Here, we report that emetine specifically releases Ca2+ from GA in HeLa and HL-1 atrial myocytes. Additionally, it has become evident that the trans-Golgi is a labile Ca2+ store that requires a continuous source of Ca2+ from either the external milieu or from the ER, to enable it to produce a detectable transient increase in cytosolic Ca2+. Our data indicates that the emetine-sensitive Ca2+ mobilizing mechanism is different from the two classical Ca2+ release mechanisms, i.e. IP3 and ryanodine receptors. This newly discovered ability of emetine to release Ca2+ from the GA may explain why chronic consumption of ipecac syrup has muscle side effects.The Golgi apparatus (GA) is a  Cells are equipped with a complex Ca2+ toolkit, which enables them to maintain cytosolic Ca2+ concentrations ([Ca2+]c) at a very low level (~100 nM). This is achieved by the concerted action of Ca2+ extrusion mechanisms localized in the plasma membrane (PM), Ca2+ storage within the lumen of intracellular compartments, and is facilitated by Ca2+ buffering proteins within the cytosol and organelle lumen1. Changes in the [Ca2+]c or within cellular organelles control different processes, e.g. excitation-contraction coupling, neurotransmission, hormone secretion, gene transcription, apoptosis and metabolism, among others2. However, high and prolonged increases of [Ca2+]c are cytotoxic3. Regarding Ca2+ control within cellular organelles, the endoplasmic reticulum (ER), or its specialized version in muscle cells called the sarcoplasmic reticulum (SR), represents the most abundant storage compartment. It possesses a well characterized ATP-dependent Ca2+ accumulation mechanisms, the sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), as well as two Ca2+ release channels; the inositol trisphosphate (IP3) receptors (IP3R) and/or the ryanodine receptors (RyR)5. SERCAs are strongly and specifically inhibited by thapsigargin and cyclopiazonic acid7. With regard to other organelles, the Ca2+ uptake and release mechanisms of mitochondria have been the subject of intense investigation in the last decade and their main molecular and functional characteristics have been unraveled in great detail9. In contrast, characterization of the Ca2+ homeostatic system of other cellular organelles, i.e. endosomes, lysosomes11, secretory vesicles12 and the Golgi apparatus (GA)15, which are characterized by a luminal acidic pH (and often collectively named “acidic Ca2+ stores”), is still poorly understood. For instance, a well characterized Ca2+ uptake mechanism in the GA is the secretory pathway Ca2+-ATPase (SPCA)17 and it has been shown that this Ca2+ pump coexists with a Ca2+/H+ exchanger18, which might have a minor role in accumulating Ca2+ given the small pH gradient between the lumen of the GA and cytosol19. As for Ca2+ release, evidence has been provided suggesting that these organelles (lysosomes in particular) possess the two-pore channel20 (TPC2), which is sensitive to nicotinic acid adenine dinucleotide phosphate (NAADP). Nevertheless, it should be noted that the role of TPC2 in Ca2+ release and its activation by NAADP has recently been challenged and the issue remains unresolved21.A tight control of cellular Ca2+ indicators (GECIs) selectively targeted to the GA sub-compartments has revealed a highly complex Ca2+ signaling toolkit in this organelle, that differs in the specialized sub-regions. The current model suggests that the cis-medial section of the GA resembles the ER in terms of its Ca2+ handling: Ca2+ release can be triggered by IP3 and, where RyRs are expressed, by caffeine; while Ca2+ uptake is catalyzed by SERCA and, in the medial-Golgi, also by SPCA17. The trans-Golgi region appears not to express IP3Rs or SERCAs and Ca2+ uptake involves the SPCA. In neonatal cardiomyocytes, the trans-Golgi appears to express RyRs and accordingly it releases Ca2+ in response to caffeine16. Indirect evidence suggests that the Ca2+ level within the GA lumen modulates vesicular trafficking and the correct sorting of proteins22.As to the GA, the recent development of genetically encoded Ca2+ leak23, we found that this alkaloid, which is also present in ipecac syrup24, was able to reduce the Ca2+ content of an undefined Ca2+ store in HeLa cells. Ipecac syrup is an emetic agent25 and its chronic consumption results in reversible myopathy and cardiomyopathy26. At the molecular level, this alkaloid acts as an inhibitor of protein synthesis27, by targeting the small subunit of ribosomes28, without actually detaching the ribosomal complex from the translocon29.While using emetine to study the well-established role of Sec61 translocon in the ER Ca2+ from the medial- and trans-regions of the GA, without affecting the ER Ca2+ content. Emetine may represent the first member of a chemical library that will enable studies on the mechanism of GA Ca2+ homeostasis and its role in Ca2+ pathophysiology.Using a variety of probes and experimental approaches, here we demonstrate that emetine is capable of mobilizing Ca2+ homeostasis, we carried out simultaneous recordings of the signals from two fluorescent indicators; Fura-2 and Mag-Fluo-4, in HeLa cells. While Fura-2 was homogeneously distributed in the cytosol and nucleus , Mag-Fluo-4 fluorescence was not distributed in a homogenous manner. Rather, a faint signal was observed in the region corresponding to the nuclear membrane, as well as reticular structures in the cytoplasmic region, while the nucleus itself was completely dark . In the presence of external [Ca2+], the combination of ATP (an IP3-generating agonist) and thapsigargin  induced a transient increase in the [Ca2+]c and this was associated with a sustained reduction in the [Ca2+]L signal , we carried out the same experiment in the absence of external [Ca2+]  and a substance that can collapse the acidic pH gradient, such as monensin (10 μM)32. In the experiments presented in Fig. 2+]c and a large reduction in the [Ca2+]L  or bafilomycin . This NH4Cl concentration was selected to avoid having an effect on the IP3-induced Ca2+ release, while the baf concentration was low enough to be specific for vacuolar H+-ATPase33. The rise in [Ca2+]c and the associated drop in [Ca2+]L induced by ATP and Tg were unaffected by pre-incubation with NH4Cl for 15 minutes . These effects can be explained by the fact that baf increases the [Ca2+]L , we searched for a more specific treatment to release Ca2+ from the lysosomal compartment. To this end, we treated HeLa cells with NAADP/AM, a membrane permeable NAADP analogue believed to be a specific lysosomal Ca2+ mobilizer via TPC2 channels36. The application of 10 nM NAADP-AM resulted in a slow but significant drop in the [Ca2+]L without any effect on the [Ca2+]c . This is a substrate of the intralysosomal protease cathepsin C, and accumulation of the hydrolysis product therein will osmotically disrupt the lysosomes+]L Fig. . Collect2+ store responding to emetine, we performed confocal microscopy experiments in HeLa cells loaded with Mag-Fluo-4, in the presence of external [Ca2+]. The perinuclear and cytosolic regions of the cells that were labelled with Mag-Fluo-4 . In the experiment presented in Fig. 37, the nucleus (H2BD3cpv)37 and the mitochondrial matrix (4mtD3cpv)38. For technical reasons, in these experiments we mainly employed His (100 µM), as the IP3-generating agonist and CPA (20 µM) as a reversible SERCA inhibitor. We first investigated the dynamics of ER [Ca2+]. As expected, His and CPA caused a very large decrease in the ΔR/R0 , the application of emetine significantly reduced the [Ca2+]n 17 and the trans compartments (GoD1cpv)16 of the GA.The experiments described so far, which have been carried out using both chemical Ca0 in both the medial-  in the medial-Golgi . To this end, we employed HL-1 cells, a cell line derived from mouse atrial myocytes, which express RyR240. Fig. 2+]n. The application of emetine did not have any effect on the [Ca2+]n when perfused either before or after caffeine , emetine caused a significant reduction in the ΔR/R0  in combination with SERCA pump inhibitors (Tg or CPA). Confocal experiments showed that emetine released Ca2+ only from the perinuclear region, where 60% of the Golgi marker colocalized with Mag-Fluo-4; a completely different picture was observed for the ER, which is distributed throughout the entire cytosol.Three different approaches, and two different cell types, support the idea that emetine is not releasing Ca2+ stores were targeted by emetine, with the knowledge that Ca2+ handling in the acidic compartment is dependent on the proton gradient generated across this membrane31. We studied the role of the acidic Ca2+ stores with the combination of ionomycin and monensin. These ionophores produced a smaller and transient [Ca2+]c response compared to the one produced by ATP and Tg, and yet caused a similar reduction in the [Ca2+]L. In addition, these ionophores inhibited the emetine-induced reduction in the [Ca2+]L. To further support the idea that emetine was targeting an acidic Ca2+ store we employed different approaches to disrupt the proton gradient of these stores, i.e. alkalization with NH4Cl42 and inhibition of the V-type H+−ATPase with bafilomycin44. This inhibitor increased the agonist-induced Ca2+ release from the ER, likely by decreasing the Ca2+ buffering activity of the acidic intracellular stores, in agreement with previous reports45; while NH4Cl did not show this effect because cellular alkalization inhibits ER Ca2+ release by decreasing the activity of SERCA pump46. We found that although bafilomycin and NH4Cl did not have the same effect on agonist induced Ca2+ release, they both decreased the emetine-induced [Ca2+]L response by increasing the pH in the intracellular acidic stores. Since these conditions do not deplete acidic Ca2+ stores within the time frame used here, we think that emetine needs an acidic environment to activate the Ca2+ mobilization mechanism. Thus, we concluded that an acidic Ca2+ store was the target of emetine.We decided then to study whether acidic Ca2+]L response. Additionally, GPN, a lysosomal disruptor47, did not decrease the [Ca2+]L response induced by emetine. Similar to previous reports48, we have observed that the effect of GPN on the [Ca2+]c and the [Ca2+]L is due to Ca2+ release from the ER. Indeed, complete depletion of the ER Ca2+ store with the combination of ATP and Tg fully abolished the effect of GPN on both the [Ca2+]c and the [Ca2+]L. These data mean that even when GPN is targeting lysosomes, its effect on [Ca2+]c and [Ca2+]L somehow reflects an action on the ER.The involvement of the endo-lysosomal system was discarded because NAADP or inhibitors of the NAADP receptor did not affect the emetine-induced [Ca17 or trans-Golgi16 cameleons as previously reported. Both compartments responded to emetine application with a clear reduction in the [Ca2+]L. Moreover, emetine still decreased the [Ca2+]L in these two GA compartments when cells were maintained in Ca2+ free extracellular medium containing EGTA, implying that emetine Ca2+ mobilization activity might involve the activation of an ion channel instead of blocking the Ca2+ loading mechanism. Since the trans-Golgi region is a labile Ca2+ store, this might explain why emetine does not produce any increase in the [Ca2+]c as