphal growth in N. crassa has been reported to rely on a tip-high [Ca2+]c gradient that is not maintained by extracellular Ca2+ influx (3)-activated Ca2+ channels  to IP3 and diacylglycerol (DAG)  . IP3 promembrane , and a smembrane . Only thgradient . Howeverin fungi . The exiallenged . In this2+. The TRP channel family comprises several subfamilies: classical (TRPC), vanilloid (TRPV), melastatin (TRPM), polycystin (TRPP), mucolipin (TRPML) and ankyrin (TRPA)  channels, which are permeable to Can (TRPA) . In N. cis YVC-1 . In Sacc cytosol  after ac cytosol  and PIP2tosol . Peak A occurred immediately upon addition of staurosporine and lasted for ∼20 minutes, and peak B, having the greatest amplitude, occurred after 35–40 minutes and lasted for ∼80 minutes. UCN-01, a natural stereoisomer of 7-hydroxystaurosporine currently in clinical trials for cancer treatment . This supports the view that peaks A and B, that occur within the first 2 hours of treatment with staurosporine, correspond to specific Ca2+ signaling events upstream of cell death and do not involve non-specific Ca2+ entry into the cytosol due to plasma membrane disruption. The third phase (C) of the staurosporine-induced Ca2+ signature coincides with a significant increase in the percentage of propidium-iodide-positive cells .The levels of staurosporine-induced cell death, as measured by staining with propidium iodide, a reporter of plasma membrane permeabilization, were only significantly increased after 3 hours of incubation with the drug , our results point to the presence of Ca2+-permeable channels in the plasma membrane that are involved in generating these [Ca2+]c transients.In th phase . We aimeignature . BAPTA aignature . This in2+ signature was evaluated by treating cells with the ER-selective Ca2+-ATPase inhibitor thapsigargin. Thapsigargin causes the depletion of ER Ca2+ and prevents Ca2+ sequestration by this organelle , which is putatively homologous to the N. crassa protein NCU08166   . In N. cm the ER  and vacum the ER . Given tl growth , we hypo2+]c dynamics in response to staurosporine treatment in cells pre-treated with the phospholipase-C-selective inhibitor U-73122 (2+]c response over the entire 5-hour timecourse was greatly suppressed (2) has been used in N. crassa to inhibit inositol monophosphatase, thus preventing the synthesis of phosphoinositides  (2+-release-activated Ca2+ channel (CRAC). We pre-treated cells with the CRAC-selective inhibitor YM-58483  , we evalYM-58483  before i reduced . Phase CN. crassa (plc-1 (ΔNCU06245) and Δplc-3 (ΔNCU09655) strains were slightly more resistant, Δplc-2 (ΔNCU01266) was substantially more resistant than the wild-type strain . The difference was also evident after 24 hours of growth; whereas the wild-type strain was fully developed with 99.9% germination and long branched and fused hyphae, the Δplc-2 knockout mutant only underwent 58.5% germination, possessed swollen cells and a few elongated branched hyphae with little cell fusion evident. The presence of some germinated hyphae in Δplc-2 knockout cultures might be explained by the redundant activity of the other phospholipase C genes, as suggested previously . At the molecular level, HACS comprises the CCH-1 and MID-1 channel  exposed to staurosporine  was more similar to that of the wild-type cells, despite a slight [Ca2+]c amplitude increase in phase B and a decrease in phase C  is considered to be a regulatory partner of CCH-1  and mycelial extension rate , consistent with both proteins acting together.A drastic increase in the c signature during phases A and B in response to staurosporine  and YO-PRO1 staining to estimate apoptosis . Deletion of fig-1 resulted in a slight increase in susceptibility to staurosporine . Thus, the upregulation of a Ca2+ influx system in the absence of CCH-1 (and possibly MID-1) is correlated with increased cell death.Given the accentuated Δosporine , we assethe drug . This wa2+ influx can be mediated by TRP channels, which are known to be regulated by the phospholipase C pathway  (2+]c signature was similar in wild-type and Δyvc-1 cells (phases A and B), but [Ca2+]c during phase C was decreased in Δyvc-1 cells . A role for this protein in extracellular Ca2+ uptake would not be expected, because the yeast homolog is localized to the vacuolar membrane and this channel mobilizes Ca2+ from the vacuole to the cytosol  . The ini-1 cells . The gro cytosol . Thus, oN. crassa undergoes programmed cell death accompanied by the manifestation of several cellular phenotypes, such as DNA fragmentation, accumulation of reactive oxygen species (ROS), ceramide synthesis, compartmentalization of cells, glutathione export and activation of multidrug resistance proteins , which trigger a Ca2+ response with a characteristic [Ca2+]c signature. Some reports have shown an increase in [Ca2+]c in response to staurosporine  genes  genes . Of the e) genes . We obseongation . Our resg hyphae . These aidiation .N. crassa, treatment with the antifungal peptide PAF  and LACS (FIG-1) mechanisms  undergo more cell death than the wild-type cells when treated with staurosporine. This is consistent with the observation that Δcch-1 and Δmid-1 cells are less tolerant to high levels of Ca2+ . In line with our results, lack of CCH-1 and MID-1 renders S. cerevisiae and Cryptococcus neoformans cells very sensitive to ER stress caused by tunicamycin or azole drugs  and release of Ca2+ from internal stores by a variety of mechanisms. The action of staurosporine includes the activation of phospholipase C, which leads to the generation of IP3 and possibly to the regulation of the TRP-like channel. The vacuoles readily sequester the excess Ca2+ in an attempt to avoid the associated deleterious effects of Ca2+. This sequestration can occur by means of a Ca2+/H+ antiport system that is indirectly blocked by the disruptive effect of bafilomycin A1 on the proton gradient by inhibiting the vacuolar H+-ATPase  sucrose was used in all experiments ; DMSO, ML204, flufenamic acid and xestospongin C ; 4-methyl-4′--1,2,3-thiadiazole-5-carboxanilide (YM-58483/BTP2) and 2-aminoethoxydiphenyl borate (2-APB) ; 1,2-bis(ortho-aminophenoxy)ethane-N,N,N′,N′-tetrasodium (BAPTA), thapsigargin, Ru360, LiCl2 and 7-hydroxystaurosporine (UCN-01) ; 1-[6--trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122)  and bafilomycin A1 .eriments . Wild-ty3 conidia onto the centre of large Petri dishes (14.2-cm diameter) containing solid minimal medium. For growth measurements in liquid minimal medium, 1×104 conidia/ml were incubated at 26°C, 100 rpm, under constant light in 96-well plates  and absorbance was monitored at 450 nm over 24 hours.Mycelial extension rates at 26°C were measured after inoculating 5 µl containing 1×102+ reporters are the most reliable option for the study of intracellular Ca2+ dynamics, and such a method was developed for filamentous fungi using the bioluminescent Ca2+ reporter aequorin that has been codon-optimized for N. crassa  at 1800 V for 5 ms. Aequorin-expressing conidia were obtained and incubated at a concentration of 2×106 cells/ml with 5 µM coelenterazine  in minimal medium. Aliquots of 100 µl were added to each well of white opaque 96-well plates and incubated for 6 hours at 26°C in the dark without agitation. The luminescence emitted was measured in relative light units (RLU) using a Bio-Tek Synergy HT  microplate reader. Owing to equipment constraints, it was not possible to convert RLU values into precise [Ca2+]c concentrations. Therefore, the luminescence results were normalized for each strain to allow direct comparisons between different experiments as follows. For each strain, 100 µl of 3 M CaCl2 in 20% ethanol was pipetted into extra wells of the 96-well plates and discharged aequorin luminescence was measured for 3 minutes. This provided a measurement of the total aequorin luminescence that could be emitted and corresponds to the maximum level of aequorin expressed for each strain in each experiment. These total aequorin discharge measurements were used to normalize the experimental RLU values for each strain [cytosolic Ca2+ levels (arbitrary units) = experimental RLU values/total emitted luminescence]. For each plot of the values of aequorin luminescence, as a readout of [Ca2+]c, quantification was performed by summing the normalized experimental values, and data are expressed as the mean±s.e.m. The RLU values of solvent DMSO control samples were subtracted from the RLU values of the staurosporine-treated samples to obtain the ‘staurosporine-induced Ca2+ amplitude of response’. Where specified, samples were pre-incubated for 15 minutes with various pharmacological agents before staurosporine (or DMSO) was added. In all instances, the volume of chemical(s) added to the wells was 10 µl (from an appropriate stock solution), to ensure good homogenization. The aequorin luminescence plots presented in this paper correspond to the average of at least three independent experiments with three to six replicates per experiment. The typical staurosporine-induced Ca2+ signature of wild-type cells was obtained from 30 independent experiments.Genetically encoded Ca. crassa . The pAB7 cells/ml. From each dilution, 5 µl was spotted onto plates containing glucose-fructose-sorbose (GFS) medium with agar supplemented with the indicated chemical. Cells were incubated at 26°C and images were obtained 72 hours later.For the spot assay, nine successive threefold dilutions were prepared for each strain starting with 6.6×106 cells/ml were cultured for 4 hours in Vogel's minimal medium at 26°C, 140 rpm, under constant light conditions, followed by the addition of staurosporine and growth for a further 2 hours. Samples were harvested by centrifugation, washed twice with PBS and incubated with 0.1 µM YO-PRO 1. After 20 minutes on ice, samples were read in a Beckman-Coulter EPICS XL-MCL . Results represent the mean±s.e.m.For the detection of apoptotic cells by flow cytometry, the fluorophore YO-PRO1  was used. Conidia at a concentration of 105 conidia/ml were placed in eight-well slide culture chambers  and incubated at 26°C. Images were captured with an ORCA-ER CCD camera  driven by the MetaMorph NX1.1 software for image acquisition. The percentage of germinated cells was calculated using ImageJ .For routine microscopy, a Nikon TE2000E inverted microscope with a 60×/1.2 NA water-immersion plan apo objective  and differential interference contrast (DIC) optics was used. For these analyses, 200-µl drops of conidial suspension in liquid Vogel's medium containing 5×106 cells/ml were incubated in eight-well slide chambers at 26°C in the dark without agitation and stained with 5 µg/ml propidium iodide (Sigma-Aldrich). After 6 hours of growth, 20 µM staurosporine or DMSO was applied and images were obtained at appropriate time-points. Micrographs were obtained using an Olympus IX81 inverted fluorescence microscope  equipped with DIC optics. Fluorescence images were acquired using a plan fluor 20×/0.45 NA objective lens, using a 100-W mercury fluorescence light source and a BP 510–550 excitation filter; fluorescence emission was filtered through a dichroic mirror DM570 . Images were acquired with a cooled CCD camera  connected to a computer running Cell F . The exposure time was adjusted to 50 ms without binning. The percentage of propidium-iodide-positive cells was quantified using ImageJ (NIH) and expressed as the mean±s.e.m.For the evaluation of propidium iodide uptake by dead cells, conidia at a concentration of 2×10P-values of ≤0.05 were considered statistically significant.Statistical analysis of the data was performed using SPSS 20 . The non-parametric Mann–Whitney test was used for comparisons between two groups.


Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga,  Haematococcus pluvialis, a freshwater species of Chlorophyta , is known to synthesize and accumulate astaxanthin when faced with stressing environmental conditions including: high or low light intensity, high salinity, elevated temperatures and nutrient starvation H. pluvialis has been performed by imaging the algae at varying growth stages with resonance-enhanced confocal Raman microscopy. Astaxanthin was found to be mostly concentrated towards the center of induced palmelloid cells and at the periphery of aplanospores H. pluvialis, little research has focused on the organization and ultrastructure of astaxanthin within oil bodies. Astaxanthin is capable of forming H- and J-aggregates in vitro where an H-aggregate consists of closely packed astaxanthin molecules which are stacked with their conjugated chains oriented parallel to one another H. pluvialis due to a characteristic shift of the C = C band in Raman spectra compared to the astaxanthin monomer Astaxanthin has been reported to accumulate in oil bodies outside the chloroplast H. pluvialis, the alga was imaged with multicontrast nonlinear optical microscopy with three distinct contrast mechanisms including; multiphoton excitation fluorescence (MPF), second harmonic generation (SHG) and third harmonic generation (THG). In a nonlinear optical microscope, the signals are generated at high laser powers which is commonly achieved by utilizing a laser with 50–500 fs duration pulses for sample excitation coupled to a conventional confocal microscope. Optical sectioning is achieved without the requirement of a confocal pinhole since the nonlinear interaction only occurs at the focal spot due to the high energy requirement of nonlinear optical processes, which results in reduction in out-of-focus bleaching. MPF is similar to confocal fluorescence except the multiphoton excitation requires a combined energy of two or three infrared photons; for example, a 1028 nm laser induces two photon absorption at 514 nm. SHG and THG differs from fluorescence as they are determined by the nonlinear optical susceptibility of the molecules constituting the material as well as by the structural symmetry of the material. SHG and THG can be generated at wavelengths away from the absorption bands of the material and, therefore, have reduced photobleaching of the sample under those imaging conditions. In addition, generation of SHG and THG differs when detected in forward or backward directions with respect to the laser beam propagation, with stronger signals occurring in the forward direction In order to determine the localization and structure of astaxanthin accumulated within χ(2)), which is a function of the constituent molecules ability to produce SHG, as well as, their organization into a non-centrosymmetric structure. For example, intense SHG occurs in collagen χ(3)), and relies on the ability of constituent molecules to produce THG, as well as their organization within the focal volume of the microscope. Unlike SHG, which requires the noncentral organization of molecules, THG requires structures that possess distinct interfaces within the focal volume. For example, chloroplasts in vivo optical investigations of photosynthetic organisms such as H. pluvialis.SHG is described by a second-order nonlinear optical susceptibility tensor,  harmonic generation microscopy δ, from the Z-axis  and the different analyzer and polarizer orientation angles as a function of the second-order nonlinear optical susceptibility ratio, SHGR.The second-order and third-order nonlinear optical susceptibility tensor component ratios were determined from PIPO SHG and PIPO THG measurements as follows e Z-axis . If one THG1R = THG2R = Similarly, by assuming that a crystal with cylindrical axis along Z′ is in the laser scanning plane, XZ, and one dominant nonlinear dipole exists along Z′, then three unique nonzero tensor components can be assumed, δ, the orientation of the crystalline axes in the XZ laser scanning frame, as well as SHGR  and a photometric sensor  in the 400–700 nm region of the electromagnetic spectrum. H. pluvialis cells were induced to accumulate astaxanthin by exposure to low light conditions of 5 µmol photons m−2·s−1 of cool-white fluorescent light.H. pluvialis cells were lifted from the agar and quickly (30 s) immobilized in an 8% polyacrylamide gel A homogeneous methanol solution containing 50 µM astaxanthin  was mixed with distilled water in a ratio of 1∶3 to induce formation of H-aggregates. The UV-Vis absorption spectrum of the mixture was recorded with an Olis-14 (upgraded Cary-14) spectrophotometer using a 1 cm Suprasil quartz cuvette  to determine which astaxanthin aggregate was formed. Formation of H-aggregates was confirmed by a large hypsochromic shift in the absorption spectra 4)2 oscillator, which provided ∼450 fs duration pulses at a wavelength of 1028 nm with a pulse repetition rate of 14.3 MHz The laser source consisted of a femtosecond Yb:KGd and one color glass filter  was placed in front of the photomultiplier tube for separation of fluorescence from the laser radiation in MPF studies of SHG and THG signal was collected in the forward direction through a home-built UV transmitting objective, then split with a dichroic mirror, and filtered with a band pass interference filter  and a color glass filter, BG-39  for SHG and F10-340  for THG. The signals were measured using photon-counting detectors .A multicontrast nonlinear optical microscope was modified for PIPO measurements as previously described SHG and THG images were collected at 132 sets of excitation and analyzer angles. The half-wave plate was rotated to 11 different polarization orientations evenly spaced between excitation angles from −90° to 90°, whereas the analyzer was rotated to 11 different evenly spaced polarization orientation angles from −90° to 90° for each excitation polarization angle. Reference frames to provide a baseline SHG and THG signal intensity were recorded every 11 measurements as an internal control.