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Ischemic stroke affects ∼795,000 people each year in the U.S., which results in an estimated annual cost of $73.7 billion. Calcium is pivotal in a variety of neuronal signaling cascades, however, during ischemia, excess calcium influx can trigger excitotoxic cell death. Calcium binding proteins help neurons regulate/buffer intracellular calcium levels during ischemia. Aequorin is a calcium binding protein isolated from the jellyfish Aequorea victoria, and has been used for years as a calcium indicator, but little is known about its neuroprotective properties. The present study used an in vitro rat brain slice preparation to test the hypothesis that an intra-hippocampal infusion of apoaequorin (the calcium binding component of aequorin) protects neurons from ischemic cell death. Bilaterally cannulated rats received an apoaequorin infusion in one hemisphere and vehicle control in the other. Hippocampal slices were then prepared and subjected to 5 minutes of oxygen-glucose deprivation (OGD), and cell death was assayed by trypan blue exclusion. Apoaequorin dose-dependently protected neurons from OGD – doses of 1% and 4% (but not 0.4%) significantly decreased the number of trypan blue-labeled neurons. This effect was also time dependent, lasting up to 48 hours. This time dependent effect was paralleled by changes in cytokine and chemokine expression, indicating that apoaequorin may protect neurons via a neuroimmunomodulatory mechanism. These data support the hypothesis that pretreatment with apoaequorin protects neurons against ischemic cell death, and may be an effective neurotherapeutic. [ABSTRACT FROM AUTHOR]

Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca2+) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca2+, we characterized Ca2+ homeostasis in P. aeruginosa PAO1 cells. By using Ca2+-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca2+ ([Ca2+]in) is 0.14±0.05μM. In response to external Ca2+, the [Ca2+]in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca2+ modulated this response. Treatment with inhibitors known to affect Ca2+ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca2+]in response, suggesting the importance of the corresponding mechanisms in Ca2+ homeostasis. To identify Ca2+ transporters maintaining this homeostasis, bioinformatic and LC–MS/MS-based membrane proteomic analyses were used. [Ca2+]in homeostasis was monitored for seven Ca2+-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca2+ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca2+-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca2+. [Copyright &y& Elsevier]

Highlights: [•] The effect of amino acids residues and explicit water molecules on the spectral properties has been studied. [•] The motion of Trp86 could change the hydrogen bond network. [•] The Natural Bond Orbital method (NBO) has been employed to analyze internal charge transfer of CLM−. [•] The greater electron density is located at O atom of 6-p-hydroxyphenyl group. It is helpful to formation hydrogen bond. [Copyright &y& Elsevier]

Highlights: [•] Coelenterazine binding and kinetics of active obelin formation are studied. [•] Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation. [•] The proper hydrogen bond network over coelenterazine is required for its activation. [•] His175 might serve as a proton shuttle during 2-hydroperoxycoelenterazine formation. [Copyright &y& Elsevier]

Background and Purpose: Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice.Experimental Approach: Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function.Key Results: Dox at 1-30 μM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions.Conclusions and Implications: Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment. [ABSTRACT FROM AUTHOR]

Background and Purpose Chronic elevation in intracellular Ca2+ concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy ( DMD). Candidate pathways mediating this Ca2+ overload involve store-operated channels ( SOCs) and stretch-activated channels ( SACs), which are modulated by the Ca2+-independent form of PL A2 ( iPLA2). We investigated the effect of doxorubicin ( Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca2+ handling and muscle function in mdx mice. Experimental Approach Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca2+ influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca2+ concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. Key Results Dox at 1-30 μM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca2+ influx in myotubes. Stimulated elevations of Ca2+ concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. Conclusions and Implications Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment. [ABSTRACT FROM AUTHOR]

Abstract: Different optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP–aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca2+-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca2+-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca2+-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca2+-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium. [Copyright &y& Elsevier]