the first stimulus, but only right after Ca2+ has been released from the ER by an agonist. We have observed that emetine increases the luminal pH as well and to assess the role of pH in the effect of emetine, we titrated pH changes with monensin. These data indicate that only a small part of the effect of emetine on the reduction of the ΔR/R0 can be explained by its associated increase in the luminal pH, the main effect of emetine is on the reduction in the [Ca2+]L.To determine whether the GA was the target for emetine, we transfected cells with either medial-43. The trans-Golgi region of HL-1 cells responded to both caffeine and emetine with a similar reduction in the [Ca2+]L. Interestingly, the trans-Golgi Ca2+ store sensitive to emetine partially overlaps with the region released by caffeine in HL-1 cells. It seems unlikely that the channel activated by emetine is the RyR2.The effect of emetine was not limited to the GA of HeLa cells. Indeed, similar results have been obtained studying the trans-Golgi region of HL-1 atrial cells, which express RyR22+ pump responsible for Ca2+ uptake by the trans-Golgi16 whereas it co-exists with SERCA in the cis/medial-GA17. The fundamental importance of SPCA in the physiology of GA has been shown by a number of studies, which have reported that reduction or ablation of SPCA causes morphological alterations in the Golgi structure, as well as the disruption and dysfunction of both vesicle trafficking and protein sorting in the secretory pathway51. However, SPCA has been reported to be expressed in other secretory vesicles, where it is required for Ca2+ homeostasis in these organelles53. Our data demonstrate that emetine is directly and specifically affecting GA. However, it is unlikely that emetine is inhibiting SPCA; but it appears to be activing a leak channel in the GA. Further experiments are needed to unveil the molecular nature behind this effect of emetine.The SPCA is a Tg-insensitive Ca2+ mobilization agent from GA, might explain the symptoms of cardiac and skeletal myopathies observed in people who are chronically consuming emetine26. This might lead to a dysfunction in Ca2+ dynamics in the GA that could result in altered Ca2+ handling in muscle cells.This new role of emetine, as a specific Ca1 (baf), histamine (His) and cyclopiazonic acid (CPA) were purchased from Sigma-Aldrich, Glycyl-L-phenylalanine-2-naphthylamide (GPN) from Santa Cruz Biotechnology, NH4Cl from Merck and Ned 19-Trans from Enzo. The acetoxymethyl (AM) ester forms of organic Ca2+ indicators Mag-Fluo-4, Fura-2, Fluo-4 were purchased from Invitrogen (Molecular Probes). NAADP-AM and BZ194 were kindly provided by Dr. Claudia Treviño from The Biotechnology Institute of UNAM 54. Emetine was dissolved in water and hydrophobic chemicals in dehydrated dimethyl sulfoxide (DMSO) in stock 1000-fold higher than the final concentration used.Adenosine triphosphate (ATP), thapsigargin (Tg), emetine, bafilomycin A2 in humid conditions. For the culture of HL-1 cells, Claycomb medium (from Sigma-Aldrich) supplemented with norepinephrine (100 µM), L-glutamine (2 mM) and the same concentration of fetal bovine serum and antibiotic as mentioned above was used.HeLa cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium with high D-glucose (4500 mg/l), L-glutamine and sodium pyruvate (110 mg/l) supplemented with 10% fetal bovine serum, and 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C and 5% of CO2+ indicators was carried out as follows: HeLa and HL1 cells were seeded onto 18 mm diameter coverslips (coated with 25 µg/mL fibronectin in 0.2% of gelatin for the HL1 cells) and transfected at 60% confluence with 1 µg DNA employing TransIT®-LT1 transfection reagent (Mirus) for HeLa cells or Lipofectamine® 2000 for HL1 cells. Experiments were performed 24 h or 48 h after transfection.Transfection of genetically encoded Ca2, 1.8 CaCl2, 6 NaHCO3, 25 HEPES and 5.5 glucose [pH 7.3 at room temperature (RT)]. Cell viability was always higher than 95% as determined by trypan blue exclusion assay and 1 × 106 cells/ml were loaded with 1 µM of each of the Ca2+ indicators, i.e. Fura-2/AM and Mag-Fluo-4/AM, to determine changes in cytosolic ([Ca2+]c) and luminal ([Ca2+]L) calcium concentrations, respectively. This loading was carried out in the dark and at RT for 2 h. At the end of this time, half a million cells were microfuged twice and the cell pellet was re-suspended in 2.5 ml of saline solution with or without CaCl2 plus 0.1 mM EGTA, as indicated. Fluorescence signals were recorded at a dwell time of 100 ms and a frequency of 2.7 Hz with excitation wavelengths of 340, 360 and 380 nm for Fura-2 and 495 nm for Mag-Fluo-4 with the same emission wavelength of 515 nm using a DeltaRAM V PTI spectrofluorometer.HeLa cells were trypsinized after reaching 80% confluence. The cell suspension was microfuged and the pellet was suspended in extracellular-like saline solution containing in mM: 121 NaCl, 5.4 KCl, 0.8 MgCl2+]c calculated from the Fura-2 signals and normalization of the Mag-Fluo-4 signals was carried out as previously described55. To discard the participation of lysosomes, cells were incubated with GPN for a time period of 10 min. Since the released naphthylamine (a product of GPN cleavage mediated by cathepsin C) interferes with Fura-2 fluorescence, cells were washed before recording. In the situation where GPN was present, we did not use the 340/380 ratio; only the 380 nm fluorescence signal was used to reflect changes in the [Ca2+]c.The c. Thus, we have normalized the 340/380 ratio using the value at time zero (ΔR/R0).The use of ionomycin and monensin to fully deplete the acidic Ca2+ indicators were excited with 488 nm laser line using a pinhole of 45 µm, and fluorescence images were collected every 1.93 s.HeLa cells were cultured onto 21 × 21 mm coverslips until they reached 80% confluence. They were then loaded with 1 µM of either Mag-Fluo-4/AM or Fluo-4/AM in the dark at RT for 2 and 1 hour, respectively. After this time, the coverslip was placed onto the chamber and bathed in extracellular-like saline (composition described above). Fluorescence recordings were obtained using a Carl Zeiss LSM700 confocal inverted microscope with a 63x oil immersion objective (1.4 N.A.). Both Ca2+ indicator fluorescence signals was carried out with ImageJ . Regions of Interest (ROIs) were drawn in two different regions: perinuclear and cytosolic. Since the Mag-Fluo-4 signal exhibited a constant exponential decrease, the first 20 images (i.e. before the perfusion of any stimulus) were fitted to this equation Y = A0exp−x/t + y0 and this was considered as the basal fluorescence (F0). Changes in the [Ca2+]L were calculated as ∆F/F0.Analysis of the CaColocalization studies of Mag-Fluo-4 and organelles, either the ER or the GA were carried out by transfecting HeLa cells with 2 µg (4 μL of Lipofectamine 2000) of either mCh-Sec61 beta  or mCherry-Golgi-7 . Transfected cells were also loaded with Mag-Fluo-4/AM, as described above. Live HeLa cell imaging was carried out with the confocal microscope to visualize the degree of colocalization between Mag-Fluo-4 and the probe for either the ER or the GA. The level of colocalization was quantified using Manders´ coefficient.et al.56. Briefly, cells seeded on coverslips were placed onto an open-topped chamber at 37 °C with modified Krebs-Ringer buffer (mKRB) containing (mM): 135 NaCl, 5 KCl, 10 glucose, 1 Mg2Cl, 1.8 CaCl2 and 10 HEPES (pH 7.4 at 37 °C). Image exposure time was 200 ms and acquisition frequency 0.5 Hz. With these probes, the ratio (R) between the fluorescence intensity emitted by the acceptor (at 540 nm) and by the donor (at 480 nm) fluorophores (upon excitation of the donor) is a function of the FRET efficiency and accordingly of the [Ca2+]. An increase or a decrease in the 540/480 emitted fluorescence ratio thus indicates an increase, or a decrease, in the [Ca2+], respectively. Data are presented as a ∆R/R0, where: ∆R is the change of the cpV/CFP emission intensity ratio at any time, and R0 is the fluorescence emission ratio at the time 0.FRET experiments were performed as described by Drago All the data represent at least three independent experiments. Figures were prepared by GraphPad Prism version 5.0. Averages are expressed as mean ± SEM  analyzed by unpaired Student´s t test, where *P < 0.05, **P < 0.01 and ***P < 0.001 are statistically significant.Supplementary file


We show, using small interfering RNA (siRNA) and vacuolar H+-ATPase (V-ATPase) inhibitors, that lysosomes sequester Ca2+ released by all IP3R subtypes, but not Ca2+ entering cells through store-operated Ca2+ entry (SOCE). A low-affinity Ca2+ sensor targeted to lysosomal membranes reports large, local increases in cytosolic [Ca2+] during IP3-evoked Ca2+ release, but not during SOCE. Most lysosomes associate with endoplasmic reticulum (ER) and dwell at regions populated by IP3R clusters, but IP3Rs do not assemble ER-lysosome contacts. Increasing lysosomal pH does not immediately prevent Ca2+ uptake, but it causes lysosomes to slowly redistribute and enlarge, reduces their association with IP3Rs, and disrupts Ca2+ exchange with ER. In a “piston-like” fashion, ER concentrates cytosolic Ca2+ and delivers it, through large-conductance IP3Rs, to a low-affinity lysosomal uptake system. The involvement of IP3Rs allows extracellular stimuli to regulate Ca2+ exchange between the ER and lysosomes.Inositol 1,4,5-trisphosphate (IP  •3 receptors (IP3Rs) selectively deliver Ca2+ to lysosomesIP•3Rs in ER membranesLysosomes associate preferentially with clusters of IP•3R-lysosome contacts required for Ca2+ uptakeLow lysosomal pH maintains the IP•2+ channels deliver Ca2+ to low-affinity lysosomal transportersER and its Ca 2+ exchanges between ER and lysosomes regulate cytosolic Ca2+ signals and lysosome behavior. Atakpa et al. show that clusters of IP3 receptors populate ER-lysosome contact sites and facilitate local delivery of Ca2+ from the ER to lysosomes.Ca Most Ca2+ signals are evoked by opening of Ca2+-permeable channels within the plasma membrane (PM) or the membranes of intracellular organelles, usually the endoplasmic reticulum (ER). In non-excitable cells, extracellular stimuli typically evoke Ca2+ signals by stimulating phospholipase C (PLC), which catalyzes formation of inositol 1,4,5-trisphosphate (IP3). Binding of both IP3 and Ca2+ to IP3 receptors (IP3Rs) causes them to open and release Ca2+ from the ER  adjacent to the PM regulates membrane potential by selectively activating Ca2+-sensitive Cl− or K+ channels  from RyRs  selectively regulates adenylyl cyclases, nitric oxide synthase, and nuclear factor of activated T cells , TPC2 (two-pore channel 2), and ATP-regulated P2X4 receptors, that allow Ca2+ release , can, in “piston-like” fashion, deliver Ca2+ from the cytosol with its low [Ca2+]c to the low-affinity uptake system of lysosomes. The involvement of IP3Rs allows cell-surface receptors, through PLC and IP3, to regulate this Ca2+ transfer and so the behavior of lysosomes.Using targeted low-affinity Ca+-ATPase (V-ATPase) maintains the luminal pH of lysosomes at about 4.5. Treatment of HEK cells with concanamycin A (CcA), a more selective inhibitor of the V-ATPase than bafilomycin A1 (2+]c (2+]c evoked by carbachol (CCh), which stimulates IP3 formation by