θ and φ was fitted using θ′ and φ′ in For each 2×2 pixel area, the variation in SHG and THG intensity as a function of THG1R between −2 and 2 and allowing THG2R and Fitting of PIPO THG data was performed by fixing Green palmella under non-inductive and inductive conditions, as well as red aplanospores were imaged with MPF, SHG and THG microscopy. Signals from the three contrast mechanisms highlight different features of the algae. Two-photon absorption of chlorophylls and carotenoids in the chloroplasts induces chlorophyll fluorescence 2 as judged by the presence of pyrenoids Second harmonic generation is observed from starch granules present in the algae at all stages . Starch The THG signal intensity of the red aplanospore  is aboutH. pluvialis which demonstrated the presence of chlorophyll, β-carotene and astaxanthin largely at the cell periphery in green-colored palmella non-induced cells, whereas in green-red colored palmella induced cells, chlorophyll remains at the cell periphery while β-carotene and astaxanthin are present in the middle of the cell These observations are similar to what is seen in resonance-enhanced confocal Raman microscopy images of In the green-colored palmella non-induced cells, correlation between all three nonlinear signals is not seen, however, there are regions where MPF and SHG or MPF and THG signals correlate. Correlation between MPF and SHG is seen where the SHG signal is generated due to starch granules present in the chloroplasts and MPF originates from the chlorophyll containing pigment-protein complexes in the chloroplasts .Correlation of MPF and THG signals is seen at all stages of cell growth. In particular, in the green-colored palmella non-induced cells  and greeIn red aplanospores , correlaH. pluvialis where astaxanthin is deposited into extra-plastidial oil bodies H. pluvialis, polarization-dependent harmonic generation was employed.Previous studies have reported high concentrations of astaxanthin at the edge of aplanospores of Haematococcus pluvialis was studied using PIPO SHG and THG microscopy. Although β-carotene and astaxanthin are both present in red aplanospores and both carotenoids have large third-order nonlinear optical properties The organizational structure of astaxanthin in red aplanospores of PIPO measurements were performed on red aplanospores. PIPO SHG and PIPO THG data are summarized by contour plots of selected pixels shown in SHGR ratio in all four regions was found to have an average value of 3.8±0.1. These ratio values are consistent with SHGR ratios measured for starch SHGR ratios are consistent in all regions studied in Fitting of the PIPO SHG data from regions i, ii, v, and vi in THG1R were found to be small. Since the two ratios, THG1R and THG2R, depend on one another, the value of THG1R was fixed between −2 and 2 while THG2R was set as a free fitting parameter.Fitting of PIPO THG data from regions iii, iv, v, and vi in THG2R in all four regions was found to have an average value of 1.1±0.1 while THG1R was found to have an average value of 0.35±0.01. THG2R for both regions is approximately 1 while THG1R for both regions is approximately 1/3. These ratios are characteristic of the symmetry properties for an isotropic material where THG1R is equal to 1/3 while THG2R is equal to 1 As mentioned, it has been hypothesized that astaxanthin within red aplanospores form H-aggregates. In order to determine if the structure of astaxanthin within H-aggregates is similar to the structure of astaxanthin found in red aplanospores, PIPO THG was performed on astaxanthin H-aggregates.H-aggregates of astaxanthin were formed by mixing a 50 µM solution of astaxanthin dissolved in methanol with distilled water at a ratio of 1∶3. The absorption spectrum of the mixture was taken as seen in H. pluvialis  for the in vitro astaxanthin H-aggregate. Since THG2R is much larger than THG1R, this means that the The H-aggregates were imaged with THG microscopy and the PIPO THG data of an H-aggregate was taken as exemplified in luvialis  which reThe PIPO THG contour plot for the astaxanthin H-aggregate in in vivo and in vitro astaxanthin structures shows that astaxanthin within oil bodies of Haematococcus pluvialis (in vitro aggregates (in vivo sample shows no long-range order. The elongated PIPO plot (in vivo.The comparative analysis of PIPO THG data between gregates  in that IPO plot  can occuHaematococcus pluvialis can be readily studied with multicontrast nonlinear optical microscopy. Under stress conditions, astaxanthin accumulates in the central region of aplanospores. Polarization-dependent THG studies of red aplanospores reveal that astaxanthin is isotropically arranged in oil bodies. In contrast, synthesized H- and J-aggregates of astaxanthin demonstrate uniaxial alignment of astaxanthin molecules. Since carotenoids tend to form H- and J-aggregates readily in aqueous solutions, this research implies that astaxanthin present in biological organisms undergoes a distinctly different way of packing likely due to the physical environment of the alga or is controlled enzymatically. PIPO SHG and THG microscopy allows convenient in vivo measurements of the dynamics of astaxanthin accumulation in algal species, and when subjected to different conditions can be used to select algae to maximize the extraction yield of carotenoids for aquaculture, nutraceutical and pharmaceutical industries.The localization of astaxanthin accumulation in


For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression. Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals. However, channel clustering is also seen in cells with relatively simple topology, suggesting it fulfills a more fundamental role in cell biology than simply orchestrating compartmentalized responses. Here, we have compared the ability of store-operated Ca  •fos and NFAT transcription factorsDispersed Orai1 channels weakly activate c-•Channel re-location to ER-PM junctions robustly activates transcription factors•Orai1-channel clustering strengthens excitation-transcription coupling In electrically silent cells, store-operated calcium channels relocate to plasma-membrane microdomains after stimulation. Samanta et al. show that such channel clustering, as opposed to a similar number of dispersed channels, augments the effects of calcium signaling through more robust activation of transcription factors. The channels open after a fall in free calcium concentration within the ER, as occurs physiologically following stimulation of cell-surface receptors that increase the levels of the second messenger inositol trisphosphate  1 and 2  pump blocker thapsigargin (2 μM) resulted in a sustained cytoplasmic Ca2+ rise, due to Ca2+ release from the stores followed by Ca2+ influx through CRAC channels  revealed an association with Syk in non-stimulated cells  alters gating such that the channel is open under resting conditions, both without the need to deplete stores and in a STIM-independent manner .2+ entry following V102C-Orai1 expression by first briefly removing external Ca2+ and then measuring the rate of rise of the cytoplasmic Ca2+ signal that occurred when external Ca2+ was readmitted  but significantly slower than that seen after challenge with a maximally effective concentration of thapsigargin in mock-transfected cells  in RBL-1 cells led to an approximate doubling of Orai1 levels A, indicaadmitted B. Compar2C-Orai1 B and 2C.channels C, consis1-cherry D was uni1-cherry D. Neitheproteins D. V102C-proteins E, and knproteins E.fos and NFAT activation induced by Ca2+ flux through V102C-Orai1 channels with that through endogenous Orai1 channels localized to ER-PM junctions. For the analysis to be meaningful, it was essential to compare gene expression for similar levels of Ca2+ entry. We therefore established the relationship between Ca2+ entry and thapsigargin concentration and from this identified a concentration of thapsigargin that generated a rate of Ca2+ influx through endogenous Orai1 channels that was identical to that evoked by V102C-Orai1. Having obtained this, we could then compare c-fos expression and NFAT activation induced by V102C-Orai1 channels with that evoked by the dose of thapsigargin that gave a similar rate of Ca2+ entry but that caused re-localization of