Abstract: Apoaequorin, a calcium-binding protein originally isolated from jellyfish is available commercially as a dietary supplement. The objective of the present study was to investigate potential adverse effects, if any, of Apoaequorin, a recombinant protein preparation, in rats following subchronic administration. For this study, Sprague–Dawley (Hsd:SD®) rats (10/sex/group) were administered via oral gavage 0 (control), 92.6, 462.9, and 926.0mg/kg body weight (bw)/day of Apoaequorin preparation, for 90days. The corresponding amount of Apoaequorin protein was 0, 66.7, 333.3 and 666.7mg/kg bw/day, respectively. Administration of the Apoaequorin preparation did not result in any mortality. There were no clinical or ophthalmological signs, body weight, body weight gain, food consumption, food efficiency, clinical pathology or histopathological changes attributable to administration of Apoaequorin. Any changes noted were incidental and in agreement with those historically observed in the age and strain of rats used in this study. Based on the results of this study, the No Observed-Adverse-Effect Level (NOAEL) for Apoaequorin was determined as 666.7mg/kg bw/day, the highest dose tested. [Copyright &y& Elsevier]

Abstract: We use here a new very low-Ca2+-affinity targeted aequorin to measure the [Ca2+] in the endoplasmic reticulum ([Ca2+]ER). The new aequorin chimera has the right Ca2+-affinity to make long-lasting measurements of [Ca2+]ER in the millimolar range. Moreover, previous Ca2+-depletion of the ER is no longer required. The steady-state [Ca2+]ER obtained is 1–2mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca2+]ER among different regions of the ER. About half of the ER had a [Ca2+]ER of 1mM or below, and the rest had [Ca2+]ER values above 1mM and in some parts even above 2mM. About 5% of the ER was also found to have high [Ca2+]ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca2+ of the ER was almost linearly dependent on the extracellular [Ca2+] between 0.1 and 3mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca2+] microdomains connecting the store operated Ca2+ channels with the ER Ca2+ pumps may be important during refilling. [Copyright &y& Elsevier]

Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF-hand calcium-binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H-bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine-type luciferases). [ABSTRACT FROM AUTHOR]

Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor–acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His–Trp–Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His–Trp–Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different. [ABSTRACT FROM AUTHOR]

Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging ( BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright. [ABSTRACT FROM AUTHOR]

The photoprotein aequorin is a calcium-dependent bioluminescent enzyme which is most widely used in biotechnology processes, but this protein is susceptible to aggregation and proteolysis degradation. Various additives such as polyols are known to enhance the stability of proteins and protect them in native folded and functional state. In this work, for study of aequorin stability, the histidine-tagged apoaequorin was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. Kinetics of light emission of purified aequorin upon addition of Ca showed a linear dependency on aequorin concentration. Furthermore, the effect of some stabilisers, such as glycerol, glucose, lactose, terehalose, sucrose and sorbitol on thermostability of recombinant aequorin was measured. Results indicate that the recombinant aequorin is very stable in phosphate buffer including 30 mM sorbitol, since after heat shock of 30 min at different temperatures, a slight decrease in activity was observed. However, flexibility and exposure of tryptophan residues of aequorin to the solvent, in the presence and absence of stabilisers, with respect to fluorescence quenching by acrylamide, indicated identical characterisation. In addition, according to limited proteolysis of aequorin demonstrating that this enzyme is sensitive to proteases as in the presence of 2 ng/ml of protease, aequorin was completely digested. In conclusion, sorbitol increases stability of aequorin with high photoactivity and not effect for flexibility and limited proteolysis of this photoprotein. [ABSTRACT FROM AUTHOR]

An Aequorin-based Film Adhesive Seedling (FAS) Ca2+ recording system was developed for monitoring Ca2+ in response to various stimuli in Arabidopsis. This system revealed stimulus- and tissue-specific Ca2+ signatures in seedlings with a simple, sensitive, and robust Ca2+ recording.Calcium ion is a versatile second messenger for diverse cell signaling in response to developmental and environmental cues. The specificity of Ca2+-mediated signaling is defined by stimulus-elicited Ca2+ signature and downstream decoding processes. Here, an Aequorin-based luminescence recording system was developed for monitoring Ca2+ in response to various stimuli in Arabidopsis. With the simple, highly sensitive, and robust Ca2+ recording, this system revealed stimulus- and tissue-specific Ca2+ signatures in seedlings. Cellular Ca2+ dynamics and relationship to Aequorin-based Ca2+ recording were explored using a GFP-based Ca2+ indicator, which suggested that a synchronous cellular Ca2+ signal is responsible for cold-induced Ca2+ response in seedlings, whereas asynchronous Ca2+ oscillation contributes to osmotic stress-induced Ca2+ increase in seedlings. The optimized recording system would be a powerful tool for the identification and characterization of novel components in Ca2+-mediated stress-signaling pathways. [ABSTRACT FROM PUBLISHER]