activating M3 muscarinic receptors in HEK cells, was increased by CcA  to an essential pore-forming subunit of the V-ATPase (ATP6V0C)  reduced 2+]c G. HoweveV-ATPase H. These embranes . We conc2+ entry across the PM through SOCE, which is stimulated when IP3 causes loss of Ca2+ from the ER  to inhibit SERCA and deplete the ER of Ca2+, restoration of extracellular Ca2+ caused a sustained increase in [Ca2+]c reflecting the activity of SOCE  to record global increases in [Ca2+]c   targeted [Ca2+]c . HeLa cem the ER , were uswere not G–S1I. Be ∼360 nM G, Ly-GG 2+-free HBS stimulated with histamine (100 μM) and then with ionomycin (10 μM with 2 mM Ca2+) to saturate the indicators. Time (h:min:s:ms) displayed in top right corner; fps, frames/s.Wide-field video  shows HeLa cells in nominally Cacoloc = 0.93 ± 0.02, n = 3; Rcoloc is Pearson’s correlation coefficient) and with the lysosomal channel, TPC2-RFP  (2+]c (reported as F/Fmax) was similar when detected with Ly-GG or with Cy-GG  or from comparable areas of cytosol (Cy-GG). Histamine caused a transient increase in Cy-GG fluorescence and a larger transient increase in Ly-GG fluorescence; the latter often came close to saturating the sensor  or cytosol-like responses . Similar analyses of SOCE revealed no disparity in the responses of Cy-GG and Ly-GG: both sensors reported similar increases in [Ca2+]c  A. In unsth Cy-GG B–2D. Thie sensor C and 2E.e sensor F and 2G. [Ca2+]c H.Figure 2+ signals might be due to lysosomal Ca2+ channels, rather than to juxtaposed IP3Rs. H1 receptors activated by histamine can, for example, stimulate accumulation of NAADP, which evokes Ca2+ release through TPC2  also caused increases in [Ca2+]c that were larger at the lysosome surface than in bulk cytosol   between wild-type cells  . We alsoisrupted  to estabhe cells A. There pe cells B and celpe cells B–3G. We  signals B–3G. In  20%–30% H. These 3R1 had been tagged by gene editing with EGFP (EGFP-IP3R1-HeLa cells)  are associated with ER, and lysosomes maintain these associations as they move . This isa cells)  to explong TIRFM , and S8.ic IP3Rs . Most lyR puncta C. This bysosomes . We obseacent ER . Some ly punctum  and S5. r parked . We also punctum A. Finallnt times , and of lysosome .Figure 4TIRFM video  of part of a HeLa cell expressing markers of ER  and lysosomes . Almost all lysosomes are associated with ER, and they maintain their association as each moves.3R1-HeLa cell expressing markers of ER  and lysosomes . As lysosomes move, they maintain close contact with ER.TIRFM video  of a small part of an EGFP-IP3R1-HeLa cell expressing markers of ER (mCherry-ER) and lysosomes (mTurquoise-LAMP1). Left panel shows merged images of lysosomes (cyan) and ER (red). Right panel shows merged images from the same region of EGFP-IP3R1 (green) and lysosomes (pseudo-colored in magenta). Lysosomes remain associated with ER, but they linger longest at ER regions where there are IP3R puncta. Note, for example, how a lysosome moves rapidly along ER to the central IP3R punctum, remains parked there for ∼60 s, and then moves rapidly away along the ER. Note also, the lysosome parked at the bright IP3R punctum at the beginning of the sequence (bottom of image), and how it then moves rapidly along ER before parking at another IP3R punctum.TIRFM video  shows part of an EGFP-IP3R1-HeLa cell expressing markers of ER  and lysosomes (mTurquoise -LAMP1). Left panel shows merged images of lysosomes (cyan) and ER (red). Right panel shows merged images of EGFP-IP3R1 (green) and lysosomes (pseudo-colored in magenta) from the same region. Note (middle of field), how a lysosome leaps between 3 different immobile IP3R puncta, parking at each, but passing quickly over intervening ER.TIRFM video  shows part of an EGFP-IP3R1-HeLa cell expressing markers of ER (mCherry-ER) and lysosomes (mTurquoise-LAMP1). Left panel shows merged images of lysosomes (cyan) and ER (red). Right panel shows merged images of EGFP-IP3R1 (green) and lysosomes (pseudo-colored in magenta) from the same region. Note, how when two lysosomes move apart, one of them then associates with an IP3R punctum and moves with it.TIRFM video  shows part of an EGFP-IP3R1-HeLa cell expressing markers of ER (mCherry-ER) and lysosomes (mTurquoise-LAMP1). Left panel shows merged images of lysosomes (cyan) and ER (red). Right panel shows merged images of EGFP-IP3R1 (green) and lysosomes (pseudo-colored in magenta) from the same region. Note, how several lysosomes visit each of the two IP3R puncta at the left side of the image.TIRFM video  shows part of an EGFP-IP3R1-HeLa cell expressing markers of ER (mCherry-ER) and lysosomes (mTurquoise-LAMP1). Left panel shows merged images of lysosomes (pseudo-colored in magenta) and ER (pseudo-colored in cyan). Right panel shows merged images of EGFP-IP3R1 (green) and lysosomes from the same region. Note, how at least 3 mobile IP3R puncta sequentially collide with the central lysosome and then remain associated with it for tens of seconds.TIRFM video  shows part of an EGFP-IP3Rs and lysosomes, we used in situ proximity ligation assays (PLA) to report whether an ER (VAP-A) and a lysosomal protein (Rab7) were located within ∼40 nm of each other    E and 5F.e, 100%) D. Hence R puncta .Figure 53Rs might contribute to formation of ER-lysosome MCSs, but two lines of evidence suggest this is unlikely. First, in HEK cells with and without IP3Rs the association of lysosomes with ER was indistinguishable whether determined by PLA  of part of a wild-type HEK cell expressing markers of ER  and lysosomes . Lysosomes associate with ER, and maintain their interactions as they move.3Rs (HEK-KO), but expressing markers of ER  and lysosomes . Just as in wild-type cells, lysosomes associate with ER, and maintain their interactions as they move.TIRFM video  of part of a HEK cell lacking IP2+ exchanges occur. Expression of either ORP1L or forms expected to stabilize (ΔORD) or destabilize (ΔORDPHDPHD) ER-lysosome MCSs , and STARD3, ORP1L, Rab7, TPC2, or phosphatidylinositol 3-phosphate (in lysosomes) . We maniome MCSs A did nots by CcA B and S6C+ signal B, but be2+ uptake mechanism in mammalian lysosomes is unknown. It has been suggested to involve a low-affinity Ca2+-H+ exchanger (CAX), but there is no known CAX in mammalian cells  to detect Ca2+ signals evoked by histamine in HeLa cells   and then delivered locally through large-conductance channels (IP3Rs and perhaps others) at a high local concentration to a low-affinity uptake system (MCU or lysosomes)   cannot dysosomes . Our resysosomes . A similex (MCU) . For mitchondria . By contysosomes . We suggsosomes) I. The ERganelles I.3Rs provide a link between extracellular stimuli and delivery of Ca2+ to lysosomes .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Colin W. Taylor  . Short tandem repeat profiling was used to authenticate the HeLa cells used  and the HEK cells lacking all three IP3R subtypes . We have not confirmed the authenticity of the HAP1 cell lines. Regular screening throughout the study established that all cell lines were free of mycoplasma.The methods used to establish EGFP-IPescribed . In briechannels . HEK celter, NY)  and supp2, and passaged every 3-4 days using TrypLE Express.HeLa and HEK293 cell lines were cultured in Dulbecco’s Modified Eagles Medium (DMEM)/F-12 with GlutaMAX supplemented with fetal bovine serum . HAP1 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) GlutaMAX with 10% FBS. All cells were maintained at 37°C in humidified air with 5% CO2+ indicators (Ly-GG or Cy-GG) or tagged proteins according to the manufacturer’s instructions using TransIT-LT1 reagent (1 μg DNA/2.5 μl reagent). For siRNA transfections, cells were plated in clear-bottomed 96-well plates  coated with poly-l-lysine (0.01% w/v). After 24 hr, cells were transfected with Silencer™ siRNA (40 nM) directed against ATP6V0C or a non-silencing control siRNA using siPORT NeoFX transfection reagent (220 ng siRNA/μl reagent). Experiments were performed 48-72 hr after transfection. The same methods were used for transfection with siRNA directed against human ORP1L  coated with human fibronectin (10 μg/ml). Cells were transfected with plasmids encoding Caan ORP1L   were LAMP1-mCherry and cytosolic G-GECO1.2. A HindIII recognition site was inserted at the 5′ end of LAMP1-mCherry using primer LAMP1F   was usedT7 promoterF, G-GECO1.2SeqM and GGECO1.2SeqE . Sequenc2, 1.5 mM CaCl2, 11.5 mM D-glucose, 11.6 mM HEPES, pH 7.3. CaCl2 was omitted from nominally Ca2+-free HBS. In some experiments, BAPTA  was added to HBS immediately before stimulation to reduce the free [Ca2+] of the HBS to < 20 nM. Fluorescence was recorded using a FlexStation III fluorescence plate-reader  (max) and minimal (Fmin) fluorescence values were determined from parallel wells after addition of Triton X-100 (0.1%) to lyse cells in the presence of either 10 mM CaCl2 (Fmax) or 10 mM BAPTA (Fmin). Fluorescence values (F) were calibrated to [Ca2+]c using a KD = 389 nM from:3-evoked Ca2+ release from saponin-permeabilized HAP1 cells was measured in cytosol-like medium (CLM) using a low-affinity Ca2+ indicator (Mag-fluo 4) trapped within the ER, as previously described for other cell types . Cells were recovered , re-suspended (∼107 cells/ml) in Mg2+-free CLM, distributed (45 μl/well) into black half-area 96-well plates and centrifuged . Mag-fluo 4 fluorescence  was recorded at 1.44-s intervals at 20°C using a FlexStation III fluorescence plate-reader. Addition of MgATP (1.5 mM) allowed Ca2+ uptake into the intracellular stores. When steady-state Ca2+ loading was achieved (∼2 min), IP3 was added with cyclopiazonic acid  to inhibit further Ca2+ uptake. IP3-evoked Ca2+ release is reported as the fractional decrease in the ATP-dependent Mag-fluo 4 fluorescence.Confluent monolayers of cells grown in a 96-well plate  were loaded with fluo 8 by incubation for 1 hr at 20°C in HEPES-buffered saline  containing fluo 8-AM (2 μM) and 0.02% Pluronic F-127. Cells were then washed and incubated in HBS for 1 h at 20°C to allow de-esterification of fluo 8-AM. HBS had the following composition: 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgClCA, USA) . Fluoresll types . Briefly2 targeted laser illumination system . Excitation light was transmitted through either a quad dichroic beam-splitter (TRF89902-QUAD) or a dichroic mirror . Emitted light was passed through appropriate filters  and detected with either an iXon Ultra 897 electron multiplied charge-coupled device (EMCCD) camera  or , a multi-line laser bank  and an iLas) or for  a Prime coloc) for comparisons of fluorophores in every pixel, or Manders’ split coefficient to identify the fraction of PLA spots that colocalized with EGFP-IP3R1  that exceeded a threshold value :For colocalization analyses, we used either Pearson’s correlation coefficient  and the nearest EGFP-IP punctum G and 7H, punctum .HeLa cells transfected with Ly-GG were washed three times in HBS, and Ly-GG fluorescence  was imaged using wide-field fluorescence microscopy (1 frame/s) at 20°C. After background correction, the Ly-GG fluorescence associated with single lysosomes was measured using single-particle tracking with the MetaMorph Track Objects plugin . A templ3/PM  . Images were acquired at 50-ms intervals with an iXon Ultra 897 EMCCD camera, corrected