endogenous channels to ER-PM junctions.Because V102C-Orai1 channels are active under resting conditions, do not form punctate-like structures in the PM and do not require STIM1, they seem to operate as a series of independent channels. We therefore compared the extent of c-fos levels had increased 24 hr after V102C-Orai1 transfection, although to a level less than that induced by thapsigargin. Constitutive Ca2+ entry through the mutant channels, integrated over many hours, is therefore sufficient to stimulate NFAT and c-fos gene expression, at least in a sizeable fraction of the cells. This was problematic for two reasons. First, we needed a low background of NFAT/c-fos expression in order to compare the relative gene expression capabilities of a defined pulse of Ca2+ entry through V102C-Orai1 with that through endogenous Orai1 channels confined to ER-PM junctions. Second, constitutive Ca2+ entry through V102C-Orai1 could lead to Ca2+-dependent inactivation of the channel, thus diminishing the ability of Orai1 to stimulate gene expression. We therefore adopted the La3+ approach that has been used to prevent constitutive Ca2+ influx through recombinant Orai1 channels following transfection with the CRAC channel activation domain of STIM1  resulted in very little basal Ca2+ influx  increases inositol trisphosphate levels, resulting in a series of cytoplasmic Ca2+ oscillations  and in low Na+-containing solution  of STIM1 interacts with a leucine-rich coiled-coil motif on the C terminus of Orai1. Mutation of leucine 273 (L273S) or leucine 276 (L276D) in this region prevents STIM-Orai interaction as well as CRAC channel activation . To test compare B and NFA compare D and 5E. compare B. Accumu compare D and 5E.ed cells D and 5E.fos expression was measured  is essential for the gating step    and examepletion D. Althountensity D. Followoccurred C and 6E. instead C. These rai1-YFP G and 6H.rai1-YFP I. Althourai1-YFP I.2+ flux in physiological solution  and taking an open probability of 0.8 . The simulated Ca2+ concentration for a line scan across the center of the junction is shown in 2+ both below the PM and at the apposed ER surface is low, as few channels are close by. However, local Ca2+ at both surfaces increases steeply close to a single channel that happens to be near the central line. The Ca2+ profile here is similar to that predicted for a single channel  for ∼40 nm. Re-localization of the low conductance CRAC channels to ER-PM junctions therefore enables a local Ca2+ signal to extend several tens of nanometers across the junction. The fraction of PM or outer ER surface membrane that experiences different local Ca2+ concentrations is plotted in 2+ concentration >1 μM. By contrast, almost all the ER in a junction experiences Ca2+ < 1 μM at 43- or 88-nm spacing. The profiles suggest that clustering enables Ca2+ sensors or detectors on the apposite ER surface to be exposed to Ca2+ levels several-fold more than the bulk level.From overexpression studies, it has been estimated that individual puncta contain ∼1,300 CRAC channels . Howevery of 0.8 , we calc channel A, indicanm apart D, mimick2+ arise E: a larg2+ arise   in DMEM with 10% fetal bovine serum and 2 mM L-glutamine and penicillin-streptomycin, as previously described (Rat basophilic leukemia (RBL-1) and HEK293 cells were bought from the ATCC and cultured  with NaOH. Ca2+-free solution contained 145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, and 0.1 mM EGTA (pH 7.4) with NaOH. For the low Na+ external solution, NaCl was reduced to 10 mM and replaced with 135 mM Tris base. Cells were alternately excited at 356 and 380 nm (20-ms exposures), and images were acquired every 2 s. Ca2+ signals are plotted as R, which denotes the 356/380 nm ratio. Further details are provided in Cahotonics . Cells wHEK293 cells expressing Orai1-YFP, V102C-Orai1-YFP, or mutants thereof were illuminated with 488-nm laser light. Light reflected from the back focal plane was detected with a ×100 oil-immersion objective, and images were captured with 1 × 1 pixel binning. YFP fluorescence was measured before and then after stimulation with thapsigargin (concentrations indicated in text) for each cell. YFP fluorescence was measured in Image J along three lines drawn across each cell, as described in NFAT1-GFP levels in the cytosol and nucleus were measured using the IMAGO charge-coupled device camera-based system from TILL Photonics, with a ×100 oil-immersion objective . Regions2+ influx through V102C-Orai1 and associated mutants from stimulating gene expression during the culture period after transfection, we used the La3+ method , cells were stimulated with thapsigargin and the percentage of cells expressing EGFP measured subsequently (∼24 hr later), as described in siRNAs against Orai1 and STIM1 were from Origene and siRNA against SYK was from Invitrogen, as reported previously .fos primary antibody (Santa Cruz Biotechnology), as described in After treatment, cells were fixed in 4% paraformaldehyde at room temperature and permeabilized with PBS/Triton 0.5%. Cells were stained overnight at 4°C with c-2+ free external solution for 5 min and then lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitors  at 4°C , as described previously  between any two of the points picked at random (88.5 nm) and (2) between any one point picked at random and its nearest neighboring point (47.5 nm).D is the diffusion constant for calcium ions (set as 300 nm2/μs), and Simulations of calcium diffusion within the microdomain were performed using the diffusion/heat equation with a source term for CRAC channel currents:z = 15) represents the cell membrane and the bottom surface (z = 0) the ER membrane; at each of these boundaries, “no flux” conditions are applied The model is set up on a 3D disc-shaped domain of the following dimensions: Results are presented as mean ± SEM. Data were compared using Student’s t test or by ANOVA for multiple groups. Differences were considered statistically significant at values of p < 0.05.


It primarily exists and is linked with signaling of the sensory neurons – sight, smell, taste, and pinealocytes. Exceptionally, it is also present and is Ca2+-modulated in t he non-neuronal cells, the sperm cells in the testes, where S100B protein serves as its Ca2+ sensor. The present report demonstrates the identification of an additional Ca2+ sensor of ROS-GC1 in the testes, neurocalcin δ. Through mouse molecular genetic models, it compares and quantifies the relative input of the S100B and neurocalcin δ in regulating the Ca2+ signaling of ROS-GC1 transduction machinery, and via immunochemistry it demonstrates the co-presence of neurocalcin δ and ROS-GC1 in the spermatogenic cells of the testes. The suggestion is that in more ways than one the Ca2+-modulated ROS-GC1 transduction system is linked with the testicular function. This non-neuronal transduction system may represent an illustration of the ROS-GC1 expanding role in the trans-signaling of the neural and non-neural systems.ROS-GC1 belongs to the Ca Its discovery and molecular characterization was a land mark event in the field of phototransduction  sense the post-illumination fall in Ca2+ and stimulate ROS-GCs to synthesize cyclic GMP at a faster rate and restore its dark level  channels, lowering the free Ca2+ sensing modulators of ROS-GC activity. While increasing Ca2+ concentrations inhibit ROS-GCs activity through GCAPs, two other Ca2+ sensors, S100B and neurocalcin δ stimulate ROS-GC1 in a Ca2+-dependent fashion  belongs to a subfamily of neuronal calcium sensor (NCS) proteins called visinin like proteins (VSNLs). Similar to other, but not all NCS proteins, it is acylated at the N-terminus by myristic acid and undergoes a classical calcium-myristoyl switch  e.g., itecoverin . Myristoid EGTA; . Althougid EGTA; , trafficid EGTA; , and witid EGTA; . The preid EGTA; , olfactoid EGTA; , 2004 anid EGTA; .2+-dependent modulator of membrane guanylate cyclase ROS-GC1 , bovine , and Wistar rat . The study was approved by the IACUC and Ethics Review Boards of all the involved Institutions.The study group consists of testes from human  mice is described in , according to the manufacturer’s protocols. The cDNA library was constructed using Advantage RT for PCR kit (BD-Bioscience) and used for the amplification of ROS-GC1 and neurocalcin δ fragments. Sequences of primers used for the amplification are listed in The procedure was carried out according to the previously published protocols . 