Gap junctions (GJs) are intercellular channels which are composed of the connexin family of proteins that allow electrical and chemical communications and synchronization in tissue ensembles. Evidence suggests that pharmaceutical modulators of these channels may have therapeutic potential or carry undesired liability. In this report, we exogenously expressed human connexin 43 (Cx43, GJA1 ) and demonstrated functionality in a 96-well flow cytometry assay detecting intercellular transfer of the calcein dye. We have designed a 384-well high-throughput method for detecting the transfer of calcium between HeLa cells expressing Cx43. In this assay, donor cells coexpress Cx43 and the α1A adrenergic Gα-coupled receptor, while recipient cells coexpress Cx43 and the cytoplasmic version of the calcium-sensitive luminescent protein aequorin enhanced by codon optimization (cytoAeq). The two cell populations were mixed, dispensed to 384-well plates, and incubated for 3 h to allow the formation of GJs. Activation of α1A by epinephrine in donor cells led to dose-dependent calcium increases in recipient cells, which were detected by measuring the intensity of aequorin luminescence. The response was dependent on the expression of Cx43 and inhibited by the GJ blocker 18α-glycyrrhetinic acid, suggesting Cx43 GJ-mediated activity. In a parallel experiment with capsaicin and the TrpV1 ion channel in place of phenylephrine and α1A, a similar magnitude of difference in the maximal calcium response was detected in both donor and recipient cells, suggesting that calcium is likely the permeant ion through the GJ. This assay may pave the way for high-throughput screening of GJ modulators for drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: The absorption and emission spectra of six different possible light emitters of coelenteramide: neutral, phenolate anion, phenolate anion with a counter ion (phenolate−–M+), amide anion, pyrazine anion, and the dianion were investigated with the CAM-B3LYP method in the gas phase, in aqueous solution, in a hydrophobic environment and in benzene. The emission spectra of the phenolate anion can be modulated by the covalent character of an oxygen metal bond between the phenolate anion and the countercation. The phenolate anion is more easily formed than the amide anion by deprotonation of the neutral in the excited state. The phenolate anion could not be deprotonated to form the dianion. To examine the charge transfer states of coelenteramide, the charge distributions for the ground and excited states of the complexes were predicted. The emission spectra were predicted in the excited state solvent reaction field. The ground state energies were determined with non-equilibrium solvation, at the excited state geometry with static solvation from the excited state. [Copyright &y& Elsevier]

Abstract: The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgi in vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70μM vs. 0.05μM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2–3μM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones. [Copyright &y& Elsevier]

Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields. Herein, we describe the development and optimization of a protein-based inhibition assay for the quantification of OH-PCBs. Specifically, the photoprotein aequorin was utilized for the detection of OH-PCBs. We hypothesized that OH-PCBs interact with aequorin, and we established that OH-PCBs actually inhibit the bioluminescence of aequorin in a dose-dependent manner. We took advantage of this phenomenon to develop an assay that is capable of detecting a wide variety of OH-PCBs with a range of detection limits, the best detection limit being 11 nM for the compound 2-hydroxy-2',3,4',5',6-pentachorobiphenyl. The viability of this system for the screening of OH-PCBs in spiked biological and environmental samples was also established. We envision the implementation of this novel bioluminescence inhibition assay as a rapid, sensitive, and cost-effective method for monitoring OH-PCBs. Furthermore, to the best of our knowledge, this is the first time aequorin has been employed to detect an analyte by the inhibition of its bioluminescence reaction. Hence, this strategy may prove to be a general approach for the development of a new generation of protein-based inhibition assays. [ABSTRACT FROM AUTHOR]