for background fluorescence, and analyzed using MetaMorph. Ly-GG was tracked to determine Ca2+ signals around single lysosomes. Photolysis of ci-IP3 releases an active, but more metabolically stable, analog of IP3  , then loaded with ci-IP 50 min) . After we group) .425: λex = 425 nm, λem = 480 nm. F488: λex = 488 nm, λem = 525 nm). Images were collected for 100 ms, with 5 min between each round of data acquisition. After background subtraction, ROIs were drawn around lysosome clusters and fluorescence ratios  were calculated from F488/F425 at each time. Results are presented as R/R0, where R0 is the fluorescence ratio recorded before stimulation.The pH within lysosomes was measured from defined ROI within single cells using a dextran-conjugated ratiometric pH indicator, fluorescein-dextran, loaded into lysosomes by endoytosis. Cells were incubated with fluorescein-dextran  for 16 hr, followed by a 4-hr chase in DMEM F12 at 37°C. The cells were then washed 3 times with HBS, and imaged immediately with alternating excitation/emission  of 0.6-1.0. The circularity criterion ensured that only roughly circular particles were selected for analysis  for the indicated PCR product. Results are reported as mean ± SD for cDNA samples independently isolated from 3 different experiments.QPCR was carried out as previously described . cDNA wain situ. The antibodies are then recognized by secondary antibodies conjugated to complementary oligonucleotides, which are amplified to incorporate a fluorescent nucleotide (Texas Red) only if the pair of secondary antibodies are less than ∼40 nm apart (A Duolink proximity ligation assay (PLA) was used to quantify interactions between proteins less than ∼40 nm apart, according to the manufacturer’s instructions. The method uses antibodies from two species (mouse and rabbit) to recognize two candidate proteins nm apart  , ligase and polymerase, and the washes between each step, were exactly as recommended by the manufacturer. Cells were then mounted in Duolink II mounting medium containing DAPI to label the nucleus. PLA products were visualized using an Olympus microscope with x60 or x100 objective, and spots were quantified using CellProfiler software. The specificity of the PLA reactions was confirmed by omission of either primary antibody (VAP-A or Rab7) and, for EGFP-IP3R1-lysosome measurements by using cells without EGFP-IP3R1 , washed with PBS, permeabilized , and incubated with primary antibodies . For ER-lysosome interactions, the primary antibodies were against VAP-A (ER) and either LAMP1 or Rab7 (both lysosomes), and for EGFP-IPFP-IP3R1 .g, 2 min) were lysed in cold medium containing protease inhibitors  and the supernatant  was used for western blotting. Proteins were separated , transferred to a polyvinyl difluoride (PVDF) membrane using an iBLOT gel-transfer system (ThermoFisher), blocked in Tris-buffered saline  containing 0.2% Tween-20 and 5% BSA for 1 hr, washed in the same medium, and incubated with primary antibody  in the blocking buffer. After washing (3 × 5 min), the membrane was incubated with secondary antibody , washed (3 × 5 min). Bands were visualized using ECL Prime western blotting detection reagent and a Syngene Pxi chemiluminescence detection system with GeneTools software.Cells isolated by centrifugation  and maximal response for each individual experiment. The individually determined pEC50 values were then pooled for statistical analysis.All statistical analyses used Prism, version 5. For analyses of concentration-effect relationships, non-linear curve-fitting to a Hill equation was used to provide values for pECn) refer to independent experiments. p < 0.05 was considered significant. The tests used are reported in the figure legends.All results are presented as mean ± SD or SEM, as appropriate. Student’s t test (for 2 variables), and one-way or two-way ANOVA with Tukey’s multiple comparison test or Bonferroni post hoc test (more than 2 variables) were used for statistical analyses. The Kolmogorov-Smirnov normality test was used to determine whether frequency distributions deviated from normality (p < 0.05) F and 2G.


The ion channel protein is massive (more than 2.2 MDa) and has a structure that has defied detailed determination until recent developments in cryo-electron microscopy revealed much of its structure at near-atomic resolution. The availability of this high-resolution structure has provided the most significant advances in understanding the function of the ion channel in the past 30 years. We can now visualise the molecular environment of individual amino acid residues that form binding sites for essential modulators of ion channel function and determine its role in Ca2+ signalling. Importantly, the structure has revealed the structural environment of the many deletions and point mutations that disrupt Ca2+ signalling in skeletal and cardiac myopathies and neuropathies. The implications are of vital importance to our understanding of the molecular basis of the ion channel’s function and for the design of therapies to counteract the effects of ryanodine receptor-associated disorders.The ryanodine receptor calcium release channel is central to cytoplasmic Ca Ca2+ is released from the stores through two classes of ion channel: the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR)1. Muscle contraction is almost entirely dependent on a massive outflow of Ca2+ from the modified ER—sarcoplasmic reticulum (SR)—through RyR channels; relaxation then follows the return of Ca2+ to the store by SERCA  pumps1. The RyR opens in response to an action potential during excitation-contraction (EC) coupling. The action potential travels along the fibre surface and throughout the muscle fibre along transverse tubule extensions of the surface membrane, which forms multiple junctions with the SR membrane2. The gap between the membranes is filled almost entirely by the cytoplasmic bulk of the RyR, which is embedded in the SR membrane 4. The signal from the action potential is transmitted to the RyR through a series of protein–protein interactions that are not yet fully defined but likely include the β1a subunit of the DHPR and the STAC3 protein6. A simpler EC coupling system exists in the heart, where an action potential induces Ca2+ entry through the cardiac DHPR (CaV1.2) and the entering Ca2+ ions activate the cardiac RyR2 in a Ca2+-activated Ca2+ release process1. The massive RyR ion channel protein is composed of four monomers of more than 500 KDa. Its efficient operation depends on a myriad of factors that interact with its many domains. Such factors include associated proteins such as calmodulin, the FK506 binding proteins, and CLIC2, all of which bind to its large cytoplasmic domain. Binding to its tiny luminal domain are triadin, junctin, and the Ca2+ binding protein calsequestrin5. Other essential factors regulating the ion channel open probability and gating behaviour include post-translational modifications such as phosphorylation, oxidation, nitrosylation, nitration, and glutathiolation8. Acquired changes in channel function and genetically transmitted modifications can severely disrupt the ion channel’s function and detrimentally alter Ca2+ signalling to lead to skeletal and cardiac myopathies that can be debilitating and often fatal. Despite the essential function of this ion channel protein, the molecular nature of its gating mechanisms and how they are disrupted in myopathies have remained elusive. Such insight required near-atomic resolution structures of the protein, which are now available with a resolution of not more than 6.1 Å following recent advances in cryo-electron microscopy (cryo-EM)16. Thus far, the more structurally stable RyR1 protein has been determined at the highest resolution of about 3.8 Å13  the Ry 1 and 2 or P2 domains; (4) the SPRY2 and 3 domain; (5) the junctional solenoid (Jsol or Handle), which also contributes to the FKBP binding pocket -dependent protein kinase II (CaMKII) phosphorylation sites; (8) the core solenoid , which contains two EF hand Ca2+ binding motifs; (9) the transmembrane domain (TMD) containing the Ca2+ pore with binding sites for ryanodine and the luminal domain of the RyR with triadin and junctin binding sites; and (10) the C terminal domain (CTD) that forms part of the Ca2+, ATP, and caffeine activation site.The enormous impact of the higher-resolution structures on our understanding of RyR function has been elegantly presented in full reviews by Meissner2+, ATP, and caffeine binding sites have been identified by comparing differences between RyR1 with Ca2+, but not ATP or caffeine, and RyR1 with ATP and caffeine bound, but without Ca2+13. All three activators bind within a small region of the protein in which the CTD comes into close approximation with the Csol domain and TMD. Ca2+ binds at the interface between the CTD and CSol. ATP binds at the junction between the CTD, the cytoplasmic extension of the S6 transmembrane helix, and the “thumb and forefinger” region adjacent to CSol. Caffeine binds between the CTD and cytoplasmic extension of the S6 helix. As predicted from many previous biochemical and physiological studies24, the ryanodine binding site is within the pore and adjacent to residue Q4933.The Ca2+ signalling in the two tissues25. Also of major importance for basic muscle physiology/biophysics are the structural changes determining ion channel gating between the open and closed states. The recent high-resolution structures of RyRs locked into open or closed conformations, in combination with previous lower-resolution structural studies, allow refined models of the ion channel gating mechanisms to be developed20. This has been no easy task, as the ion channel opening and closing require the coordination of more than 40 domains and the transmission of allosteric changes from both cytoplasmic domains and luminal domains to the channel gating mechanism. As stated by Samsó18, “the RyR is the quintessential allosteric machine”. Detailed summaries of RyR1 and RyR2 domain structures are given in several recent publications25. In addition, defined locations of essential ATP, caffeine, and Ca2+ binding sites in RyR1 are reported by des Georgeset al.13, and these observations provide a basis for predictions of the location of similar sites in the RyR2 protein27. It became apparent that the channel can exist in a variety of conformations, not simply “open or closed”. Indeed, priming changes in the C-terminal activation module can occur without altering pore structure, and there may be a hierarchy of pore structure changes that depend on binding of Ca2+, ATP, or caffeine that have not yet been determined but can produce incremental changes in channel open probability13. It is worth noting that the structure of the open channel may continue to be further refined as optimal conditions, including the lipid content of preparative solutions, are developed for cryo-EM structural studies16.Of particular importance to muscle physiology are the substantial differences between the structures of the skeletal (RyR1) and cardiac (RyR2) isoforms, which explain many of the functional differences between the proteins and their role in Ca28 and arrhythmia in cardiac muscle29. Locating the mutated residues in the full-length channel structure has been