150 mg To visualize the immunoreactive bands SuperSignal blaze chemiluminescent substrate (Pierce) was used according to the manufacturer’s protocol. Signal was detected by exposing the blot to Kodak X-ray film for 15 s. Then the X-ray film was scanned and processed using Photoshop 6.0 software.Paraffin sections of the testes fixed in 4% paraformaldehyde were used for immunohistochemical detecting of ROS-GC1 and NCδ. To block the non-specific binding the sections were first washed with TBS  and incubated in blocking solution consisting of TBS buffer containing 0.05% Tween 20 (TBS-T) and 1% BSA for 1 h at room temperature. After washing with TBS-T the sections were incubated with the respective primary antibodies for 60 min at 37°C and washed four times (15 min each) in TBS-T. Anti ROS-GC1 antibodies were diluted 1:200 and anti neurocalcin δ, 1:100. AP-conjugated anti-rabbit IgG, diluted 1:200 and NBT/BCIP as the substrate were used for detection. Controls included detection reactions carried out under identical conditions except that the primary antibodies were replaced by preimmune serum.g and then the supernatant at 10,000 g and finally, at 40,000 g. The resulting pellet represented the membrane fraction.Membrane fraction of the testes was isolated according to the protocol described previously . The tis2+ concentration with precalibrated Ca2/EGTA solutions (Molecular Probes). The reaction was initiated by the addition of a substrate solution  and continued by incubation at 37°C for 10 min. The reaction was terminated by the addition of 50 mM sodium acetate (pH 6.2) followed by heating in a boiling water-bath for 3 min. The amount of cyclic GMP formed was determined by radioimmunoassay . Sequencing of all amplified products confirmed their identities with ROS-GC1 or NCδ cDNAs. The ROS-GC1 fragments represented indeed sequence coding for the kinase-like domain, and the NCδ fragment had sequence identical with the predicted part of NCδ coding region. Amplification of uninterrupted coding sequences for ROS-GC1 and NCδ documented that the RNA used was not contaminated with genomic DNA. Thus both, ROS-GC1 and NCδ transcripts are present in human, bovine, and rat testes.The presence of ROS-GC1 and NCδ transcripts was analyzed in human, bovine, and rat testes. Using total RNA isolated from these tissues individual cDNA libraries were constructed and used for amplification of specific fragments of ROS-GC1 and NCδ cDNAs by polymerase chain reaction. For the amplification, distinct, species-specific primers for ROS-GC1 or NCδ were designed. These primers corresponded to the sequences located within so called “kinase-like domain” of ROS-GC1 cDNA and for NCδ they overlapped with the 5′- and 3′- ends of its coding sequence. The amplification yielded fragments of 553, 548, and 566 bp of the human, bovine, and rat ROS-GC1, respectively, and 526 bp fragment of neurocalcin δ  was identical to that observed previously for ROS-GC1 expressed in photoreceptor outer segments . The pattern of this staining was identical to that observed for bovine testes . Visual analysis allowed localizing the staining to spermatogenic cells, especially to primary spermatocytes and spermatids. The majority of primary spermatocytes , spermatids  and single spermatogonias  were immunostained in all species tested, human, rat, and bovine. To verify specificity of the ROS-GC1 immunostaining a control reaction was performed in which ROS-GC1 antibody was omitted and substituted with the preimmune serum. Under these conditions no labeling was observed .Detailed analyses of the localization of ROS-GC1 and NCδ in the mammalian testes were performed by immunocytochemistry. In human and rat testes ROS-GC1 immunostaining was randomly distributed in the seminiferous tubules  and compared with that in bovine testis . NCδ immunoreactivity was observed in all types of germinal cells: spermatogonias , spermatocytes  and spermatids . Extremely strong staining was observed in spermatids localized close to seminiferous tubule lumen. No labeling was observed when the sections were incubated with preimmune rabbit serum instead of the NCδ antibody, attesting to the specificity of the staining .In a similar manner the expression of NCδ in the human and rat testes was analyzed  strongly indicating that they co-localize with each other. Notably, the intensities of the immunostaining for ROS-GC1 or NCδ increase with the maturation of the spermatogenic cells.The results presented clearly demonstrate that both ROS-GC1 and NCδ are expressed in the mammalian testes and in the germinal cells. Importantly, the patterns of ROS-GC1 and NCδ immunoreactivities are identical in the species analyzed . Half-maximal stimulation (EC50) was observed at ~0.8 μM NCδ in all three species tested. The maximal stimulation of ~2.2-fold above the basal value was at 2 μM NCδ. Importantly, the stimulatory profiles of guanylate cyclase in the analyzed membranes were very similar to those observed for the recombinant ROS-GC1 ]. To test this prediction, the particulate fractions of the testes from wild type and NCδ+/- mice were isolated in the presence of 100 μM Ca2+ and tested for guanylate cyclase activity in the presence and absence of Ca2+. The reason for isolating the membranes in the presence of Ca2+ was that NCδ exhibits the property of “calcium-myristoyl switch” which means that in the presence of Ca2+ it is membrane bound and therefore would be present in the membrane fraction (2+ (1 mM EGTA was present in the assay mixture) was 58 ± 7 pmol cyclic GMP min-1 (mg prot)-1 for the wt and 63 ± 8 pmol cyclic GMP min-1 (mg prot)-1 for the NCδ+/- mice . However, the cyclase activity determined in the presence of Ca2+ was strongly dependent on the mice genotype. With 1 μM Ca2+ present in the assay mixture the activity was 248 ± 17 pmol cyclic GMP min-1 (mg prot)-1 for the wild type mice and 148 ± 16 pmol cyclic GMP min-1 (mg prot)-1 for the NCδ+/- mice . These results, as predicted, demonstrate that the Ca2+-dependent NCδ-modulated ROS-GC1 signaling pathway in the mice with one copy of NCδ gene deleted (NCδ+/-) is about half as active as in the wild type mice. To further validate that the lowering of the Ca2+-dependent cyclase activity in the membranes of NCδ+/- mice is the exclusive consequence of lower NCδ expression, 4 μM exogenous NCδ was added to the wild type and NCδ+/- membranes and the cyclase activity was assessed in the presence of 1 μM Ca2+. The cyclase activity in the wild type membranes increased only minimally, from 248 to 273 ± 25 pmol cyclic GMP min-1 (mg prot)-1but in the NCδ+/- membranes, from 148 to 258 ± 28 . Thus, the activity achieved was practically the same for both types of membranes. These results demonstrate that addition of exogenous NCδ to the NCδ+/- membranes restores the guanylate cyclase catalytic activity and brings it to the level of activity in the wild type membranes. The slight activity increase in the wild type membranes can be explained by a partial loss of the native NCδ during the membrane preparation procedure. Together these results demonstrate that functional ROS-GC1-NCδ-Ca2+ transduction system is operative and is the functional transduction element in the testes.Because the experiments described above were conducted on the native membranes of the testes but with exogenously supplied NCδ, they prove the ability of ROS-GC1 to respond to NCδ but they do not prove that these two together form a functional unit. To determine this, the mouse model with deletion of one copy of the NCδ gene, NCδnot born ]. It wasfraction . The gua2+-dependent modulators of ROS-GC1 activity are present in the testes. Taking advantage of the availability of two types of genetically modified mice, NCδ+/- and S100B-/- we attempted comparing the relative inputs of NCδ and S100B into regulation of ROS-GC1 transduction machinery.Our previous investigation has shown that S100B is expressed in the testes  and the +/- and S100B-/- mice, their membrane fractions were isolated in the presence of 100 μM Ca2+ and assessed for guanylate cyclase activity. The results of this experiment are shown in Figure 6. In the absence of Ca2+ all three types of membranes exhibited comparable activity of ~60 pmol cyclic GMP min-1 (mg prot)-1  and, as expected, the activity was not affected by the addition of NCδ or S100B . However, when the cyclase activity was assayed in the presence of Ca2+ the values differed between the genotypes: 250 pmol cyclic GMP min-1 (mg prot)-1 for the wild type, 150 pmol cyclic GMP min-1 (mg prot)-1 for the NCδ+/- and 200 pmol cyclic GMP min-1 (mg prot)-1 for the S100B-/- . These values describe the ROS-GC1 activity stimulated together by NCδ and S100B in the case of the wild type membranes; stimulated by half of wild type amount of NCδ and S100B in the case of NCδ+/- membranes; and stimulated by NCδ only in the case of