Mitochondria have a very large capacity to accumulate Ca2+ during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca2+ ]M (mitochondrial [Ca2+ ]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca2+ ]M during prolonged stimulation has been previously precluded by the high Ca2+ affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca2+ ] in the millimolar range for long periods of timewithout problems derived from aequorin consumption. We show in the present study that addition of Ca2+ to permeabilized HeLa cells triggers an increase in [Ca2+ ]M up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5- 1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca2+ ] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca2+ ]M levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca2+ uniporter largely reduced the rate of [Ca2+ ]M increase, but the final steady-state [Ca2+ ]M reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca2+ ]M without problems derived from aequorin consumption. [ABSTRACT FROM AUTHOR]

Abstract: Background: In recent years, as our understanding of the various roles played by Ca2+ signaling in development and differentiation has expanded, the challenge of imaging Ca2+ dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains. Scope of review: In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca2+ imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell. Major conclusions: These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca2+ signaling. The improvement in the spatial resolution that results from specifically targeting a Ca2+ reporter has helped to reveal how a ubiquitous signaling messenger like Ca2+ can regulate coincidental but different signaling events within an individual cell; a Ca2+ signaling paradox that until now has been hard to explain. General significance: Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca2+ signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling. [Copyright &y& Elsevier]

Mitochondria have a very large capacity to accumulate Ca2+ during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca2+]M (mitochondrial [Ca2+]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca2+]M during prolonged stimulation has been previously precluded by the high Ca2+ affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca2+ ] in the millimolar range for long periods of timewithout problems derived from aequorin consumption. We show in the present study that addition of Ca2+ to permeabilized HeLa cells triggers an increase in [Ca2+]M up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5- 1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca2+ ] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca2+]M levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca2+ uniporter largely reduced the rate of [Ca2+]M increase, but the final steady-state [Ca2+]M reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca2+]M without problems derived from aequorin consumption. [ABSTRACT FROM AUTHOR]

Chromaffin cells are an excellent model for stimulus-secretion coupling. Ca entry through plasma membrane voltage-operated Ca channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca signal. The endoplasmic reticulum amplifies the cytosolic Ca ([Ca]) signal by Ca-induced Ca release (CICR) and helps generation of microdomains with high [Ca] (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca from HCMD and stop progression of the Ca wave towards the cell core. On the other hand, the increase of [Ca] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca] decreases rapidly and mitochondria release the Ca accumulated in the matrix through the Na/Ca exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus-secretion coupling while avoiding propagation of the Ca signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Abstract: Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3mg of His-Cys4-aequorin from 2L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-acitivated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for α-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01–100ng/ml and the sensitivities were similar to that using aequorin-labeled specific antibody and amino-biotinylated aequorin. [Copyright &y& Elsevier]

Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo. [ABSTRACT FROM PUBLISHER]

Abstract: Available methods to measure mitochondrial [Ca2+] ([Ca2+]M) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca2+]M values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca2+-affinity dye rhod-5N provides [Ca2+]M values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca2+-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5mM. Addition of Ca2+ buffers containing between 4.5 and 10μM [Ca2+] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca2+]M up to the 100μM–1mM range, which were dependent on mitochondrial membrane potential. Ca2+ release from mitochondria was largely dependent on [Na+]. We have then used rhod-5N loaded cells to investigate the [Ca2+]M response to agonist stimulation at the single-cell and subcellular level. The [Ca2+]M peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25μM. In the presence of the Ca2+ uniporter stimulator kaempferol, the [Ca2+]M peaks induced by histamine were also highly variable, and the mean [Ca2+]M peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca2+] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca2+]M peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca2+]M increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus. [Copyright &y& Elsevier]

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47–57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca2+-sensitive photoprotein widely used as a reliable Ca2+ reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT–aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca2+ concentrations. Plant cells incubated for just 5 min with TAT–aequorin responded to different environmental stimuli with the expected Ca2+ signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca2+ concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca2+ dynamics rapidly and effectively in plant cells. [ABSTRACT FROM PUBLISHER]