difficult in the past but is now possible with the high-resolution cryo-EM RyR structures20. Although such mutations can occur at almost any region in the protein, there are specific “hot spots”. MH occurs when RyR1 is activated by a volatile anaesthetic and leads to a rapid rise in core body temperature and organ failure unless treated30. MH mutations generally occur in central (Bsol) and N-terminal clusters in regions that would impact on pore opening and lead to uncontrollable Ca2+ release in the presence of anaesthetics20. CCD is characterised histologically by cores of inactive tissue in the centre of muscle fibres that lead to progressive muscle weakness28. RyR1 mutations causing CCD are clustered in the C-terminal Ca2+ binding site at the interface between the CTD and CSol, where they can directly affect the activation mechanism20. Mutations in the cardiac RyR2 that lead to arrhythmias are generally located in the pore, transmembrane, and central domain regions, which are directly involved in channel activation and gating20.Mutations in RyRs can lead to severe genetic conditions, including malignant hyperthermia (MH) and central core disease (CCD) in skeletal muscle2+ signalling will help in understanding the molecular process involved in regulating the ion channel gating mechanisms. In addition, the structure of potential drug binding sites opens the possibility of rational design of therapeutics to correct defective RyR channel function and restore healthy Ca2+ signalling32. As an aside, the high-resolution structural studies have provided evidence supporting early insightful hypotheses that the functional consequences of some of the disease-causing mutations are caused by “unzipping” of essential inter-domain interactions and thereby favour the open unstable conformation of the channel35.Importantly, the detailed three-dimensional (3D) locations of disease-causing mutations in RyR1 and RyR2 and determination of structural changes associated with mutations that lead to disrupted Ca33. X-ray crystal structures have been docked into the 3D envelope of RyR calculated from cryo-EM data to help interpret the high-resolution structures reported for RyR111. This is further discussed inet al.36.It is worth noting that X-ray crystallography has been used to predict the structure of individual elements within the RyR. X-ray crystal structures of isolated RyR domains at resolutions of less than 2 Å have been obtained and have defined the structures of ligand binding sites and also revealed structural changes induced by ligand binding or caused by mutations5. However, as the hydrophilic luminal environment extends into the highly charged domains of the channel pore, the actual amount of the protein exposed to the luminal solution is greater than the luminal loops that extend beyond the generalised membrane boundary. Nevertheless, it is remarkable that the luminal domain is relatively very small given the importance of luminal conditions in determining the open probability of RyR1 and RyR2 channelin vivo andin vitro. The open probability of the RyR channel is very sensitive to changes in luminal [Ca2+], and changes in the RyR’s sensitivity to luminal Ca2+ underlie the defects in Ca2+ signalling that produce skeletal and cardiac myopathies38. In addition, the luminal domains provide the RyR binding sites for the associated regulatory proteins triadin and junctin  membrane and the SR. This will reveal the RyR1 structure when it is associated with other proteins that are essential for EC coupling such as the DHPR α and β subunits, STAC3, and junctophilin. For those in the field, it is a continual frustration that we know these proteins are essential for EC coupling and we can construct models for their interactions but that we will never fully understand skeletal EC coupling until we have determined their 3D associations within the intact cell. This next step awaits the development of high-resolution tomography techniques, possibly cryo-EM tomography


In mammals, the Ca2+ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP3 generated by PLCζ induces Ca2+ release from the intracellular Ca2+ store through IP3 receptor, termed IP3-induced Ca2+ release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047–1.7 μM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca2+ and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca2+ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca2+ wave. [IP3] stayed at the elevated level with small peaks followed after Ca2+ spikes through Ca2+ oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca2+ spike, suggesting that Ca2+-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca2+ oscillations in fertilized eggs.In most species, fertilization induces Ca Unfertilized eggs, which are arrested at different stages of meiosis in different species, are “activated” and released from the arrest by fertilization2. In mammals, egg activation is triggered by a periodic series of Ca2+ transients, known as Ca2+ oscillations4. The response in mammalian eggs lasts for several hours and involves relatively low frequency, large amplitude Ca2+ increases5. The multiple increases in [Ca2+] are essential for completion of all the events of egg activation in mammals6.In most species, rises in cytosolic Ca2+ transient occurs some minutes after sperm-egg fusion7. The Ca2+ oscillations in mammalian eggs appear to be a result of Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ release channel (IP3R) located on the intracellular Ca2+ stores8. A sperm-specific phospholipase Cζ (PLCζ)9, which produces IP3 via hydrolysis of phosphatidyl 4,5-bisphosphate (PIP2), is reported as an egg-activating sperm factor10 in mammalian species. The microinjection of complementary RNA (cRNA) encoding PLCζ11 or recombinant PLCζ proteins12 into unfertilized mouse eggs triggers characteristic Ca2+ oscillations like those observed at fertilization. Sperm from transgenic mice with significantly reduced expression of PLCζ display a premature termination of Ca2+ oscillations following in vitro fertilization13. PLCζ shows extremely high Ca2+ sensitivity for its enzymatic activity compared with other PLC isoforms, with 70% maximal activity at 100 nM Ca2+ 12. Therefore, it has been considered that basal cytosolic Ca2+ in the fertilized egg can stimulate PLCζ to produce an amount of IP3 sufficient to trigger the initial release of Ca2+, which has not been confirmed experimentally, since single cell imaging using fluorescent IP3 indicators, such as green fluorescent protein (GFP)-fused to pleckstrin homology domain (GFP-PHD)14 and fretino-215, failed to clearly detected IP3 concentration ([IP3]) changes evoked in fertilized mouse eggs17.The first Ca2+ 20, there will be further increase in IP3 production when [Ca2+] start to increase. This positive feedback has been proposed to play a central role for the generation of the upstroke of Ca2+ transients22. Except for PLCζ, members of each of the PLC families are expressed in eggs, and PLCβ1 is reported to contribute generation of Ca2+ transients23 and in theory any of these could be involved in modulating Ca2+ oscillations. On the other hand, the positive feedback regulation of Ca2+ acting directly on the IP3R has been proposed to drive regenerative Ca2+ increases24. Simultaneous detection of [Ca2+] and [IP3] is necessary to figure out the contributions of Ca2+-induced IP3 production from PLCs and Ca2+ release form IP3R for the generation of fertilization-induced Ca2+ transients.Because all PLC isoforms including PLCζ are activated by Ca3 sensor proteins, designated as IRIS-2s, to visualize IP3 dynamics in fertilized mouse eggs. The novel IP3 sensors possess an improved dynamic range compared with the previous sensor, IRIS-125. A high IP3 binding affinity variant, IRIS-2.3, can be successfully used to monitor [IP3] changes naturally induced in fertilized mouse eggs. IRIS-2s contain enhanced green fluorescent protein (EGFP) and Halo-protein with tetramethylrhodamine (TMR) ligand as FRET donor and acceptor, respectively. To monitor [Ca2+] and [IP3] changes simultaneously, we also developed a new Ca2+ sensor protein, designated as DYC3.60, which has enhanced cyan fluorescent protein (ECFP) as a solo fluorophore. The pair of IRIS-2s and DYC3.60 contains a new set of fluorophores for dual-FRET imaging, and real time monitoring with IRIS-2s and DYC3.60 provide us insights into the mechanism underlying the generation of Ca2+ oscillations in mouse fertilized eggs.In the present study, we developed novel fluorescent resonant energy transfer (FRET)-based IP3 sensors composed of HaloTag protein (Promega), IP3 binding domain (IP3BD) of mouse IP3R125, and mEGFP  ligand (Promega) as a FRET acceptor for mEGFP. Amino acid residues 224–575 and 224–579 of mouse IP3R1 were used for IRIS-2 and IRIS-2.3, respectively  for IP3 binding have been replaced in IRIS-2, as a negative control25  showed greater TMR emission (565 nm) and lesser EGFP emission (510 nm)  was three times larger than that of IRIS-1   . Figure 3 dependence of the emission ratio of IRIS-2TMR and IRIS-2.3TMR. The Kd value of IRIS-2.3TMR 25.We constructed novel IPing Fig. . The rel2+ indicator with single fluorophore to avoid fluorescent overlapping with IRIS-2s. We introduced a non-fluorescent mutation (Y145W26) into a yellow fluorescent protein, cp173Venus, of YC3.6027  reduced after addition of Ca2+ in DYC3.60   Fig. . IRIS-2Tons Fig. . These c3] increase precedes [Ca2+] rise as same as our previous report with IRIS-125 ; IRIS-1-Dmut: 3.75 ± 0.5 (n = 4); p = 0.008, Student’s t-test), suggesting that IRIS-1 works as a significant IP3 buffer. We also tested IRIS-2 expressing eggs for in-vitro fertilization assay and found IRIS-2 expressing eggs had normal Ca2+ spikes after fertilization .To detect IPion Fig. . Howeverent Fig. . On the ggs Fig. . During P3] Fig. . Three i 0.5 n = ; p = 0.03] changes and [Ca2+] changes during the first Ca2+ transient evoked after fertilization. To detect the initial [Ca2+] changes experimentally, we used Fura-2, whose affinity is higher than that of Indo-5F , as a Ca2+ indicator to detect the timing of the onset of the first Ca2+ transient as precise as possible. As shown in Fig. 3] rise preceded the onset of the initial step of the first Ca2+ transient for 2.7 ± 2.4 min in 11 of 13 eggs. The initial [IP3] increase should initiate Ca2+ release from IP3R. IP3 and Ca2+ are the co-agonist of IP3R, and open probability of IP3R markedly increase with Ca2+ in the presence of IP332. Thus, Ca2+-induced Ca2+ release (CICR) from IP3R should have major role to produce initial Ca2+ spike. The first Ca2+ transient observed in IRIS-2.3-expressing eggs was composed of two steps separated by a shoulder point  is able to induce Ca2+ spike in unfertilized mouse eggs48. Our measurements showed the same results that the amount of IP3 produced in mouse eggs is small even after the fertilization because only IRIS-2.3, which shows the highest IP3 sensitivity (Kd = 47 nM) among the IP3 sensors developed, could detect [IP3] increases at the onset of the first Ca2+ transients.The new FRET sensors enabled imaging of [IP3R has a bell-shaped calcium response curve: the open probability of IP3R is activated by low [Ca2+] and inhibited by high [Ca2+]32. Based on this finding, De