Photoprotein Aequorin Research Articles

Monitoring ER Ca2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca2+, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca2+ dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca2+. Here we describe a robust and quantitative method based on the Ca2+ indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca2+ affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca2+ such that it conforms with the [Ca2+] values found in the ER and it slows the consumption of the probe by Ca2+. This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1h) ER Ca2+ measurements. GAP1 targeted to the ER allows monitoring of resting [Ca2+]ER and Ca2+ dynamics in intact cells stimulated with IP3-produced agonists. In addition, GAP1 can record Ca2+ mobilization in permeabilized cells challenged with IP3. We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca2+]ER.

Suppression of Contraction Raises Calcium Ion Levels in the Heart of Zebrafish Larvae.

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca2+ levels, and of the heart rate.

Quantitative Analysis of Plant Cytosolic Calcium Signals in Response to Water Activated by Low-Power Non-Thermal Plasma.

Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca2+ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca2+ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca2+ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.

Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+ -Binding Photoprotein Aequorin.

Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+ , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.

Chiral deaza-coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin.

The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.

Engineering aequorin to improve thermostability through rigidifying flexible sites

The photoprotein aequorin has been widely used as a bioluminescent label in various biological and analytical techniques. Here, rigidifying flexible sites as a protein engineering approach was used to design the aequorin variants with high thermostability. Based on two simple approaches, B-factor analysis and replacement of glycine residues in α-helix, three mutations were created in the Gly14 and Glu156 positions. The studies showed that the bioluminescence activity of the G14A mutant was slightly increased, however, it was decreased for the E156N mutant. Also, the G14N mutant showed no significant changes compared to the wild type. Structural studies indicated that E156N mutation became much more flexible than the wild type, although G14A and G14N didn't significantly differ. Irreversible thermoinactivation experiments were done upon incubation of aequorin variants at representative temperatures ranging from 333 to 348 K. The inactivation rate constants (kinact) at 333 K were determined to be 0.009 min−1, 0.01 min−1, and 0.013 min−1 for wild type, G14N, and E156N variants, respectively. In contrast, a value of 0.004 min−1 was observed for the G14A mutant. Detailed analysis of the inactivation process showed that the greater thermostability of the G14A mutant is due to the higher activation energy (Ea) and subsequently more values of ∆H‡ and ∆G‡ at a given temperature. However, E156N mutation decreased the thermostability of apoaequorin, and the G14N mutant showed no significant changes. According to molecular dynamics simulation, it seems that the higher thermostability of G14A mutant is due to a local increase in the number of van der Waals interactions, when compared the wild type. Moreover, E156N mutation decreased the thermal stability by increasing flexibility in the C terminal region and losing some contacts of this fragment with other parts of the protein.

Identification of Chemical and Pharmacological Chaperones for Correction of Trafficking-Deficient Mutant Cyclic Nucleotide-Gated A3 Channels.

Trafficking deficiency caused by missense mutations is a well known phenomenon that occurs for mutant, misfolded proteins. Typically, the misfolded protein is retained by the protein quality-control system and degraded by the endoplasmic reticulum-associated protein degradation pathway and thus does not reach its destination, although residual function of the protein may be preserved. Chemical and pharmacological chaperones can improve the targeting of trafficking-deficient proteins and thus may be promising candidates for therapeutic applications. Here, we report the application of a cellular bioassay based on the bioluminescent calcium reporter aequorin to quantify surface expression of mutant CNGA3 channels associated with the autosomal recessively inherited retinal disease achromatopsia. A screening of 77 compounds enabled the identification of effective chemical and pharmacological chaperones that result in a 1.5- to 4.8-fold increase of surface expression of mutant CNGA3. Using selected compounds, we confirmed that the rescue of the defective trafficking is not limited to a single mutation in CNGA3. Active compounds and our structure-activity correlated data for the dihydropyridine compound class may provide valuable information for developing a treatment of the trafficking defect in achromatopsia. SIGNIFICANCE STATEMENT: This study describes a novel luminescence-based assay to detect the surface expression of mutant trafficking-deficient CNGA3 channels based on the calcium-sensitive photoprotein aequorin. Using this assay for a compound screening, this study identifies novel chemical and pharmacological chaperones that restore the surface localization of mutant trafficking-deficient CNGA3 channels. The results from this work may serve as starting point for the development of potent compounds that rescue trafficking deficiencies in the autosomal recessively inherited retinal disease achromatopsia.