Abstract: The calcium ion is probably one of the most studied second messenger both in plant and animal fields. A large number of reviews have browsed the diversity of cytosolic calcium signatures and evaluated their pleiotropic roles in plant and animal cells. In the recent years, an increasing number of reviews has focused on nuclear calcium, especially on the possible roles of nuclear calcium concentration variations on nuclear activities. Experiments initially performed on animal cells gave conflicting results that brought about a controversy about the ability of the nucleus to generate its own calcium signals and to regulate its calcium level. But in plant cells, several converging scientific pieces of evidence support the hypothesis of nucleus autonomy. The present review briefly summarizes data supporting this hypothesis and tries to put forward some possible roles for these nucleus-generated calcium signals in controlling nuclear activity. [Copyright &y& Elsevier]

Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The 'Ca signature' is the combination of changes in all Ca signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca signatures were induced before toxicity was evident, we propose that intracellular free Ca may serve as an early biomarker of exposure to environmental pollution. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene. [ABSTRACT FROM AUTHOR]

Abstract: Ca2+ signalling is important for controlling gene transcription. Changes of the cytosolic Ca2+ ([Ca2+]C) may promote migration of transcription factors or transcriptional regulators to the nucleus. Changes of the nucleoplasmic Ca2+ ([Ca2+]N) can also regulate directly gene expression. [Ca2+]N may change by propagation of [Ca2+]C changes through the nuclear envelope or by direct release of Ca2+ inside the nucleus. In the last case nuclear and cytosolic signalling can be dissociated. Phosphatidylinositol bisphosphate, phospholipase C and cyclic ADP-ribosyl cyclase are present inside the nucleus. Inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) have also been found in the nucleus and can be activated by agonists. Furthermore, nuclear location of the synthesizing enzymes and receptors may be atypical, not associated to the nuclear envelope or other membranes. The possible role of nuclear subdomains such as speckles, nucleoplasmic reticulum, multi-macromolecular complexes and nuclear nanovesicles is discussed. [Copyright &y& Elsevier]

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death (PCD) in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species (ROS) has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium (DPI), indicating the involvement of NADPH oxidase(s) in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum (La3+). Therefore, ROS production occurs downstream of DHS-induced Ca2+ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La3+ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms. [ABSTRACT FROM PUBLISHER]

The cellular prion protein (PrPC) is a cell-surface glycoprotein mainly expressed in the CNS. The structural conversion of PrPC generates the prion, the infectious agent causing transmissible spongiform encephalopathies, which are rare and fatal diseases affecting animals and humans. Despite decades of intensive research, the mechanism of prionassociated neurodegeneration and the physiologic role of PrPC are still obscure. Recent evidence, however, supports the hypothesis that PrPC may be involved in the control of Ca2+ homeostasis. Given the universal significance of Ca2+ as an intracellular messenger for both the life and death of cells, this possibility may help explain the complex, often controversial, dataset accumulated on PrPC physiology, and the events leading to prion-associated neuronal demise. In this study, we have compared local Ca2+ movements in cerebellar granule neurons (CGN) derived from wild-type (WT), or PrPknockout (KO), mice, by means of the Ca2+-sensitive photoprobe, aequorin, genetically targeted to specific intracellular domains and delivered to CGN by lentiviral vectors. The use of an aequorin that localizes to the cytosolic domains proximal to the plasma membrane has allowed us to demonstrate that there was a dramatic increase of store-operated Ca2+ entry in PrP-KO CGN compared to WT neurons. Notably, this phenotype was rescued upon restoring PrPC expression. The Ca2+-phenotype of PrP-KO neurons can in part be explained by the lower expression of two major Ca2+-extruding proteins, namely the plasma membrane and the sarco-endoplasmic reticulum Ca2+-ATPases. The lower sarco-endoplasmic reticulum Ca2+-ATPase content may also contribute to explain why PrP-KO CGN accumulated less Ca2+ in the endoplasmic reticulum than the WT counterpart. [ABSTRACT FROM AUTHOR]