Young and Keizer reported a mathematical model to reproduce Ca2+ oscillations with constant [IP3]24. In this and previous reports, we showed sustained [IP3] increase during Ca2+ oscillations in HeLa cells and fertilized mouse eggs25 27 was generated using the site-directed mutagenesis. The FRET acceptor of YC3.6027 was replaced with cp173Venus-Y145W to produce DYC3.60. IRIS-2, IRIS-2.3 and DYC3.60 cDNAs were cloned into the NheI and XbaI sites of pcDNA3.1 zeo(+) (Invitrogen) for the expression in HeLa cells. The cDNAs were cloned into the XbaI site of pTNTTM (Promega) with extended poly(A) tail (57 residues) and synthesized cRNAs were injected into mouse oocytes.The FRET donor and acceptor of IRIS-1 were replaced with mEGFP and Halo-protein (Promega), respectively, to produce IRIS-2. Amino acid residues 224–575 of mouse IP3R1 in IRIS-2 were replaced with amino acid residues 224–579 of mouse IP3R1 to produce IRIS-2.3. The Y145W mutant52. The expressed proteins were purified on a HiTrap heparin HP column  as described previously53.The full-length cDNA of IRIS-2 was isolated from pcDNA3.1 zeo(+)-IRIS-2 by using NheI and XbaI sites and was cloned into the XbaI site of baculovirus transfer vector pFastBac1 (Invitrogen). The recombinant baculovirus was used for the large-scale expression of IRIS-2 in Sf9 cells as described previouslyHeLa cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum. HeLa cells were transfected with expression vectors by transfection reagent (Mirus TransIT). One day after the transfection, cells were used for imaging experiments.Plasmids carrying IRISs or DYC3.60 were digested by NdeI, and linearized DNA fragments were purified with Wizard SV Gel and PCR clean-up Kit (Promega). They were used as the templates for RNA transcription by T7 polymerase using T7 mMESSAGEmMACHINE Kit (Ambion). RNA was purified using RNeasy MinElute Cleanup Kit (Qiagen) and stored at −80 °C until use.54 supplemented with 4 mg/ml BSA (Sigma) at 37 °C (5% CO2) for >5 h for capacitation and acrosome reaction55.Full grown immature oocytes were collected from the follicles in the ovaries of female mice 47–49 h after the injection of pregnant mare serum gonadotropin. Isolated oocytes were freed from cumulus cells mechanically by pipetting in M2 medium, and then cRNAs of IRIS-1, IRIS-2, IRIS-2.3, DYC3.60 or dKeima570 were injected as described below. Sperm was collected from the caudal epididymides and were incubated in M16 medium2. Only eggs maturated normally to metaphase II with the first polar body were used in the following experiments. After loaded with 2 μM of Indo-5F or Fura-2 for 30 min in the M2 medium, eggs were freed from the zona pellucida by brief treatment with acidic Tyrode’s solution (pH 2.5)56 for insemination. Sperm was added during imaging experiments.RNA solutions were diluted to 130 ng/μl with the intracellular medium . Immature oocytes were injected with 20 pl of RNA solutions and incubated in the M16 medium for 16 h at 37 °C with 5% COAfter loading HeLa cells with 10 μM Indo-5F-AM (AnaSpec), imaging was performed under the constant flow (2 ml/min) of the balanced salt solution containing 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.3 mM CaCl2, and 10 mM glucose as an imaging media at 37 °C through an inverted microscope  with a cooled charge-coupled device (CCD) camera  and a 40x, 1.35 NA, oil-immersion objective (Olympus). For the fluorescent images of IRIS-1 and Indo-5F, an emission splitter  was used with a light source exchanger  on the IX71 inverted microscope. Sequential excitation of IRIS-1 and Indo-5F was performed by using a 450-nm dichroic mirror and two excitation filters (a 425–445 nm filter for IRIS-1 and a 360-nm filter for Indo-5F). Emissions from IRIS-1 and Indo-5F were split with a 460–510-nm filter (for IRIS-1 and Indo-5F), a long-path 520-nm (for IRIS-1) barrier filter, and two 505-nm dichroic mirrors equipped in W-view.2+ and IP3 were visualized with sets of Indo-5F and IRIS-1, Indo-5F and IRIS-2, Indo-5F and IRIS-2.3, Fura-2 and IRIS-2.3, or DYC3.60 and IRIS-2.3, respectively. Sequential excitation of Ca2+ and IP3 indicators was performed by using dichroic mirrors (a 400-nm mirror for Indo-5F and a 450-nm mirror for IRIS-1 and DYC3.60 and a 505-nm mirror for IRIS-2 and IRIS-2.3) and excitation filters (a set of 340 and 380-nm filters for Fura-2 and a 380-nm filter for Indo-5F and a 425–445 nm filter for IRIS-1 and DYC3.60 and a 460–490-nm filter for IRIS-2 and IRIS-2.3). Emissions from Ca2+ and IP3 indicators were split with emission filters (a set of 400–420 and 460–510-nm filters for Indo-5F and a set of 460–510 and 525–565 filters for IRIS-1 and DYC3.60 and a 510–550 filter for Fura-2 and a set of 510–550 and 573–613-nm filters for IRIS-2 and IRIS-2.3TMR), and three filter exchangers .Eggs were incubated with M2 buffer at 37 °C on IX81 inverted microscope. CaImage acquisition was performed with MetaFluor (Molecular Devices). Data analysis was performed with MetaFluor and Igor Pro (WaveMetrics) softwares. The EGFP/TMR emission ratio (IRIS-2s), the ECFP/Venus emission ratio (IRIS-1s), the dKeima570/ECFP emission ratio (DYC3.60), the 420–440 nm/460–510 nm emission ratio (Indo-1) and the ratio of 510–550 nm emission excited at 340 nm and 510–550 nm emission excited at 380 nm (Fura-2) were defined as R. ∆R was defined as R - Rbase, where Rbase is the basal level of R. Baseline drift in each experiment was corrected with subtracting the trend line which is calculated with the line around the beginning of each experiment.3 P3/PM (caged), Enzo Life Science). The uncaging stimulation was done with extra light source (mercury lamp) equipped in IX81, filtered by a 333–348-nm filter and a 400-nm dichroic mirror, illuminated the cells through 20x, 0.50 NA, water-immersion objective (Olympus).HeLa cells transfected with IRIS-2 and DYC3.60 were loaded with 10 μM membrane permeable caged-IPSupplementary FiguresSupplementary Video 1


We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.Ca Although calcium signals are known to be critical for many cellular processes, how signaling elicits specific functions remains unclear. In visually striking work, Duan et al. reveal that networks of cytoplasmic nanocourses orchestrate cell activity by directing site-specific calcium signals. Therefore, stimuli must induce different Ca2+ signals to engage specific cellular responses, such as, for example, contraction or relaxation of smooth muscles as well as their switch from contractile to migratory-proliferative phenotypes, which additionally requires changes in gene expression2. However, despite the extraordinarily detailed mapping of the temporal characteristics of both unitary and macroscopic Ca2+ signals across a variety of cell types8, how cells deliver the diverse range of site- and function-specific Ca2+ signals necessary to coordinate the full panoply of cellular processes remains enigmatic2.Cells select for one or a combination of distinct functions through Ca2+ store is the sarco/endoplasmic reticulum (S/ER)2, which is known to be a contiguous organelle, from its origin at the outer nuclear membrane (ONM) to the periphery of the cell. Yet the S/ER delivers Ca2+ signals with clear diversities of form and function5. In arterial smooth muscles, for example, the current consensus is that relaxation is mediated by highly localised Ca2+ sparks that recruit Ca2+-activated potassium channels to promote plasma membrane hyperpolarization, while contraction is triggered by propagating global Ca2+ waves9, with adjustments to gene expression presumed to be governed by the spatiotemporal patterns of global Ca2+ transients that gain unrestricted entry to the nucleoplasm across the nuclear envelope (NE) and its invaginations14.The primary intracellular Ca2+ may exist within the SR, filled by spatially segregated subtypes of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps and mobilised through similarly segregated subtypes of Ca2+ release channel17, including ryanodine receptors (RyRs) 1, 2 and 322. This led to an alternative proposal, that different Ca2+ signals may arise in distinct cytoplasmic spaces demarcated by junctions between the SR and its target organelles24. Hitherto, direct visualisation of Ca2+ signalling within junctional complexes of the SR has not been achieved, so little more than speculation has guided such considerations on functional signal segregation within cells23.However, in smooth muscles it has long been suggested that multiple, spatially segregated and independently releasable subcompartments of Ca2+ transporters and release channels. This extends from the plasma membrane to NE invaginations, delivering highly localised Ca2+ flux, with path lengths on the nanoscale at all points. Cells may thus support unforeseen levels of network activity.Here we identify a cell-wide network of distinct cytoplasmic nanocourses, demarcated both by the SR and by different types of SR resident Ca2+ ATPase subtype 2b (SERCA2b), while RyR2s are incorporated in the extraperinuclear SR, where SERCA are few, with SERCA2a and RyR3 clusters heavily restricted to the deepest perinuclear SR19. Adding to this, we now identify within the same image series a distinct subtype of SERCA pump, SERCA1  and of S/ER CaCA1 Fig. , which tCA1 Fig.  appear t2+ signals and a membrane permeant DNA marker (Draq5) to identify the boundary of the nucleus. By adjusting the threshold for fluorescence detection, it was evident that a network of narrow tributaries of cytoplasm ≤500 nm wide penetrated the nucleus, perhaps reflecting the path of nuclear invaginations. The cytoplasmic nanocourses within the boundary of the Draq5 labelled nucleus exhibited markedly higher levels of Fluo-4 fluorescence than the surrounding nucleoplasm, and could be equally well distinguished from any aspect of the wider cytoplasm, which in turn and invariably exhibited higher basal fluorescence than the nucleoplasm  set to detect these cytoplasmic nanocourses within the boundary of the nucleus, we frequently observed variegated, region-specific differences in Fluo-4 fluorescence intensity in the bulk cytoplasm (beyond the boundary of the nucleus), i.e. highly localised, time-dependent and asynchronous fluctuations in Fluo-4 fluorescence intensity were evident across the wider cell. Therefore, we carried out deconvolution of all Fluo-4 images within each time series acquired. This revealed a cell-wide network of well-defined cytoplasmic nanocourses  and perinuclear (452 ± 32 nm) nanocourses  were evident despite the limited temporal resolution at the optimal sampling frequencies used here . Transitions from basal to the highest level of fluorescence intensity  varied in duration from ~2 s to ≥10 s, with even longer dwell times evident for lower frequency, low intensity sub-states. The asynchronous activity, spatial characteristics and pharmacology of hotspots suggests that these events most likely reflect low level, basal Ca2+ flux (leak) from the SR via RyRs. However, while RyRs can remain open for many seconds, the fastest gating events are on the millisecond time scale29. Therefore, the development of confocal systems with higher temporal and spatial resolution is required before we can measure the kinetics of hotspots of Ca2+ flux characterised here with precision and