Evidence of Calcium Signaling and Modulation of the LmrS Multidrug Resistant Efflux Pump Activity by Ca2 + Ions in S. aureus.

Calcium ions (Ca2+) play a pivotal role in eukaryote cell signaling and regulate many physiological functions. Although a similar role for Ca2+ in prokaryotes has been difficult to demonstrate, there is increasing evidence for Ca2+ as a cell regulator in bacteria. The purpose of this study was to investigate Ca2+ signaling and the effect of Ca2+ on the Staphylococcus aureus multidrug resistant efflux pump LmrS. We hypothesized that antibiotics act by increasing Ca2+ concentrations, which in turn enhance the efflux activity of LmrS. These Ca2+ transients were measured by luminometry in response to various antibiotics by using the photoprotein aequorin reconstituted within live bacterial cells. Efflux associated with LmrS was measured by the increase in fluorescence due to the loss of ethidium bromide (EtBr) from both S. aureus cells and from E. coli cells in which the lmrs gene of S. aureus was expressed. We found that addition of antibiotics to cells generated unique cytosolic Ca2+ transients and that addition of CaCl2 to cells enhanced EtBr efflux whereas addition of Ca2+ chelators or efflux pump inhibitors significantly decreased EtBr efflux from cells. We conclude that antibiotics induce a Ca2+ mediated response through transients in cytosolic Ca2+, which then stimulates LmrS efflux pump.

Cytotoxicity of Oleandrin Is Mediated by Calcium Influx and by Increased Manganese Uptake in Saccharomyces cerevisiae Cells.

Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.

Manganese Suppresses the Haploinsufficiency of Heterozygous trpy1Δ/TRPY1Saccharomyces cerevisiae Cells and Stimulates the TRPY1-Dependent Release of Vacuolar Ca2+ under H₂O₂ Stress.

Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn2+ surplus. Using cells expressing the Ca2+-sensitive photoprotein aequorin we found that Mn2+ augmented the Ca2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn2+-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn2+ activate TRPY1 in the response to oxidative stress.

Development of a novel, sensitive cell-based corin assay

Corin (atrial natriuretic peptide-converting enzyme, EC 3.4.21) is a transmembrane serine protease expressed in cardiomyocytes. Corin exerts its cardioprotective effects via the proteolytic cleavage and activation of pro-atrial natriuretic peptide (pro-ANP) to ANP. We recently described an ANP reporter cell line stably expressing the ANP receptor, a cGMP-dependent cation channel used as a real-time cGMP biosensor, and the Ca2+-sensitive photoprotein aequorin. Here, we describe the generation of a novel reporter cell line expressing the calcium biosensor GCaMP6 instead of aequorin. In contrast to the luminescence-based assay, ANP stimulation of our novel GCaMP6 reporter cell resulted in stable, long-lasting fluorescence signals. Using this novel reporter system, we were able to detect pro-ANP to ANP conversion by purified, soluble wildtype corin (solCorin), but not the active site mutant solCorin(S985A), resulting in left-shifted concentration-response curves. Furthermore, cellular pro-ANPase activity could be detected on HEK 293 cells after transient expression of wildtype corin. In contrast, corin activity was not detected after transfection with the inactive corin(S985A) variant. In supernatants from cardiomyocyte-derived HL-1 cells pro-ANP to ANP conversion could also be detected, while in HL-1 corin knockout cells no conversion was observed. These findings underline the role of corin as the pro-ANP convertase. Our novel fluorescence-based ANP reporter cell line is well-suited for the sensitive detection of corin activity, and may be used for the identification and characterization of novel corin modulators.

The Claudin Family Protein FigA Mediates Ca2+ Homeostasis in Response to Extracellular Stimuli in Aspergillus nidulans and Aspergillus fumigatus.