Abstract: Cross-talk between subcellular organelles is essential for cellular Ca2+ homeostasis. We have studied the effects of knocking down STIM1, the Ca2+ sensor of the endoplasmic reticulum (ER), on several homeostatic Ca2+-handling mechanisms, including plasma membrane Ca2+ entry and transport by ER, mitochondria and nucleus. We have used targeted aequorins to selectively measure calcium fluxes in different organelles. Actions of STIM1 were extremely selective, restricted to store operated Ca2+ channels (SOC) and Ca2+ uptake by the ER. No interactions with uptake or release of Ca2+ by mitochondria or nucleus were detected. Ca2+ exit from the ER, including passive leak, release via inositol 1,4,5-trisphosphate and ryanodine receptors, was unaffected. STIM1 knock-down inhibited ER Ca2+ uptake in intact but not in permeabilized cells, suggesting a privileged calcium entry-calcium refilling (CECR) coupling between plasma membrane SOC and ER calcium pump in the intact cell. As a result a large part of the entering Ca2+ is taken up into the ER without reaching the bulk cytosol. The tightness of CECR, as measured by the slope of the stimulus-signal strength function, was comparable to classic excitation–response coupling mechanisms, such as excitation–contraction, excitation–secretion or excitation–transcription coupling. [Copyright &y& Elsevier]

Abstract: Aequorin is a Ca2+-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O2 to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca2+ of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca2+ and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays. [ABSTRACT FROM AUTHOR]

Legume-rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals - flavonoids - before infection. Host plants sense counterpart rhizobial signalling molecules - Nod factors - through transient changes in intracellular free-calcium. Here we investigate the potential involvement of Ca2+ in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae. By using aequorin-expressing rhizobial strains, we monitored intracellular Ca2+ dynamics and the Ca2+ dependence of nod gene transcriptional activation. Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca2+ that were essential for the induction of nod genes. Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca2+ variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca2+ signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca2+ signalling event. These findings demonstrate a key role played by Ca2+ in sensing and transducing plant-specific flavonoid signals in rhizobia and open up a new perspective in the flavonoid-NodD paradigm of nod gene regulation. [ABSTRACT FROM AUTHOR]

We have investigated the dynamics of the free [Ca] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca] ([Ca]] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca pumps with thapsigargin largely blocked Ca uptake by the granules in Ca-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca pump inhibitor benzohydroquinone induced a rapid release of Ca from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca pumps is essential to maintain the steady-state [Ca]. Both inositol 1,4,5-trisphosphate (InsP) and caffeine produced a rapid Ca release from the granules, suggesting the presence of InsP and ryanodine receptors in the granules. The response to high-K depolarization was different in both cell types, a decrease in [Ca] in PC12 cells and an increase in [Ca] in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca] in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca through SERCA-type Ca pumps and can release it through InsP and ryanodine receptors, supporting the hypothesis that secretory granule Ca may be released during cell stimulation and contribute to secretion. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the kinetics of mitochondrial Ca2+ influx and efflux and their dependence on cytosolic [Ca2+] and [Na+] using low-Ca2+-affinity aequorin. The rate of Ca2+ release from mitochondria increased linearly with mitochondrial [Ca2+] ([Ca2+]M). Na+-dependent Ca2+ release was predominant al low [Ca2+]M but saturated at [Ca2+]M around 400μM, while Na+-independent Ca2+ release was very slow at [Ca2+]M below 200μM, and then increased at higher [Ca2+]M, perhaps through the opening of a new pathway. Half-maximal activation of Na+-dependent Ca2+ release occurred at 5–10mM [Na+], within the physiological range of cytosolic [Na+]. Ca2+ entry rates were comparable in size to Ca2+ exit rates at cytosolic [Ca2+] ([Ca2+]c) below 7μM, but the rate of uptake was dramatically accelerated at higher [Ca2+]c. As a consequence, the presence of [Na+] considerably reduced the rate of [Ca2+]M increase at [Ca2+]c below 7μM, but its effect was hardly appreciable at 10μM [Ca2+]c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca2+]M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca2+ buffering, and comparison of our results with data on total mitochondrial Ca2+ fluxes indicate that the mitochondrial Ca2+ bound/Ca2+ free ratio is around 10- to 100-fold for most of the observed [Ca2+]M range and suggest that massive phosphate precipitation can only occur when [Ca2+]M reaches the millimolar range. [Copyright &y& Elsevier]

