Use Of Aequorin Research Articles

Retrospective on the development of aequorin and aequorin‐based imaging to visualize changes in intracellular free [Ca2+

In this review, we take a retrospective look at the discovery and utilization of the Ca(2+) -sensitive bioluminescent protein complex, aequorin. We do consider the contribution it has made to our understanding of the natural phenomenon of bioluminescence, but it is in the application of extracted and purified aequorin as a reporter of Ca(2+) dynamics in living cells, which is arguably its major contribution to biological and biomedical science. Following its extraction, purification, and subsequent availability in the mid-1960s, aequorin became the intracellular reporter of choice until it was replaced in the late 1970s by easier-to-use fluorescence-based reporters. From the mid-1980s onwards, however, aequorin-based Ca(2+) imaging underwent a renaissance following the cloning of the aequorin gene and the emergence of routine techniques to target and express it exogenously in plant and animal systems. The development of aequorin as a tool continues as spectral varieties are being developed that allow simultaneous imaging of Ca(2+) dynamics in different cellular organelles and microdomains. We predict that further developments in the use of aequorin, as well as other bioluminescent proteins, will continue, especially in the areas of regenerative medicine and whole organism imaging.

The Use of Aequorin and Its Variants for Ca2+ Measurements

Ca(2+)-sensitive photoproteins are ideal agents for measuring the Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because they can be modified to include specific targeting sequences. Aequorin was the first Ca(2+)-sensitive photoprotein probe used to measure the [Ca(2+)] inside specific intracellular organelles in intact cells. Aequorin is a 22-kDa protein produced by the jellyfish Aequorea victoria. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group is released and a photon is emitted. Aequorin has become widely used for intracellular Ca(2+) measurements because it offers many advantages: For example, it can be targeted with precision, functions over a wide range of [Ca(2+)], and shows low buffering capacity. In this article we describe the main characteristics of the aequorin probe and review the reasons why it is widely used to measure intracellular [Ca(2+)].

Method for rapid conjugation of recombinant photoprotein aequorin with streptavidin and application as a universal detection reagent for binding assays

A binding assay utilizes the highly specific molecular recognition properties of a biological macromolecule, e.g. an antibody, receptor or DNA/RNA probe for the detection/quantification of a target analyte in a complex mixture. The recognition event is linked to a signal-producing system. Because biomolecules can be easily labeled with biotin, the biotin–streptavidin interaction has been established as a general system for linking molecular recognition with signal generation. To this end, we report a rapid and simple method for conjugation of the highly detectable recombinant photoprotein aequorin with streptavidin, thus generating a universal reagent for binding assays. The method is based on the use of aequorin fused to a hexahistidine tag at the amino terminus. Thiol groups were introduced to aequorin whereas streptavidin was derivatized with maleimide groups. The conjugate was purified in a single step by immobilized metal ion affinity chromatography, thus avoiding laborious chromatographic procedures. The performance of aequorin–streptavidin conjugate was tested in a DNA hybridization assay as a model. The limit of detection was 0.3 pmol l −1 and the analytical range extended up to 500 pmol l −1. The CV was about 8%. Besides the high detectability, the assay is rapid (completed in just 25 min) due to the flash-type (3 s) bioluminescent reaction of aequorin, which avoids substrate incubation steps that are common to binding assays employing enzyme labels.

An immunoassay for Leu-enkephalin based on a C-terminal aequorin-peptide fusion.