thus confirm whether they truly represent unitary Ca2+ release through RyRs. Nevertheless, it is clear that the Ca2+ signalling machinery of subplasmalemmal, extraperinuclear, perinuclear and nuclear nanocourses incorporates unique receptor components, conferring different nanocourses with the capacity to deliver discrete spatially- and functionally-segregated signals. Supporting the view that the wider network of cytoplasmic nanocourses may represent a circuit for cell-wide communication, LysoTracker Red labelled endolysosomes migrated through this network of cytoplasmic nanocourses  Maurocalcine-induced myocyte relaxation was not accompanied by concomitant falls in Ca2+ flux into extra/perinuclear nanocourses. If anything, asynchronous Ca2+ flux continued within these nanocourses, with perhaps slight increases in activity but no evidence of cell-wide signal propagation . Consistent with the spatial separation of RyR1s  in the absence  associating with NE proteins such as emerin and making the nuclear periphery generally silencing39. However, interestingly, these marks segregate in differentiated arterial myocytes with the H3K9me2/3 both still at the outer limits of the nucleus but depleted with respect to BAF, and the nuclear invaginations rich with H3K9me2  reduced the expression of two genes of interest (identified by RNAseq), one encoding the DNA mismatch repair protein MutL homolog 1 (Mlh1), which can be repressed through interaction with H3K9me242, and another encoding the S100 calcium binding protein A9 (S100a9), which can be repressed by BAF43. That said, further investigation into the role of nuclear invaginations in regulating gene expression will undoubtedly reveal greater complexities of gene regulation, given that individual, acutely isolated smooth muscles possess different types 45. Irrespective of cellular region, increases in Ca2+ flux induced by Angiotensin II were abolished by prior depletion of SR stores with thapsigargin, block of RyRs with tetracaine and by pre-incubation with the cyclic ADP-ribose antagonist 8-bromo-cADPR  evokes a global Ca2+ wave and contraction of acutely isolated pulmonary arterial myocytes48. Given that cADPR preferentially activates RyR1s and RyR3s49 but can only sensitise RyR2s to CICR44, it therefore seems likely that cADPR accumulation within or proximal to extraperinuclear nanocourses in response to Angiotensin II, serves to activate local subpopulations of RyR1s and/or RyR3s49 while delivering concomitant sensitisation of RyR2s to`CICR44, that permits subsequent initiation of a propagating Ca2+ signal and thus myocyte contraction. This is in stark contrast to the effect of low concentrations of cADPR (10 μM), the intracellular dialysis of which preferentially releases Ca2+ from RyR1s on the peripheral SR proximal to the plasma membrane, to thus evoke membrane hyperpolarization and vasodilation17. Furthermore, the response to Angiotensin II remained unaffected in the presence of the inositol  trisphosphate receptor (IP3R) antagonist 2APB (not shown). This is in accordance with the fact that IP3Rs do not couple by CICR to RyRs in pulmonary arterial myocytes51, and suggests that this segregation of RyRs from IP3Rs might be conferred by the targeting of RyR2/RyR3s to the SR that demarcates cytoplasmic nanocourses.As one might expect, increases in Caons Fig. , there wons Fig. . This isDPR Fig. , which i2+-binding protein calmodulin is tethered proximal to the SR membranes that line myofilament arrays52, rather than being freely diffusible in the cytoplasm. All relevant path lengths from the SR to myofilaments must therefore be on the nanoscale too. Multiple coordinated actions may thus be delivered by signal segregation between distinct nanocourse networks, enabling nanocourse-specific delivery of Ca2+ signals with distinct temporal characteristics  is consistent with the finding that the functional Cacs Figs. .2+ signalling  A switch in dependency of Angiotensin II-induced Ca2+ transients from RyRs to IP3Rs  Loss of the ‘nuclear buffer barrier’53 that opposed direct Ca2+ flux into the nucleoplasm in acutely isolated cells  of lamin/emerin positive nuclear invaginations Fig. ; althoug3Rs Fig. ; (ii) Unlls Fig. . Accordions Fig. , consistyRs Fig.  and prev60, by the constraints on Ca2+ diffusion imposed by SR junctional membranes and by the strategic positioning of different types of Ca2+ transporters and release channels targeted to them, through unique kinetics, affinities for Ca2+ and mechanisms of regulation23. Ca2+ flux through RyR1s located within PM-SR nanocourses evoked relaxation with no evidence of cell-wide signal propagation, confirming the existence of a functional ‘superficial buffer barrier’32. Distinct nanocourses rich in RyR2s and RyR3s carried rapid, propagating Ca2+ signals that crossed the entire cell from pole to pole triggering myocyte contraction, yet these signals did not enter those nanocourses demarcated by PM-SR junctions, which constitute the superficial buffer barrier. Therefore, RyR2 subtype designation determines the capacity for rapid signal propagation across specific nanocourses, allowing on the one hand cell-wide synchronous actions as required, but perhaps also conferring the capacity for the onward transfer of a fraction of released Ca2+ through SERCA and RyRs that feed functionally distinct subsets of nanocourses, such as nuclear invaginations. Invaginations of the NE confer further segregated and diverse networks of cytoplasmic nanocourses that project deep into the nucleoplasm, which break down themselves into multiple subtypes based on emerin-BAF and lamin A-H3K9me2 clusters, are served by a unique pairing of ONM resident Ca2+ pumps (SERCA1) and release channels (RyR1) and carry Ca2+ signals with different spatiotemporal characteristics when activated. That S100A9 expression was attenuated by blocking RyRs and upon loss of nuclear invaginations in culture, strongly supports the view that regulated Ca2+ flux across the ONM into nuclear nanocourses might contribute additional levels of genome regulation by, for example, segregating specific chromatin types for cycles of reactivation in differentiated cells and providing a path for related gene repression during phenotypic modulation56.We have identified a cell-wide network of cytoplasmic nanocourses delineated by membrane-membrane nanojunctions of the SR, which provide discrete lines of communication that span the entire cell. Functional specification is therefore determined, as predicted by computer models2+ release site to targeted signalling complexes must be on the nanoscale, with picolitre volumes of cytoplasm lying within the boundaries of each nanocourse60. Relatively small net increases in local Ca2+ flux (1–2 ions per picolitre) will therefore be sufficient to raise the local concentration into the affinity ranges of most cytoplasmic Ca2+ binding proteins60. Ca2+ binding proteins may thus operate as local ‘switches’ that coordinate nanocourse-specific functions, the probability of moving from OFF to ON determined by changes in unitary rather than macroscopic Ca2+ flux. Significantly, coincident Ca2+ flux can thus be triggered in two distant parts of the cell at the same time, to coordinate, for example, myocyte relaxation and associated gene expression regulation. This draws obvious parallels  Act 1986. All experiments have complied with all relevant ethical regulations for animal testing and research. Adult male Sprague Dawley rats (~300 g) were sacrificed by cervical dislocation. Skeletal muscle, brain, heart and lungs were removed and placed on ice in physiological salt solution (PSS) of the following composition (mmol/L): 130 NaCl, 5.2 KCl, 1 MgCl® reagent according to the manufacturer’s instructions . Reverse transcription was carried out using 6 μg RNA and 200 U Moloney murine leukaemia virus  and PCR was performed on 1 μl cDNA with 1 U/μl TAQ DNA polymerase . All primer sequences were checked against the GenBank and no cross-reactivity was found. The RT-PCR products over 40 cycles of amplification were resolved by electrophoresis in 1% agarose gels and visualised under UV illumination using an image capture system .For end point PCR, total RNA was extracted from second and third order branches of the pulmonary arterial tree, heart, brain and skeletal muscle using TRIzolg) and 25 µl added to a MicroAmpTM Fast Optical 96-Well Reaction Plate (Greiner bio-one), the reaction plate sealed with an optical adhesive cover (Applied Biosystems) and the plate centrifuged . The reaction was then run on a sequence detection system (Applied Biosystems) using AmpliTaq Fast DNA Polymerase, with a 2 min initial step at 50 °C, followed by a 10 min step at 95 °C, then a 15 s step at 95 °C which was repeated 40 times. Then a dissociation stage with a 15 s step at 95 °C followed by 20 s at 60 °C and a 15 s step at 95 °C. Data were normalised to a housekeeping gene . Negative controls included control cell aspirants for which no reverse transcriptase was added, and aspiration of extracellular medium and PCR controls. None of the controls produced any detectable amplicon, ruling out genomic or other contamination.For qPCR RNA from pulmonary arterial smooth muscle was extracted using the High Pure RNA Tissue Kit (Roche) following the manufacturer’s guidelines and the concentration determined using the Nanodrop 1000 spectrophotometer (ThermoScientific). cDNA synthesis was carried out using the Transcriptor High Fidelity cDNA synthesis Kit (Roche) following the manufacturers’ instructions. For qPCR analysis, 2.5 µl of cDNA in RNase free water was made up to 25 µl with FastStart Universal SYBR Green Master , Ultra Pure Water  and fwd and rev primers (Origene) for the genes encoding MutL homolog 1 (Mlh1) and S100 calcium binding protein A9 (S100a9). Samples were then centrifuged , predicted product size 340 bp, Antisense GTC AGC TAG TTG CCT TGT CCC TG (nucleotide position: 3423–3401); SERCA2a Sense CCT GTC CAG TTA CTC TGG GTC (nucleotide position: 2569–2589), predicted product size 694 bp, Antisense GCT AAC AAC GCA CAT GCA CGC (nucleotide position: 3262–3242); SERCA2b Sense GCC AAC ATT GCC TAT TCA GTG GCA C (nucleotide position: 4382–4406) predicted product size 501 bp, Antisense GGA TGT TGG AAG GCA TTG GAG G (nucleotide position: 4882–4861); SERCA3 Sense CTC WGA GAA CCA GTC ACT GCT GCa (nucleotide position: 2879–2901) predicted product size 264 bp, Antisense GCT CYC AGG ATT TAC TTC AGG TCCb (nucleotide position: 3142–3119). Primer sequences were checked against the GenBankTM, and no cross-reactivity was found.Commercial primers used were as follows:BioRad; catalogue number: 10025636). Mlh1 primer, Rat, Unique Assay ID: qRnoCID0007985 ; Rat lamin A primer Rn_Lmna_1_SG QuantiTect Primer Assay ; PBGD primer PCR SYBR Green Assay . Ipo8 Quantitect Primer Assay .Rat s100a9 primer PrimePCR™ SYBR® Green Assay . The supernatants were removed and their protein concentrations determined.Tissues samples were rapidly frozen in liquid nitrogen. Small segments of tissue were ground to a powder under liquid nitrogen and homogenised in an appropriate volume of ice-cold buffer  Triton, pH 7.4) using a motor-driven pestle. The homogenate was kept on ice for 30 min and then centrifuged  from second order pulmonary artery smooth muscle and mircrosomal