The claudin family protein Fig1 is a unique fungal protein that is involved in pheromone-induced calcium influx and membrane fusion during the mating of Saccharomyces cerevisiae and Candida albicans. Whether and how Fig1 regulates Ca2+ homeostasis in response to extracellular stimuli is poorly understood. Previously, we found Aspergillus nidulans FigA, a homolog of Fig1 in S. cerevisiae, similar to the high-affinity calcium uptake system, is required for normal growth under low-Ca2+ minimal medium. In this study, using the calcium-sensitive photoprotein aequorin to monitor cytosolic free calcium concentration ([Ca2+]c) in living cells, we found that the FigA dysfunction decreases the transient [Ca2+]c induced by a high extracellular calcium stress. Furthermore, FigA acts synergistically with CchA (a high-affinity Ca2+ channel) to coordinate cytoplasmic Ca2+ influx in response to an extracellular Ca2+ stimulus. Moreover, FigA mediates ER stress-induced transient [Ca2+]c in the presence or absence of extracellular calcium. Most importantly, these [Ca2+]c responses mediated by FigA are closely related to its conserved claudin superfamily motif, which is also required for hyphal growth and asexual development in A. nidulans. Finally, the function of FigA in Aspergillus fumigatus, the most common airborne human fungal pathogen was studied. The result showed that the two FigA homologous in A. nidulans and A. fumigatus have a large degree of functional homology not only in asexual development but also in regulating transient [Ca2+]c. Our study expands the knowledge of claudin family protein FigA in Ca2+ homeostasis in response to extracellular stimuli.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans.

The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

CdTe quantum dots with green fluorescence generated by bioluminescence resonance energy transfer from aequorin

The need for external excitation sources limits the utility of quantum dots (QDs) in multiplexed detection schemes and in in vivo imaging, because it can lead to strong background by surface illumination and tissue autofluorescence. In this work, the authors describe the use of oxidized dextran as a support to conjugate the photoprotein aequorin to QDs in order to obtain self-illuminating QDs and an efficient QD-based bioluminescence (BL) resonance energy system. On addition of Ca2+, BL is generated by immobilized aequorin and transferred to the QDs which thereby become photoexcited. Hence, these QDs will fluoresce without being excited by an external light source and therefore have the typical merits (such as very low background) of bioluminescent systems. The half-life of the BL of aequorin peaking at 460 nm is 1.6 s, and that of the QD-conjugated aequorin (peaking at 528 nm) is 6.4 s. We perceive that by labeling antibodies with these nanocomposites, highly advanced multiplex immunoassays will become possible.

A putative mitochondrial calcium uniporter in A. fumigatus contributes to mitochondrial Ca2+ homeostasis and stress responses

Ca2+ uptake into mitochondria plays a central role in cell physiology by stimulating ATP production, shaping cytosolic Ca2+ transients and regulating cell survival or death. Although this system has been studied extensively in mammalian cells, the physiological implications of Ca2+ uptake into mitochondria in fungal cells are still unknown. In this study, a bi-directional best-hit BLASTP search revealed that the genome of Aspergillus fumigatus encodes a homolog of a putative mitochondrial Ca2+ uniporter (MCU) and a mitochondrial carrier protein AGC1/MICU1 homolog. Both putative homologs are mitochondrially localized and required for the response to azole and oxidative stress such that the loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress, suggesting a role in environmental stress adaptation. Overexpressing mcuA restores the azole-resistance phenotype of the ΔagcA strain to wild-type levels, but not vice versa, indicating McuA plays a dominant role during these stress responses. Using a mitochondrially targeted version of the calcium-sensitive photoprotein aequorin, we found that only mcuA deletion leads to dysfunctional [Ca2+]mt and [Ca2+]c homeostasis, suggesting that McuA, but not AgcA, contributes to Ca2+ uptake into mitochondria. Further point-mutation experiments combined with extracellular Ca2+ chelator treatment verified that two predicted Ca2+-binding sites in McuA are required for Ca2+ uptake into mitochondria and stress responses through the regulation of [Ca2+]c homeostasis.