Recently we demonstrated that the fusion of an octapeptide to the C-terminus of a cysteine-free mutant of aequorin showed no inhibitory effect on the luminescence activity of the photoprotein. This observation is of particular importance when the use of aequorin as a label in the development of immunoassays for peptides whose activity lies in their C-terminal region or the epitope for antibody recognition is at their C-terminus is desired. In the case of opioid peptides, antibodies are directed toward their C-terminus as they differ from each other at this terminus. The goal of this study was to develop an immunoassay for Leu-enkephalin, a mammalian opioid peptide, using a C-terminal aequorin-peptide fusion protein. For that, the N-terminus of Leu-enkephalin was genetically fused to the C-terminus of a cysteine-free mutant of aequorin. It was observed that the C-terminal conjugated aequorin maintained its luminescence activity. An immunoassay for Leu-enkephalin was then developed using the aequorin-Leu-enkephalin fusion protein as a labeled analyte in a competitive as well as in a sequential binding mode. It was demonstrated that aequorin can be used as a label in peptide assays in which it is critical that the peptide's C-terminus be free for activity and/or for antibody recognition.

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis.

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.

Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell.

In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.

Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.

Measurement of Intracellular Calcium in Cell Populations Loaded with Aequorin: Neurokinin-1 Responses in U373MG Cells

Changes in intracellular calcium concentration are important in mediating a wide variety of physiological responses. Recently there has been renewed interest in the use of aequorin, a protein from jellyfish that emits light when calcium is bound, to measure calcium levels in cells. We have loaded populations of cells from the human glioma line, U373MG, with aequorin. Lysis of aequorin-loaded but not control cells with detergent resulted in a luminescence signal that was dependent on extracellular calcium. Aequorin-loaded cells responded to substance P, histamine, or the calcium ionophore, ionomycin, with an increase in luminescence. Signals in response to detergent, ionomycin, or substance P could be detected up to 48 h after cells were loaded with aequorin. Other neurokinin-1 agonists but not agonists at neurokinin-2 or neurokinin-3 receptors produced luminescence signals. Neurokinin-1 antagonists inhibited the substance P-induced signal. The aequorin-loading procedure worked well with U373MG cells but not with AR42J, CHO, IMR-90, or WI-38 cells.

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Intact frog skeletal muscle fibers were injected with the Ca2+ indicator fura-2 conjugated to high molecular weight dextran (fura dextran, MW approximately 10,000; dissociation constant for Ca2+, 0.52 microM), and the fluorescence was measured from cytoplasm (17 degrees C). The fluorescence excitation spectrum of fura dextran measured in resting fibers was slightly red-shifted compared with the spectrum of the Ca(2+)-free indicator in buffer solutions. A simple comparison of the spectra in the cytoplasm and the in vitro solutions indicates an apparently "negative" cytoplasmic [Ca2+], which probably reflects an alteration of the indicator properties in the cytoplasm. To calibrate the indicator's fluorescence signal in terms of cytoplasmic [Ca2+], we applied beta-escin to permeabilize the cell membrane of the fibers injected with fura dextran. After treatment with 5 microM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca2+, ATP), whereas the 10-kD fura dextran only slowly leaked out of the fiber. It was thus possible to estimate calibration parameters in the indicator fluorescence in the fibers by changing the bathing solution [Ca2+] to various levels; the average values for the fraction of Ca(2+)-bound indicator in the resting fibers and the dissociation constant for Ca2+ (KD) were, respectively, 0.052 and 1.0 microM. For the comparison, the KD value was also estimated by a kinetic analysis of the indicator fluorescence change after an action potential stimulation in intact muscle fibers, and the average value was 2.5 microM. From these values estimated in the fibers, resting cytoplasmic [Ca2+] in frog skeletal muscle fibers was calculated to be 0.06-0.14 microM. The range lies between the high estimates from other tetracarboxylate indicators (0.1-0.3 microM; Kurebayashi, N., A. B. Harkins, and S. M. Baylor. 1993. Biophysical Journal. 64:1934-1960; Harkins, A. B., N. Kurebayashi, and S. M. Baylor. 1993. Biophysical Journal. 65:865-881) and the low estimate from the simultaneous use of aequorin and Ca(2+)-sensitive microelectrodes (< 0.04-0.06 microM; Blatter, L. A., and J. R. Blinks. 1991. Journal of General Physiology. 98:1141-1160) recently reported for resting cytoplasmic [Ca2+] in frog muscle fibers.

Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery.

1. We hypothesized that the homogeneity of intracellular [Ca2+] ([Ca2+]i) varies and is regulated in arterial smooth muscle. 2. We evaluated this hypothesis by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which theoretically can measure focal increases in [Ca2+]i, (2) fura-2, which is predominantly a measure of mean cytoplasmic [Ca2+], and (3) myosin light chain phosphorylation and force, which reflect increases in [Ca2+] near the contractile apparatus. 3. From the differences in the observed aequorin and fura-2 signals, we developed an index of the relative degree of [Ca2+]i homogeneity as the ratio of the aequorin signal and fura-2 signal. 4. Stimulation with intermediate concentrations of histamine (1 and 10 microM) or high [K+]o (25 and 40 mM) increased [Ca2+]i and contractile stress. Relative [Ca2+]i homogeneity, estimated from the aequorin/fura-2 ratio, remained similar to levels observed in unstimulated tissues. 5. Higher concentrations of histamine (100 microM) also increased [Ca2+]i and stress, but the aequorin/fura 2 ratio declined , indicating increased [Ca2+]i homogeneity. Similarly, the aequorin/fura-2 ratio decreased when extracellular Ca2+ was removed. 6. Stimulation with histamine in low extracellular [Ca2+] transiently increased [Ca2+]i and the aequorin/fura-2 ratio. Similarly, in tissues treated with low extracellular [Ca2+], restoration of extracellular Ca2+ transiently increased both [Ca2+]i and the aequorin/fura-2 ratio. Although both of these experiments demonstrated a transient decrease in [Ca2+]i homogeneity, only histamine stimulation led to increased myosin light chain phosphorylation and force. These results indicate that the focal increases in [Ca2+]i observed with histamine stimulation and Ca2+ restoration occurred in different cellular regions. 7. Addition of caffeine (20 mM) increased [Ca2+]i and [cAMP], but this was not accompanied by sustained increased myosin light chain phosphorylation or contraction. Phosphorylation of myosin light chain kinase did not appear to underlie the lack of increase in myosin light chain phosphorylation. Rather, caffeine induced a sustained increase in the aequorin/fura-2 ratio, suggesting that caffeine inhibits smooth muscle contraction by localizing increases in [Ca2+]i to a region distant from the contractile apparatus. 8. These data suggest that there can be transient and sustained focal increases in [Ca2+]i. Aequorin detected increased [Ca2+]i in small regions of the cytoplasm during release from and refilling of the intracellular Ca2+ store and with caffeine stimulation. Dual use of aequorin and fura-2 permits determination of relative [Ca2+]i homogeneity in smooth muscle.

Endoplasmic reticulum: a dynamic patchwork of specialized subregions.

Endoplasmic reticulum: a dynamic patchwork of specialized subregions.

Ca2+]i in single isolated cardiac cells: a review of recent results obtained with digital imaging microscopy and fura-2.

The use of fluorescent Ca2+ indicators to observe [Ca2+]i transients in voltage-clamped single cells has many advantages over previous methods, such as the use of aequorin in multicellular preparations, for studying excitation-contraction coupling. In the studies reviewed in this article, [Ca2+]i in single isolated mammalian ventricular myocytes was observed through the use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were generally studied with the use of inverted microscopes equipped with ultraviolet epifluorescence illumination, intensified silicon intensifier target cameras (ISIT), and (or) a photomultiplier tube. Analysis of subcellular patterns of fura-2 fluorescence was performed by digital analysis of the images obtained with the ISIT camera. Variation of membrane voltage and exposure of cells to ryanodine (which was assumed to selectively block the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The main results of these studies are the following. (1) In any population of enzymatically isolated heart cells, there are (i) mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; (ii) spontaneously contracting cells, which have a relatively elevated [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous, propagating waves of high [Ca2+]i; and (iii) cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes

Intracellular calcium was monitored by the use of aequorin in voltage-clamped oocytes of Xenopus laevis. Injection of inositol trisphosphate (IP3) into oocytes elicited slowly rising and decaying aequorin/calcium signals and produced oscillatory chloride membrane currents. These responses did not depend upon extracellular calcium, since they could be elicited in calcium-free solution and after addition of cobalt or lanthanum to block calcium channels in the surface membrane. We conclude that IP3 causes the release of calcium from intracellular stores in the oocyte. Injections of calcium gave aequorin and membrane current responses that were more transient than those seen with IP3.

Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells

A change of pH did not modify the sensitivity of aequorin to Ca 2+, but an increase of pH enhanced the Ca 2+ sensitivity of the myofilaments of a skinned canine cardiac Purkinje cell. The tension-pCa curve did not present any hysteresis when a given [free Ca 2+] was reached from a higher versus from a lower [free Ca 2+] in the presence of pH 6.60, 7.10 or 7.40. A rapid variation of pH in either direction failed to induce Ca 2+ release from the sarcoplasmic reticulum (SR). The proton ionophores CCCP and gramicidin also failed to induce Ca 2+ release from the SR. Increase of pH from 7.10 to 7.40 enhanced Ca 2+ accumulation into the SR and, there-by, augmented the Ca 2+ content of the SR. Consequently, the amplitude of a subsequent Ca 2+ release triggered by a rapid increase of [free Ca 2+] at the outer surface of the SR was increased. Conversely, a decrease of pH from 7.10 to 6.60 diminished the Ca 2+ accumulation into the SR, the Ca 2+ content of the SR and the amplitude of a subsequent Ca 2+-induced release of Ca 2+ from the SR. In addition, the optimum [free Ca 2+] for triggering Ca 2+-induced release of Ca 2+ was shifted to higher [free Ca 2+] values by a decrease of pH from 7.40 to 7.10 or 7.10 to 6.60. This may help to explain the enhancement of the aequorin light transient during acidosis in the intact cardiac muscle inasmuch as acidosis may increase the [free Ca 2+] trigger at the outer surface of the SR by inhibiting Na +-Ca 2+ exchange across the sarcolemma.

Calcium and cardiovascular function: Intracellular calcium levels during contraction and relaxation ofmammalian cardiac and vascular smooth muscle as detected with aequorin

Calcium ion (Ca++) plays a central role in excitation-contraction coupling of both cardiac and vascular smooth muscles and have important functional interactions with other cations, including sodium, potassium, and magnesium. Ca++ transients associated with contraction-relaxation cycles of the heart and vasculature can now be recorded directly by use of aequorin, a bioluminescent protein that emits light when it combines with Ca++. After microinjection or chemical loading of aequorin into the sarcoplasm, light output provides an index of intracellular [Ca++]. In cardiac muscle, intracellular Ca++ increases more quickly than tension and decreases toward basal levels by the time peak tension is reached. The calcium transients of working myocardium in both human subjects and other mammalian species appear to be dominated by the release and uptake of Ca++ from intracellular stores under all conditions studied. Drugs and disease states produce marked changes in the amplitude and time course of the Ca++ transient and the corresponding contractile response. In vascular smooth muscle, there are stimulus-specific patterns in intracellular Ca++ associated with tonic contractions. Although Ca++ is related to tension development, the relationship appears to be more complex than that in cardiac muscle. As a result, tension development cannot be used as an index of free Ca++ levels in vascular smooth muscle. Selection of the most effective therapy to reverse a tonic contraction in states of spasm or hypertension may depend on the specific stimulus that caused the increased tone.