S100B-/- membranes. Thus, the difference between ROS-GC1 activity in testes of the wild type mouse and the activity in the genetically modified mice (100 and 50 pmol pmol cyclic GMP min-1 (mg prot)-1 for the NCδ+/- and S100B-/-, respectively) describes the Ca2+-dependent stimulatory effects lost due to the absence of half of the NCδ present in the wild type testes or to the total absence of S100B. Higher loss of activity resulting from deletion of one copy of NCδ gene than resulting from deletion of two copies of S100B gene indicates that NCδ is more potent than S100B in modulating the Ca2+-dependent ROS-GC1 activity. One could argue, however, that this comparison might not be totally reflective of the in vivo contributions of NCδ and/or S100B, because there is a possibility that some of these proteins were lost during the membrane preparation process. To settle this possibility the ROS-GC1 activity was measured in the presence of individually added NCδ or S100B.Testes were removed from the wild type, NCδFigure 6B: panel “+ncalc δ”) resulted in a small increase of the cyclase activity exhibited by the membranes isolated from the wild type testes (from ~250 to ~270 pmol cyclic GMP min-1 (mg prot)-1 and isolated from the S100B-/- testes [from ~200 to ~220 pmol cyclic GMP min-1 (mg prot)-1]. This non-significant to minimal increase of ROS-GC1 catalytic activity in these membranes indicates that if any NCδ was lost during the membrane preparation, it was only the insignificant amount. For the NCδ+/- membranes the increase was significant, from ~150 to ~260 pmol cyclic GMP min-1 (mg prot)-1. Importantly, addition of 4 μM NCδ brought the ROS-GC1 activity in these membranes very close to the ROS-GC1 activity in the wild type membranes. Because only residual amount of NCδ was lost during membrane preparation, as concluded from the wild type and S100B-/- membranes, practically all the increase of activity in the NCδ+/- membranes accounts for the activity lost due to the expression of only half of the NCδ present in the membranes from wild type testes.Addition of 4 μM NCδ  it increased by ~40 pmol cyclic GMP min-1 (mg prot)-1 in the wild type and by 25 pmol cyclic GMP min-1 (mg prot)-1in the NCδ+/- membranes indicating that some S100B was lost in membranes preparation. The increase was ~80 pmol cyclic GMP min-1 (mg prot)-1for the S100B-/- membranes [from 205 to 282 pmol cyclic GMP min-1 (mg prot)-1] and this increase of activity in the S100B-/- membranes accounts for the activity lost due to the loss of S100B expression in the testes. Comparing the activity loss because of the absence of half of NCδ with the activity loss due to the total absence of S100B expression shows that neurocalcin δ is more significant contributor to ROS-GC1 Ca2+-dependent activity than S100B. It could be estimated that NCδ contributes about 75% to ROS-GC1 Ca2+-dependent activity and S100B, about 25%.When the ROS-GC1 activity was assayed with 4 μM S100B added to the membranes  What is the precise morphological residence of any of these transduction systems in the testicular cells? (3) What is the exact stoichiometry of these components in the given cell types? (4) What are the mechanistic details of the operational modes of this/these ROS-GC transduction pathways? (5) What is the physiological significance of these pathways to the testicular function, endocrine, fertility, including spermatogenesis; (6) what is the interplay between the Ca2+-modulated ROS-GC1 signaling pathway and ANF-dependent ANF-RGC pathway, another cyclic GMP generating machinery functioning in the mammalian testes  What is the exact composition of the transduction system: does it represent a single pathway, composed of one ROS-GC1 and two Can testes  and fina2+-modulated signal transduction model for any of the specific physiological function/s of the testes. However, a preliminary signal transduction scheme is conceptualized where [Ca2+]i modulation of ROS-GC1 activity can occur through three modes. MODE 1 involves interaction of ROS-GC1 with its Ca2+ sensors, NCδ and S100B. The ROS-GC is stimulated in [Ca2+]i – dependent manner and generates cyclic GMP. MODE 2 involves interaction of the ROS-GC1 with GCAP1 which is also present in the testes –NCδ, S100B – and with GCAP1. The ROS-GC activity oscillates between the GCAP1-dependent state and CD-GCAPs-dependent state. In these states the cyclase is a bimodal Ca2+ transduction switch. And it functions according to the principles established for the synapse region between the photoreceptor and ON-bipolar cells (Figure 7. Neurocalcin δ targets the ROS-GC1 region V836–D856 (962–N981and I1030–Q1041 (445–L456 and L503–1522(Handicapped with these informational gaps, it is difficult to construct a proper Cae testes . It is rntration . Thus, tar cells . This mo836–D856 ; S100B, 30–Q1041 ; GCAP1, L503–1522.2+Basal state – low Ca: Ca2+-free GCAP1 stimulates ROS-GC1 catalytic activity. Cyclic GMP formed in response to this stimulation opens some of the CNG channels present in the sperm membranes (2+ ions entering the sperm through the open CNG channels are bound by neurocalcin δ and/or S100B. It is important to note that the half-maximal activation of ROS-GC1 by GCAP1 occurs at 707 nM Ca2+ (2+ concentration at which neurocalcin δ and S100B are able to activate ROS-GC1 with approximately half-maximal effectiveness. With increasing Ca2+ concentrations GCAP1 inhibits ROS-GC1 activity while neurocalcin δ and S100B stimulate it. Active state – Ca2+-bound NCδ and/or S100B further stimulate ROS-GC1 and cyclic GMP is formed in higher quantities leading to massive opening of CNG channels and influx of Ca2+ (Signal termination: when cyclic GMP and [Ca2+]i reach the optimal level necessary for sperm membrane hyperpolarization and physiological function (e.g., capacitation and/or acrosome reaction; 2+ influx.embranes . At leas nM Ca2+ , the Ca2 of Ca2+ . In this2+ sensitivity to cyclic GMP synthesis. The stimulatory arm of the switch operated by NCδ and S100B is essential for timely opening of the CNG channels by cyclic GMP produced and vast influx of Ca2+ necessary for the acrosome reaction. In this stimulatory arm NCδ appears to be a significantly more important player than S100B. The inhibitory arm operated by GCAP1 stops the cyclic GMP synthesis and therefore Ca2+ influx. Our findings demonstrate that ROS-GC1 transduction system and its bimodal Ca2+ modulation is not unique to neurosensory and neurosensory-linked systems and has a more widespread role in general Ca2+-dependent signaling and may be a part of the fertilization machinery.Co-expression of NCδ, S100B, and GCAP1 with ROS-GC1 in mammalian spermatogenic cells confers bimodal CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significant mitochondrial function impairment is known to result from cardiac ischemia reperfusion injury (IR) precipitated by cardiopulmonary bypass during heart surgery.We sought to determine the effect of different ischemia time spans in cardiac IR on mitochondrial respiratory chain (RC) function, inner membrane polarization and Ca2+homeostasis.Wistar rat hearts were harvested and divided into 4 groups of stop-flow induced warm global IR: 0, 15, 30 and 40 min of ischemia followed by 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records . Subsarcolemmal mitochondria (SSM) were isolated and analyzed regarding electron transport chain (ETC) coupling using a Clark-type electrode (polarography), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+induced swelling and Ca2+-uptake and release (Ca2+-sensitive electrode).IR in general depressed LV contractility irrespective of ischemia duration. In contrast, increasing length of ischemia time highly significantly promoted ETC uncoupling at complex I-V and II-IV in state 3 respiration, respectively. Membrane potential showed a distinct hyperpolarization in IR30/30 and IR40/30 compared to the other groups (p < 0.0001), continuously wearing off after CCCP-induced uncoupling. Regarding Ca2+-induced swelling, light transmission of IR40/30 SSMs started to differ significantly (p < 0.04) from IR0/30 after 6.5 min of Ca2+-addition, swiftly followed by IR15/30 (8.5 min) and 30/30 (16.5 min). All effects were delayed by app. 3.6 min by pyruvate addition in parallel assays also halving recorded swellings. Ca2+-uptake revealed slower rates and greater spans in IR15/30 and IR30/30 (p < 0.005) whereas Ca2+-release was delayed for ischemia an duration ≤30 min (p < 0.0001).Longer ischemia duration in IR injury gradually impairs SSMs in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Therefore, ischemia time should be considered an important factor influencing IR experiment-derived conclusions.


GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload‐induced Ca2+ release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1f/f and cardiac‐specific Rac1 knockdown (Rac1ckd) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1f/f mice. Remarkably, I/R‐induced ventricular arrhythmia was significantly decreased in Rac1ckd compared to Rac1f/f mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R‐induced ventricular arrhythmia. Ca2+ imaging analysis showed that in response to a 6 mM external Ca2+ concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca2+ waves in Rac1f/f cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R‐induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1ckd mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.Rac1 is a small  Under normal physiological conditions, a small Ca2+ influx through the L‐type Ca2+ channel upon membrane depolarization activates the RyR2 channel, resulting in a large Ca2+ release from the SR and subsequent muscle contraction 2+ leak 2+ release and leak mediated by RyR2, a term previously defined as store overload‐induced Ca2+ release (SOICR), leading to spontaneous Ca2+ waves in cardiomyocytes, which induce cardiac arrhythmias 2+ in cardiomyocytes 2+ homeostasis and promotes ventricular arrhythmia in the ischaemic heart remains elusive.Ryanodine receptor 2 (RyR2), an intracellular Cackd) mice were subjected to myocardial I/R. Rac1 activation, ROS production and oxidation of RyR2 were determined, SOICR in cardiomyocytes were analysed. Our data show for the first time that inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia during myocardial I/R. Thus, Rac1 may represent a potential therapeutic target for the treatment of cardiac arrhythmia in ischaemic heart disease.In this study, we hypothesized that inhibition of Rac1 protects against ventricular arrhythmia during myocardial I/R through decreases in ROS, RyR2 oxidation and SOICR. To test this hypothesis, cardiac‐specific Rac1 knockdown . In these mice, exon 1 of Rac1 gene is flanked by loxP sites. The generation of cardiac‐specific Rac1 knockdown mice  was carried out by breeding Rac1f/f with cardiac‐specific α‐myosin heavy chain (α‐MHC) promoter controlled Cre recombinase overexpression transgenic mice (α‐MHC‐Cre), resulting in deletion of Rac1 gene in adult cardiomyocytes as previously described Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health  and all experimental protocols were approved by Animal Use Subcommittee at the University of Western Ontario.Rac1f/f and Rac1ckd male mice (3–4 months old) were anaesthetized with ketamine  and xylazine . The adequacy of anaesthesia was monitored by the absence of withdrawal reflex to tail pinch. Subsequently mice were intubated and artificially ventilated with a respirator . The left coronary artery was occluded by positioning a 8‐0 suture around it with a PE‐50 tubing. After 45 min., the PE tubing was removed to allow reperfusion for either 60 min.  or 15 min. (for free thiol content measurement). To study the effect of Rac1 inhibition, some Rac1f/f and Rac1ckd mice were treated with saline or a Rac1 inhibitor NSC23766  for 30 min. before I/R Myocardial I/R were performed as previously described The ECG was monitored 5 min. before and throughout the entire I/R by limb lead I, with needle electrodes inserted subcutaneously in mice and recorded with PowerLab Chart 7.0  as previously described After I/R, infarct size was determined as described previously Rac1 activity was measured by GTP‐bound Rac1 GTPase through specific protein interaction with the Pak1 protein‐binding domain  according to the manufacturer's instructions Protein lysates were subjected to separation on a 10% SDS‐PAGE gel, followed by electrotransfer to nitrocellulose membranes. Blots were probed with specific antibodies against Rac1 (Active Rac1 Pull‐Down and Detection Kit), RyR2 , α‐actinin  respectively. Signals were detected by the enhanced chemiluminescence detection method and quantified by densitometry using FluorChem 8000 software .f/f and Rac1ckd mice were isolated and perfused with digestion buffer containing 50 μg/ml of liberase TH (Roche) through a Langendorff system. After enzymatic digestion, healthy and rod‐shaped cardiomyocytes were incubated with buffer containing increasing concentrations of Ca2+ (12.5 μM–1 mM).Adult cardiomyocytes were enzymatically isolated from the heart as described before f/f and Rac1ckd mice were loaded with 1 μM Fura‐2AM  for 30 min. at room temperature. To inhibit Rac1 activity, Rac1f/f myocytes were perfused with a Rac1 inhibitor, NSC23766 (10 μM) 2, 125 mM NaCl, 5 mM KCl, 6 mM glucose, 1.2 mM MgCl2 and 25 mM Hepes (pH7.4) and stimulated at 0.5 Hz for 20 sec. CaCl2 was added to the bath to final extracellular Ca2+ concentrations  followed by caffeine (10 mM) treatment to assess SR Ca2+ content.Ventricular cardiomyocytes isolated from Rac1Changes in superoxide production from myocardium after I/R were detected with a fluorescent indicator dihydroethidium  Ketamine and xylazine were purchased from Bioniche Animal Health Canada Inc.  and Bachem AG, Switzerland respectively. NSC23766 was from Calbiochem . All other reagents if unspecified were obtained from Sigma, St. Louis, MO, USA.anova followed by Newman–Keuls or Bonferroni post‐hoc test for multigroup comparisons. P < 0.05 was considered statistically significant.Data are presented as mean ± SEM. Results were analysed using one or two‐way ckd mice were generated through breeding of alpha‐myosin heavy chain (MHC)‐Cre mice with Rac1f/f mice and baseline cardiac function was not significantly different between adult Rac1ckd and Rac1f/f mice as we previously reported ckd and Rac1f/f mice were subjected to 45 min. of myocardial ischaemia followed by 1 hr of reperfusion (I/R). Hearts were isolated, and Rac1‐GTP, total Rac1 and α‐actinin protein levels were determined using Western blot analysis. Myocardial Rac1 activity was induced after I/R in Rac1f/f mice. The response was diminished in Rac1ckd mice . Furthermore, at 6 mM external Ca2+ concentration, over 50% of Rac1f/f cardiomyocytes exhibited SOICR while the occurrence of SOICR was seen in only 20–30% of Rac1ckd and NSC23766‐treated Rac1f/f cells  stimulation at the end of 6 mM external Ca2+ treatment was significantly lower in Rac1ckd and NSC23766‐treated Rac1f/f cells  stimulation at 1 and 6 mM extracellular Ca2+ concentrations , spontaneous intracellular Ca2+ oscillations were observed in both Rac1f/f and Rac1ckd cardiomyocytes, indicating induction of SOICR. However, the threshold extracellular Ca2+ to induce SOICR in Rac1ckd cardiomyocytes was doubled. Furthermore, the frequency and amplitude of SOICR were significantly decreased in Rac1ckd cardiomyocytes. Similar results were observed in Rac1f/f cardiomyocytes treated with a pharmacological Rac1 inhibitor NSC23766. Our results demonstrated that Rac1 promotes the occurrence of SOICR in cardiomyocytes, which induces ventricular arrhythmia.To understand the mechanism by which Rac1 activation induces ventricular arrhythmia, SOICR, that is, spontaneous SR Ca2+ leak in cardiomyocytes f/f hearts after I/R but was inhibited in Rac1ckd mice. To assess thiol oxidation of RyR2, monobromobimane labelling assay was employed. Our data show that the amount of free thiols in RyR2 labelled by monobromobimane was significantly decreased after myocardial I/R in Rac1f/f mice. Knockdown of Rac1 restored the amount of free thiols of RyR2. Our results suggest that Rac1 activation promotes ROS generation and RyR2 oxidation, leading to SOICR in cardiomyocytes during myocardial I/R. This study is limited to address the role of Rac1 in RyR2‐mediated cardiac Ca2+ handling. Further studies are required to examine whether Rac1 affects other key players including Na+/Ca2+ exchanger, SERCA and L‐type Ca2+ channels in cardiomyocytes during myocardial I/R.RyR2 is sensitive to redox modifications and oxidation of RyR2 by ROS generation induces SR Caet al. generated a mouse model that overexpresses a constitutively active mutant of Rac1, V12Rac1 in cardiomyocytes (RacET) phox plasma membrane to cytosol ratio, which is critical to NADPH oxidase activity. The study showed that Rac1 overexpression results in a ROS‐independent Ca2+ dyshomeostasis and cardiac dysfunction 2+ and SR Ca2+ content, the Rac1 knockdown cardiomyocytes are more resistant to Ca2+ overload during I/R injury. Data from our study are consistent with an important role of endogenous Rac1 in ROS‐dependent modulation of Ca2+ handling in cardiomyocytes.Oberhofer In summary, this study showed that myocardial I/R activates Rac1 and induces ventricular arrhythmia. Inhibition of Rac1 decreases ROS production, RyR2 oxidation, and cardiac arrhythmia during I/R. Furthermore, SOICR, an important mechanism of arrhythmogenesis can be effectively inhibited by Rac1 inhibitor NSC23766 in cardiomyocytes. Collectively, our study demonstrated that inhibition of Rac1 activation protects against arrhythmogenesis during myocardial I/R, and may represent a new paradigm for the treatment of cardiac arrhythmia in ischemic heart disease.This study was supported by operating grants to Q.F. from the Canadian Institutes of Health Research , to S.M.S. from CIHR (MOP‐64453) and to G.W. from National Natural Science Foundation of China (#81270176 and #81570254). L.Z. was a visiting PhD student from Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Q.F. was a Career Investigator of the Heart Stroke Foundation of Ontario.The authors declare that there are no conflicts of interest.S.M.S., G.W. and Q.F. contributed to conception and design of research; L.Z., X.L., L.G., and Y.W. performed experiments and analysed data. L.Z., L.G., S.M.S. and Q.F. interpreted results; L.Z. and L.G. prepared figures; L.Z. drafted manuscript; S.M.S., G.W. and Q.F. approved final version of manuscript; S.M.S. and Q.F. edited and revised manuscript.Video S1. Normal uniform contractions elicited by pacing isolated adult Rac1f/f cardiomyocytes.Video S2. Spontaneous waves in isolated adult Rac1f/f cardiomyocytes in response to Ca2+ overload.Click here for additional data file.