fractions of lysates from second order pulmonary arterial smooth muscle, brain and skeletal muscle homogenates were separated on 10% bis-acrylamide gels  and blotted onto nitrocellulose membranes. Blots were probed with sequence-specific antibodies for SERCA1 . Every blot was also probed with a polyclonal antibody for actin to ensure equal protein loading  alongside the SERCA antibodies. Detection was performed with horseradish peroxide-conjugated secondary antibodies using the ECL system .4 + 3% C6FeK4N6·3H2O  in 0.1 M sodium cacodylate buffer for 1 h at 18–24 °C followed by three 10 min washes with distilled water. The samples were then dehydrated in increasing concentrations of acetone . In the final process of dehydration, the samples underwent 3 washes in 100% acetone. The artery rings were then resin-infiltrated in increasing concentrations  of TAAB 812 resin mix  and left in pure resin overnight in obscurity. The blocks were finally resin-embedded in moulds and polymerised in an oven at 60 °C for 8 h. For standard (2D) electron microscopy imaging, 80-nm sections were cut from the embedded sample blocks on a Reichert Ultracut-E microtome  using a diamond knife  and were collected on uncoated 100- and 200-mesh copper grids . The sections were post-stained with 1% uranyl acetate  and Reynolds lead citrate  for 3 and 4 min, respectively. Electron micrographs at various magnifications were obtained with a Jeol JEM-1010 high resolution transmission electronic microscope .Secondary and tertiary branches of the pulmonary artery were dissected and immediately immersed in fixative solution, 4 °C. The primary fixative solution contained 2.5% glutaraldehyde + 4% paraformaldehyde  in 0.1 M sodium cacodylate buffer . The artery rings were then washed five times (30 min per wash) in 0.1 M sodium cacodylate and left overnight at, 4 °C. In the process of secondary fixation, the tissue rings were fixed with 1% OsO2+ solution of the following composition (mM): 110 NaCl, 5 KCl, 2 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO, 15 NaHCO3, 0.16 CaCl2, 0.5 EDTA, 10 glucose, 10 taurine and 10 Hepes, pH 7.4. After 10 min the arteries were placed in the same solution containing 0.5 mg/ml papain and 1 mg/ml bovine serum albumin and kept at 4 °C overnight. The following day 0.2 mmol/L 1,4-dithio-DL-threitol was added to the solution, to activate the protease, and the preparation was incubated for 1 h at room temperature (22 °C). The tissue was then washed 3× in fresh low Ca2+ solution without enzymes, and single smooth muscle cells were isolated by gentle trituration with a fire-polished Pasteur pipette. Cells were stored in suspension at 4 °C until required.Single arterial smooth muscle cells were isolated from secondary and tertiary order branches of the pulmonary arterial tree. Briefly, arteries were dissected out and placed in low Ca2+ imaging by confocal microscopy, freshly isolated pulmonary arterial smooth muscle cells were suspended in nominally Ca2+ free solution with a cell density of 1000,000–2000,000 cells per ml. 25 μl of cell suspension was plated on Fluorodishes  and cultured for 7 days. For immunocytochemistry, the same amount of cell suspension was added to a 13 mm circular coverslip sat in a 12-well plate. Cells were allowed to adhere for 45 min at room temperature in a cell culture hood. 2 ml of culture medium of following composition , 1% Penicillin-Streptomycin) was then added to each Fluorodish or each well of the 12-well plate and the cells were incubated at 37 °C with air + 5% CO2. FBS was added to enhance cell growth and proliferation61, while antibiotics were added to reduce the chance of infection. The culture medium was replaced every 3 days for the best outcomes. Proliferating cells were identified by shape, morphology and reductions in myosin heavy chain labelling62.For CaoC, permeabilized by three 5 min washes with 0.6% Triton X-100 in phosphate-buffered saline (pH 7.4) followed by 20 min wash with blocking solution . The cells were incubated overnight at 4 °C with the sequence-specific antibodies for SERCA1 (1:500), SERCA2a (1:500) and SERCA2b (1:500). In addition, paired samples of cells were incubated overnight at 4 °C with affinity-purified rabbit anti-RyR1 , anti-RyR2  and anti-RyR3 . To visualise trans-nuclear membrane proteins, cells were incubated overnight at 4 °C with the sequence-specific antibodies for A-type lamin (1:200), emerin (1:200), H3K9me2 (1:200), BAF (1:50). Coverslips were washed four times with blocking solution and incubated with goat anti-mouse Alexa448-conjugated secondary antibodies , donkey anti-mouse IgG (H + L) highly cross-adsorbed , Alexa Fluor® 546 goat anti-mouse IgG (H + L), highly cross-adsorbed, goat anti-rabbit Texas Red-conjugated secondary antibody , donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody , or donkey anti-goat IgG (H + L) cross-adsorbed secondary antibody . To visualise nuclei the coverslips were incubated with 4-,6-diamidino-2-phenylindole  for 15 min at room temperature. Then, the coverslips were washed 3 times with phosphate-buffered saline and 2 times with phosphate-buffered saline containing 0.1% Tween-20. After 5 min of air drying the coverslips were attached to slides by anti-fading mountant  octane). For controls, the primary antibody was omitted.Cells were placed onto poly-D-lysine-coated coverslips, fixed using ice-cold methanol for 15 min at −20 Images were acquired and processed by either:(1) A Deltavision imaging system  consisting of an Olympus IX70 inverted microscope with an Olympus PlanApo 60×, 1.40 n.a. oil immersion objective and a Photometric CH300 charge-coupled device camera. Z section (0.2 µm) stacks were taken through cells. Images were deconvolved using Softworx acquisition and analysis software .x,y and 150 nm in z. Estimated resolution with these settings is x,y = 170 nm, z = 540 nm. Image processing and 3D rendering was carried out using Imaris . Images were deconvolved using Huygens Essential .(2) A Nikon A1R + confocal system via a Nikon Eclipse Ti inverted microscope with a Nikon Apo 63× λS DIC N2, 1.25 n.a. oil immersion objective . Images were deconvolved, with pinhole at 0.7 Airy units, and pixel settings of 60 nm in Primary antibodies used were as follows:17, a kind gift from F. Wuytack, University of Leuven, Belgium. SERCA2b, rabbit polyclonal, raised against residues 1032–1043 of pig SERCA2b17, a kind gift from F. Wuytack, University of Leuven, Belgium. SERCA3, rabbit polyclonal, raised against residues 29–39 of mouse SERCA3 (Abcam).SERCA antibodies—SERCA1, mouse monoclonal, raised against residues 199–505 of rabbit SERCA1 (Abcam). SERCA2a, rabbit polyclonal, raised against residues 989–997 of pig SERCA2a63. RyR2, anti-RyR2 raised against the peptide residues 1344–136564. RyR3, anti-RyR3 raised against the peptide residues 4236–433665. All were a kind gift from S. Fleicher, Vanderbilt University, TN, USA.RyR antibodies—RyR1, affinity-purified rabbit anti-RyR1 raised against the peptide residues 4476–4486http://www.glennmorris.org.uk/mabs/WCIND.htm.NE proteins—Emerin polyclonal antibody were a kind gift from Glenn Morris and outcomes confirmed by use of commercial emerin polyclonal antibodies . Thoroughly characterised, see 66.Lamin A polyclonal antibody , specificity previously confirmedHistone H3 di methyl K9 . Di-Methyl-Histone H3 Lys9 polyclonal antibody . Epigenetic mark antibodies were selected by loss of labelling in yeast with enzymes that confer the marks deleted.Secondary antibodies used were as follows:Texas Red-conjugated goat anti-rabbit secondary .Alexa Fluor® 488-AffiniPure goat anti-rabbit IgG (H + L) .Alexa Fluor® 546 goat anti-mouse IgG (H + L), highly cross-adsorbed .Alexa Fluor® 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed .Alexa Fluor 568 donkey anti-rabbit IgG (H + L) highly cross-adsorbed, .18. Thus, data are presented as the mean ± SEM of the volume of labelling per μm3 of a given region (μm3 per μm3).3D rendered images of pulmonary arterial smooth muscle cells were obtained using Volocity software  and subdivided into three defined volumes that excluded the DAPI labelled nucleus, namely the perinuclear (the area of the cell located within 1.5 µm of the DAPI labelled nucleus), the subplasmalemmal (the area of the cell located within 1.5 µm of the plasma membrane) and the extra-perinuclear regions (the remaining volume of the cell). The volumes occupied by these three defined regions were measured and then the density of fluorescent labelling in that region determined by dividing the volume of protein labelling (e.g. for SERCA2a) within a given region by the volume of that region as described in detail previously2+-loaded sarcoplasmic reticulum of acutely isolated cells was identified by incubation (30 min) with 5 μM Calcium Orange  and/or 1μM ER-tracker  at 37 °C, washed 5 times with indicator-free PSS and allowed to equilibrate for at least 45 min at 18–22 °C prior to experimentation.The Ca2+ concentration was reported by Fluo4 fluorescence.For cytoplasmic calcium, cells were incubated for 30 min with 5 μM Fluo-4-AM  in PSS at 18–22 °C, and then washed 5 times with Fluo4-AM free PSS and allowed to equilibrate for at least 45 min prior to experimentation. Intracellular Ca−1). Note: greater spatial resolution is afforded by the Nikon A1R + due, in part, to the fact that the integration time is equal to the pixel time . Deconvolution was completed with pinhole at 0.7 Airy units, and pixel settings of 60 nm in x,y and 150 nm in z. After deconvolution under these conditions,  the estimated resolution is XY = 230 nm and Z = 582 nm. We excluded from analysis any cell that exhibited contraction or other movement artefact sufficient to alter the focal plane and/or compromise our ability to take reliable measurements of regions of interest. Images were deconvolved using Huygens Essential .In each case the Fluo-4 fluorescence ratio was recorded at 22 °C with a sampling frequency of 0.5 Hz, using a Nikon A1R + confocal system via a Nikon Eclipse Ti inverted microscope with a Nikon Apo 40 × λS DIC N2, 1.25 n.a. water immersion objective . Draq 5  was used to identify the nucleus. Experiments were processed with ImageJ software . Note, each image file was assessed manually to insure accuracy and consistency of measurement within the regions of interest specified. Images were acquired using the Galvano scanner (400 Hz). Laser power was < 3% (offset = 0), Hv ≈120 V, image size 1024 × 1024 (16 bit), frame time = 2 s, sampling frequency 0.47 Hz (1.1 frames s67 was completed according to manufacturer’s (Advanced cell diagnostics) instructions, described in the RNAscope® Fluorescent Multiplex Kit Manual. All probes were supplied by Advanced Cell Diagnostics Srl. Via Calabria, 15 20090 Segrate (Milan), Italy: RNAscope probe for Rat Mlh1 ID; 81685 ; RNAscope probe for Rat S100a9 ID: 94195 .RNAscope®Data are presented as the mean ± SEM. For data shown in Fig. For data shown in Figs. Fluo-4, AM, cell permeant (Molecular Probes®) 10 × 50 µg . Calcium Orange . DRAQ5™ Fluorescent Probe Solution . LysoTracker™ Red DND-99—Special Packaging . ER-Tracker™ Blue-White DPX, for live-cell imaging . MitoTracker™ Green FM . Thapsigargin ; Maurocalcine . Angiotensin II ; Tetracaine .Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10