Model of calcium movements during activation in the sarcomere of frog skeletal muscle

A model of calcium movement during activation of frog skeletal muscle is described. The model was based on the half sarcomere of a myofibril and included compartments representing the terminal cisternae, the longitudinal sarcoplasmic reticulum, the extramyofibrillar space, and the myofibrillar space. The calcium-binding proteins troponin, parvalbumin, and calsequestrin were present in appropriate locations and with realistic binding kinetics. During activation a time-dependent permeability in the terminal cisternal wall led to calcium release into the myoplasm and its diffusion through the myoplasm longitudinally and radially was computed. After adjustment of three parameters, the model produced a myoplasmic free-calcium concentration that was very similar to those recorded experimentally with calcium indicators. The model has been used to demonstrate the importance of parvalbumin in the relaxation of skeletal muscle, to describe the time course and magnitude of calcium gradients associated with diffusion across the sarcomere, and to estimate the errors associated with the use of aequorin as an intracellular calcium indicator in muscle.

Measurements of intracellular calcium concentration in heart muscle: The effects of inotropic interventions and hypoxia

The use of aequorin as an intracellular calcium indicator in ventricular muscle is described. If the increase of intracellular calcium concentration associated with each contraction (the calcium transient) is measured during inotropic interventions, it is possible to distinguish two classes of inotropic intervention. One class leads to changes in the calcium transient which parallel the changes in tension. The second class leads to changes in the calcium transient and tension which are different in magnitude or direction. In this latter class, changes in the sensitivity of the contractile proteins to calcium are occurring and represent an important part of the inotropic mechanism. When oxidative phosphorylation is inhibited in an isolated mammalian papillary muscle, tension declines but the amplitude of the calcium transients is unaffected. Intracellular acidosis caused by lactate production associated with the increased rate of glycolysis is the probable mechanism of this decline in tension. When both oxidative phosphorylation and glycolysis are inhibited, both the calcium transient and the developed tension decline rapidly to zero. This profound contractile failure may be a consequence of a decline in the free energy of hydrolysis of ATP so that the sarcoplasmic reticulum can no longer accumulate calcium. Hypoxic contractures occur in the absence of significant increases in resting [Ca2+]i and are probably due to rigor produced by the low [ATP].

Use of aequorin to study excitation--contraction coupling in mammalian smooth muscle.

Considerable information is available for intact skeletal and cardiac muscle cells on the role of changes in intracellular Ca2+ (Cai2+) levels during excitation-contraction coupling. Cai2+ levels seem to be similarly important in smooth muscle. However, only two published reports describe the use of indicators to directly measure [Cai2+] in intact smooth muscle cells. One described the use of murexide in molluscan smooth muscle, the other the use of aequorin in single amphibian cells. Their results differed: very long calcium transients were described for the molluscan muscle, compared with very brief ones for the amphibian cells. We present here the first description of the relationship between calcium transients and contraction of whole, mammalian smooth muscle injected with aequorin. The Ca2+ transient we observed has a slow rise, a long duration, and there was no delay between the peak of the Ca2+ transient and the onset of force. This contrasts with the amphibian findings but resembles the molluscan data. We observed a delay between the onset of the increase in [Cai2+] and the onset of the rise in tension which might be explained, at least in part, by the presence of a threshold [Cai2+] for concentration.

Preparation and use of aequorin for rapid microdetermination of Ca 2+ in biological systems.

THE photoprotein aequorin, extracted and purified from the luminescent jellyfish Aequorea aequorea (syn. A. forskalea) (Fig. 1), is triggered to emit visible light in physiological conditions1 by a trace of Ca2+ (and to a less extent by Sr2+, but by no other factor). The combined features of specificity, sensitivity (better than 0.01 µg or better than 10−7 M Ca2+)2,3, speed (faster than a few ms)3–5, non-toxicity and insensitivity of the luminescence to most enzyme inhibitory substances1 offer unique advantages for detecting the presence or changes in concentration of Ca2+ in biological systems—for example, single muscle fibres6–11, isolated mitochondria12,13, single neurones14—and for investigating the action of poisons such as cyanide, tetrodotoxin and so on, on biological processes12–14.