Xylopia frutescens Aubl. , is used in folk medicine as antidiarrheal. The essential oil from its leaves (XF-EO) has been found to cause smooth muscle relaxation. Thus, the aim of this study was to investigate the spasmolytic action by which XF-EO acts on guinea pig ileum.The components of the XF-EO were identified by gas chromatography-mass spectrometry. Segments of guinea pig ileum were suspended in organ bath containing modified Krebs solution at 37 °C, bubbled with carbogen mixture under a resting tension of 1 g. Isotonic contractions were registered using kymographs and isometric contractions using force transducer coupled to an amplifier and computer. Fluorescence measurements were obtained with a microplate reader using Fluo-4.+ channel positive modulator. In the presence of CsCl (non-selective K+ channel blocker), the relaxant potency of XF-OE was not altered, indicating a non-participation of these channels. Moreover, XF-EO inhibited CaCl2-induced cumulative contractions in a depolarizing medium nominally without Ca2+ and relaxed the ileum pre-contracted with S-(-)-Bay K8644 in a concentration-dependent manner, thus, was confirmed the inhibition of Ca2+ influx through Cav1 by XF-EO. In cellular experiments, the viability of longitudinal layer myocytes from guinea pig ileum was not altered in the presence of XF-OE and the Fluo-4-associated fluorescence intensity in these intestinal myocytes stimulated by histamine was reduced by the essential oil, indicating a [Ca2+]c reduction.Forty-three constituents were identified in XF-EO, mostly mono- and sesquiterpenes. XF-EO has been found to cause relaxation on guinea pig ileum. The essential oil inhibited in a concentration-dependent manner both CCh- and histamine-induced phasic contractions, being more potent on histamine-induced contractions as well as antagonized histamine-induced cumulative contractions in a non-competitive antagonism profile. XF-EO relaxed in a concentration-dependent manner the ileum pre-contracted with KCl and histamine. Since the potency was smaller in organ pre-contracted with KCl, it was hypothesized that XF-OE would be acting as a K2+ influx blockade, which results in [Ca2+]c reduction leading to smooth muscle relaxation.Spasmolytic action mechanism of XF-EO on guinea pig ileum can involve histaminergic receptor antagonism and Ca Diarrhea is characterized by change in bowel movements with symptoms of increment in water content, volume and frequency of stool . It is tXylopia frutecens Aubl. is a tree commonly known in Brazil as “embira”, “semente-de-embira” and “embira-vermelha” c. Experiments were performed in triplicate.The pellets were obtained as described earlier, cultured in 96-well plates (40.000 cells per well) and stabilized in incubator for 24 h for cell adhesion. After this process, the culture medium of each well was discarded and 50 μL of Fluo-4  was added and let rest for 40 min at 37 °C in COncubator . After tp < 0.05. IC50 and EC50 values were calculated with nonlinear regression c in the initial 10 s, staying reduced  throughout the observation period, showing a significant Fluo-4-associated fluorescence intensity decrease  Figs.  and 7. Ail Figs.  and 7. Ise Figs.  and 7. Tse Figs.  and 7.FiXylopia frutescens Aubl. (XF-EO) shows spasmolytic action on guinea pig ileum. Moreover, the most significant finding concerns the characterization of relaxant action mechanism, which includes histaminergic receptor antagonism and possible blockade of Cav1 which reduces cytosolic Ca2+ concentration ([Ca2+]c) leading to smooth muscle relaxation.This study demonstrated for the first time that the essential oil from leaves of 2-adrenergic receptor antagonist, Ca2+ channel blockers and calmodulin inhibitors c decrease is required to promote relaxation on smooth muscle [2+ concentration in several models of smooth muscles [2+]c availability possible due to CaV1 blockade, but no evidence of this has been shown. Thus, we aimed to provide evidence of [Ca2+]c reduction by the essential oil in myocytes isolated from ileum longitudinal layer and, so, better characterize its action mechanism. Initially, cell viability was evaluated using the XF-EO maximal concentration used in functional level. In 2 h of XF-EO exposure to the cells was not observed cell death (Fig. 2+, was added to the preparations containing ileum myocytes and the resultant Fluo-4-associated fluorescence was quantified in XF-EO presence. The essential oil reduced the great Fluo-4-associated fluorescence emitted in histamine presence, which indicates [Ca2+]c reduction of 97 % approximately (Figs. 2+]c to play its spasmolytic role.The c to promote the spasmolytic action observed in tissue experiments. Further complementary studies on this essential oil in vivo protocol are needed to pursue the search for a potential antidiarrheal agent.In conclusion, the spasmolytic action of the essential oil from


NUP98 and PHF23, resulting from a cryptic t translocation, demonstrating, for the first time, that NUP98-PHF23 is a novel recurrent (2.6 %) abnormality in pediatric CN-AML.The genomic landscape of children with acute myeloid leukemia (AML) who do not carry any cytogenetic abnormality (CN-AML) is particularly heterogeneous and challenging, being characterized by different clinical outcomes. To provide new genetic insights into this AML subset, we analyzed through RNA-seq 13 pediatric CN-AML cases, corroborating our findings in an independent cohort of 168 AML patients enrolled in the AIEOP AML 2002/01 study. We identified a chimeric transcript involving  Childhood acute myeloid leukemia (AML) is a heterogeneous disease with current survival rates of approximately 60–70 %. Cytogenetics, recurrent molecular abnormalities, and early response to treatment are the main factors influencing outcome . HoweverNUP98) and PHD finger protein 23 (PHF23) genes, resulting from a cryptic translocation t , and bioinformatic analysis was performed as previously described . In 2  harbored internal tandem duplication of FLT3 (FLT3-ITD), this latter abnormality being chosen because we previously reported a strong association between NUP98-NSD1 rearrangement and FLT3-ITD [NUP98-PHF23 fusion gene. Overall, 2 out of 139 CN-AML cases were found to harbor NUP98-PHF23  leading to the fusion between NUP98 and PHF23 in all cases , with a distinct HOX gene-expression pattern [So far, many NUP98-rearrangements have been found to be associated with both de novo and therapy-related AML but also with T-cell acute lymphoblastic leukemia with over 28 different partner genes . Recentl pattern .PHF23 have never been previously described in children with AML. Located on the reverse strand of 17p13.1, PHF23 encodes for a protein containing a plant homeodomain (PHD) finger [NUP98-PHF23 has been demonstrated to impair the differentiation of myeloid progenitor cells and promote leukemia development in vitro and in vivo [NUP98-PHF23 are sensitive to disulfiram, an FDA-approved drug, demonstrating the feasibility of targeting this oncoprotein [Conversely, chromosomal rearrangements and/or mutations of ) finger  involved in vivo . Cells eNUP98-PHF23 fusion, demonstrating that this genomic aberrancy is not exceptional (tentative frequency of 2.6 %) in pediatric CN-AML. These findings enforce the role of epigenetic regulators in pediatric AML and suggest novel therapeutic targets for this disease.In summary, we identified, for the first time in childhood AML, a