Improving the luminescence properties of aequorin by conjugating to CdSe/ZnS quantum dot nanoparticles: Red shift and slowing decay rate

Changing the properties of photoprotein aequorin such as the wavelength emission and decay half-life by using bioluminescence resonance energy transfer (BRET) phenomenon is the main aim in this paper. BRET system was set up with CdSe/ZnS quantum dot nanoparticles as an acceptor molecule and photoprotein as an energy donor molecule. Quantum dots are semiconductor nanoparticles with very interesting optical properties, including broad excitation spectra, narrow and the symmetric band width emission spectra, tunable by their sizes, compositions, negligible photo-bleaching and good chemical and photo-stability. In this QD-BRET system, aequorin is conjugated to the carboxyl groups on quantum dot surface by EDC/NHS chemistry as cross linker. Bioluminescence energy generates by aequorin upon adding Ca2+ and transfers to the quantum dots in a radiationless manner and emits at a longer wavelength. The determined bioluminescent parameters for this method included aequorin activity, emission spectra and decay half-life time. In fact, this spectrum tuning strategy resulted in a change in bioluminescent properties of photoprotein, therefore, the maximum emission wavelength shifted from 455 to 540nm and the decay time increased from 3.76 to 12.11s. Nowadays, photoproteins with different characteristics are capable of being employed as a reporter in multi-analyte detections and in vivo imaging.

Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans.

The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans Additionally, the hypersensitivity of the ΔcnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca(2+) concentration ([Ca(2+)]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca(2+)]c, but a deficiency in both cnaA and cchA switches the [Ca(2+)]c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the ΔcnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the ΔcnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7:e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca(2+)]c Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca(2+) homeostasis in response to different environments.

A new low-Ca2+ affinity GAP indicator to monitor high Ca2+ in organelles by luminescence

We have recently described a new class of genetically encoded Ca2+ indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca2+ indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine. We describe here a novel application of GAP as a low affinity bioluminescent indicator, suitable for measurements of [Ca2+] in ER or in Golgi apparatus. We used the low affinity variant, GAP1, which carries mutations in two EF-hands of aequorin, reconstituted with coelenterazine n. In comparison to previous bioluminescent aequorin fusions, the decay rate of GAP1 was decreased 8 fold and the affinity for Ca2+ was lowered one order of magnitude. This improvement allows long-term measurements in high Ca2+ environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca2+ concentrations in the range from 400 to 600μM. ER could be emptied of calcium by stimulation with ATP, carbachol or histamine in intact cells, and by challenge with inositol tris-phosphate in permeabilized cells. GAP1 was also targeted to the Golgi apparatus where it was able to precisely monitor long-term calcium dynamics. GAP1 provides a novel and robust indicator applicable to bioluminescent high-throughput quantitative assays.

Enabling Aequorin for Biotechnology Applications Through Genetic Engineering.

In recent years, luminescent proteins have been studied for their potential application in a variety of detection systems. Bioluminescent proteins, which do not require an external excitation source, are especially well-suited as reporters in analytical detection. The photoprotein aequorin is a bioluminescent protein that can be engineered for use as a molecular reporter under a wide range of conditions while maintaining its sensitivity. Herein, the characteristics of aequorin as well as the engineering and production of aequorin variants and their impact on signal detection in biological systems are presented. The structural features and activity of aequorin, its benefits as a label for sensing and applications in highly sensitive detection, as well as in gaining insight into biological processes are discussed. Among those, focus has been placed on the highly sensitive calcium detection in vivo, in vitro DNA and small molecule sensing, and development of in vivo imaging technologies. Graphical Abstract.

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca2+ concentrations in the mutant strain Aspergillus awamori 66A, containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity ofsporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 microM) stimulated spore germination: the number of germinating spores was 2-3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 microM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca2+ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 microM) were shown to cause an instantaneous increase in Ca2+ concentrations, compared to the control and lower amphotericin concentration (2.5 microM). Ca2+ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.