The Ca2+-dependent response to oxidative stress caused by H2O2 or tert-butylhydroperoxide ( tBOOH) was investigated in Saccharomyces cerevisiae cells expressing transgenic cytosolic aequorin, a Ca2+-dependent photoprotein. Both H2O2 and tBOOH induced an immediate and short-duration cytosolic Ca2+ increase that depended on the concentration of the stressors. Sublethal doses of H2O2 induced Ca2+ entry into the cytosol from both extracellular and vacuolar sources, whereas lethal H2O2 shock mobilized predominantly the vacuolar Ca2+. Sublethal and lethal tBOOH shocks induced mainly the influx of external Ca2+, accompanied by a more modest vacuolar contribution. Ca2+ transport across the plasma membrane did not necessarily involve the activity of the Cch1p/Mid1p channel, whereas the release of vacuolar Ca2+ into the cytosol required the vacuolar channel Yvc1p. In mutants lacking the Ca2+ transporters, H2O2 or tBOOH sensitivity correlated with cytosolic Ca2+ overload. Thus, it appears that under H2O2-induced or tBOOH-induced oxidative stress, Ca2+ mediates the cytotoxic effect of the stressors and not the adaptation process. [ABSTRACT FROM AUTHOR]

Abstract: The dynamics of mitochondrial [Ca2+] ([Ca2+]M) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca2+]M are fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with different methods. We have made here a systematic comparison of the response of several fluorescent dyes, rhod-2 and rhod-FF, and two Ca2+-sensitive proteins, aequorin and pericam. Our results show that measurements obtained with aequorin and pericam are consistent in terms of dynamic Ca2+ changes. Instead, fluorescent dyes failed to follow Ca2+ changes adequately, especially during repetitive stimulation. In particular, measures obtained with rhod-2 or rhod-FF evidenced the previously reported Ca2+-dependent inhibition of mitochondrial Ca2+ uptake, but data obtained with aequorin or pericam under the same conditions did not. The reason for the loss of response of fluorescent dyes is unclear. Loading with these dyes produced changes in mitochondrial morphology and membrane potential, which were small and reversible at low concentrations (1–2μM), but produced large and prolonged damage at higher concentrations. In addition, cells loaded with low concentrations of rhod-2 suffered large changes in mitochondrial morphology after light excitation. Our results suggest that [Ca2+]M data obtained with these dyes should be taken with care. [Copyright &y& Elsevier]

Abstract: Assay technologies that measure intracellular Ca2+ release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca2+ sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M1 receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays. [Copyright &y& Elsevier]

Cuscuta reflexa induces a variety of reaction in its hosts. Some of these are visual reactions, and it is clear that these morphological changes are preceded by events at the molecular level, where signal transduction is one of the early processes. Calcium (Ca2+) release is the major second messenger during signal transduction, and we therefore studied Ca2+ spiking in tomato during infection with C. reflexa. Bioluminescence in aequorin-expressing tomato was monitored for 48 h after the onset of Cuscuta infestation. Signals at the attachment sites were observed from 30 to 48 h. Treatment of aequorin-expressing tomato leaf disks with Cuscuta plant extracts suggested that the substance that induced Ca2+ release from the host was closely linked to parasite haustoria. [ABSTRACT FROM AUTHOR]

Abstract: STIM1 and Orai1 are the main players in capacitative calcium entry (CCE). STIM1 senses [Ca2+] inside the endoplasmic reticulum (ER) and, when it decreases, opens Orai1, a store-operated calcium channel (SOC) in the plasma membrane that promotes Ca2+ entry and increases cytosolic Ca2+. The final destination of the entering Ca2+ is the ER, which refills very efficiently (capacitatively) with it. We propose here that SERCA is the third element of CCE, to which is tightly coupled to favour rapid Ca2+ pumping from the high Ca2+ microdomains, generated at the SOC''s mouth, to the ER. We find that, on depletion of the intracellular Ca2+ stores, SERCA co-localizes with STIM1 at puncta. Adequate coupling of CCE and ER Ca2+ pumping requires correct proportions of STIM1, Orai1 and SERCA. Overexpression of Orai1 decreased modestly Ca2+ entry, but produced a dramatic fall of Ca2+ uptake into ER, which was rescued by STIM1 co-expression or by increasing external Ca2+. In permeabilized cells, Ca2+ uptake into the ER was indistinguishable in the Orai1-expressing and in the control cells. We propose that excess Orai1 uncouples SERCA from Ca2+ entry in the intact cell by disturbing the fine topology of Ca2+ pumping complexes within the ER-plasma membrane junctions. [Copyright &y& Elsevier]

Abstract: The novel Ca2+ channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca2+ transients ([Ca2+]c) that regulate the production of amyloid beta (Aβ). Its mutated channel P86L-CALHM1 has been associated to Alzheimer’s disease (AD). Using cytosolic- and mitochondrial-targeted aequorins, we have investigated here whether mitochondria sense with similar or different kinetics the Ca2+ entering into Hela cells and the Ca2+ released from the endoplasmic reticulum (ER), in control and in cells transfected with CALHM1 and P86L-CALHM1. We have shown that mitochondria sense Ca2+ entry in the three cell types; however, the [Ca2+]c and mitochondrial Ca2+ transients [Ca2+]m had substantially slower kinetics in cells expressing P86L-CALHM1. Mitochondria also sensed the ER Ca2+ released by histamine, but in CALHM1 and P86L-CALHM1 cells the kinetics was faster than that of control cells. Data are compatible with the idea that mutated CALHM1 may cause mitochondrial Ca2+ overload, suggesting how these cells may become more vulnerable to apoptotic stimuli. [Copyright &y& Elsevier]

Abstract: Studies performed in animals have highlighted the major role of sphingolipids in regulating the balance between cell proliferation and cell death. Sphingolipids have also been shown to induce cell death in plants via calcium-based signalling pathways but the contribution of free cytosolic and/or nuclear calcium in the overall process has never been evaluated. Here, we show that increase in tobacco BY-2 cells of the endogenous content of Long Chain Bases (LCBs) caused by external application of d-erythro-sphinganine (DHS) is followed by immediate dose-dependent elevations of cellular free calcium concentration within the first minute in the cytosol and 10min later in the nucleus. Cells challenged with DHS enter a death process through apoptotic-like mechanisms. Lanthanum chloride, a general blocker of calcium entry, suppresses the cellular calcium variations and the PCD induced by DHS. Interestingly, dl-2-amino-5-phosphopentanoic acid (AP5) and [(+)-dizocilpine] (MK801), two inhibitors of animal and plant ionotropic glutamate receptors, suppress DHS-induced cell death symptoms by selectively inhibiting the variations of nuclear calcium concentration. The selective action of these compounds demonstrates the crucial role of nuclear calcium signature in controlling DHS-induced cell death in tobacco cells. [Copyright &y& Elsevier]

In this issue of Cell Chemical Biology , Navas-Navarro et al. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca 2+ signals within the endoplasmic reticulum (ER) of living animals. [ABSTRACT FROM AUTHOR]

Summary: Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca2+]c) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca2+]c was detected in vivo by luminometry, and [Ca2+]c elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca2+]c during cold exposure. Temperature-induced changes in [Ca2+]c were found to be more dependent on the change in temperature (dT dt−1) than on the absolute value of temperature. [Ca2+]c increased only at cooling rates higher than 8°Cmin−1, indicating that an overall cellular [Ca2+]c increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called ‘calcium signature hypothesis’. [Copyright &y& Elsevier]

Abstract: Mitochondria play a pivotal role in intracellular Ca2+ signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca2+ handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca2+ transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca2+ sensors, respectively. Both methods revealed a consistent picture of Ca2+ uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca2+ levels. The big future challenge is to understand the functional impact of such Ca2+ signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory. [Copyright &y& Elsevier]

Abstract: Mitochondrial Ca2+ transport was initially considered important only in buffering of cytosolic Ca2+ by acting as a “sink” under conditions of Ca2+ overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca2+ ([Ca2+]m) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a “parallel activation model” whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca2+] that was relayed by the mitochondrial Ca2+ transporters into the matrix to give an increase in [Ca2+]m. This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca2+]m in living cells that proved [Ca2+]m changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca2+], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca2+] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca2+ is finely tuned to the demands and function of the individual organ. [Copyright &y& Elsevier]