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1.
The low-affinity fluorescent Ca2+ indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca2+ transient (Δ[Ca2+]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly track Δ[Ca2+]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (ΔF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with furaptra; thus, these indicators do not track Δ[Ca2+] with kinetic fidelity. In contrast, the ΔF time course of Mag-fluo-4 was identical to furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Δ[Ca2+]. Mag-fluo-4's ΔF has a larger signal/noise ratio than furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Δ[Ca2+] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg2+, the amplitude of the Mag-fluo-4 signal, and its calibration in Δ[Ca2+] units, is likely to be more sensitive to variations in [Mg2+] than furaptra's. 相似文献
2.
A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals 总被引:6,自引:0,他引:6
Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators. 相似文献
3.
《Cell calcium》2018
We describe the construction of a simplified, inexpensive lattice light-sheet microscope, and illustrate its use for imaging subcellular Ca2+ puffs evoked by photoreleased i-IP3 in cultured SH-SY5Y neuroblastoma cells loaded with the Ca2+ probe Cal520. The microscope provides sub-micron spatial resolution and enables recording of local Ca2+ transients in single-slice mode with a signal-to-noise ratio and temporal resolution (2 ms) at least as good as confocal or total internal reflection microscopy. Signals arising from openings of individual IP3R channels are clearly resolved, as are stepwise changes in fluorescence reflecting openings and closings of individual channels during puffs. Moreover, by stepping the specimen through the light-sheet, the entire volume of a cell can be scanned within a few hundred ms. The ability to directly visualize a sideways (axial) section through cells directly reveals that IP3-evoked Ca2+ puffs originate at sites in very close (≤a few hundred nm) to the plasma membrane, suggesting they play a specific role in signaling to the membrane. 相似文献
4.
《Cell calcium》2015,57(6):457-466
Mitochondrial Ca2+ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics. Recently developed GCaMP genetically-encoded Ca2+ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca2+ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca2+ dynamics in culture, revealing Ca2+ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca2+ indicators, our results show that mitochondrial Ca2+ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca2+ dynamics on cytosolic Ca2+ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca2+ increase using mito-GCaMP5G and a synthetic organic Ca2+ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca2+ signaling pathway, our results demonstrated that the mitochondrial Ca2+ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease. 相似文献
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Upon the B cell antigen receptor (BCR) ligation Ca2+ mobilization is induced, which is essential for activation of downstream signaling molecules such as MAP kinase. Although synthetic fluorescent chelators such as Fluo-4 and Indo-1 are widely used for Ca2+ measurement upon BCR ligation, they are leaked or unfavorably localized into some organelles with time post loading. To solve these problems, we introduce a genetically encoded fluorescent indicator cameleon which is a fluorescence resonance energy transfer (FRET)-based indicator comprising two fluorescent proteins (CFP and YFP) and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). Here, we demonstrate that cameleon as well as a conventional synthetic Ca2+ indicator enables Ca2+ measurement by flow cytometry clearly upon BCR ligation. In addition, confocal microscopy analysis allows us to detect cameleon-based Ca2+ mobilization in a single cell upon BCR ligation. 相似文献
8.
GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology.The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change.In this study,we determined crystal structures of Ca2+-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its parent version GCaMP5G.Our analyses reveal the structural basis for the outperformance of this newly developed Ca2+indicator.Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and increased fluorescence intensity common to all three versions of GCaMP6.Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity,maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling.Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators. 相似文献
9.
Michiko Yamasaki-Mann Angelo Demuro Ian Parker 《The Journal of biological chemistry》2010,285(32):25053-25061
In addition to its well established function in activating Ca2+ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca2+ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca2+ signals via changes in ER Ca2+ store content, by imaging Ca2+ liberation through inositol trisphosphate receptors (IP3R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca2+] was transiently elevated by applying voltage-clamp pulses to induce Ca2+ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca2+ signals evoked by strong photorelease of IP3, and increased numbers of local Ca2+ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca2+ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca2+ on the IP3R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca2+ may modulate IP3R function, in addition to simply determining the size of the available store and the electrochemical driving force for release. 相似文献
10.
Marnie L. Olson Susan Chalmers John G. McCarron 《The Journal of biological chemistry》2010,285(3):2040-2050
Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). Local Ca2+ release from an InsP3 receptor (InsP3R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP3R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP3-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP3R. Mitochondria may regulate InsP3-mediated Ca2+ signals by operating between or within InsP3R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP3-mediated Ca2+ release by operating within (local) or between (global) InsP3R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F0, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP3R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨM to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by ∼62%. The “fast” Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP3-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level and that mitochondria regulate both local and global InsP3-mediated Ca2+ signals. 相似文献
11.
《Cell calcium》2015,57(6):504-512
Imaging with Ca2+-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca2+ signaling. The spatiotemporal evolution of intracellular free Ca2+ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca2+ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca2+ imaging data, and can be exploited to investigate endogenous Ca2+ buffer properties. Here we extended the use of Ca2+ fluorometry in the characterization of Ca2+ binding molecules within cells, building on a method of titration of intracellular Ca2+ binding sites in situ with measured amounts of Ca2+ entering through voltage-gated Ca2+ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca2+ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca2+ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca2+ buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca2+ inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca2+-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca2+ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca2+ binding species. This method offers a versatile approach to the study of endogenous Ca2+ binding molecules in their physiological milieu. 相似文献
12.
Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells. 相似文献
13.
Jingzhe Guo Jiangman He Katayoon Dehesh Xinping Cui Zhenbiao Yang 《Plant physiology》2022,188(4):2253
As a universal second messenger, calcium (Ca2+) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca2+ signature is hampered by limited tools for simultaneously monitoring dynamic Ca2+ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca2+ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators. We uncovered several Ca2+ signatures in three types of Arabidopsis cells in response to internal and external cues, including rapid oscillations of cytosolic Ca2+ and apical plasma membrane Ca2+ influx in fast-growing Arabidopsis pollen tubes, the spatiotemporal relationship of Ca2+ dynamics in four subcellular compartments of root epidermal cells challenged with salt, and a shockwave-like Ca2+ wave propagating in laser-wounded leaf epidermis. These observations serve as a testimony to the wide applicability of the CamelliA lines for elucidating the subcellular sources contributing to the Ca2+ signatures in plants.A toolset for simultaneous imaging of Ca2+ dynamics in subcellular compartments has uncovered unrecognized Ca2+ signatures in Arabidopsis cells in response to developmental and external cues. 相似文献
14.
Barbara Landolfi Silvana Curci Lucantonio Debellis Tullio Pozzan Aldebaran M. Hofer 《The Journal of cell biology》1998,142(5):1235-1243
Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals. 相似文献
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16.
Ruotian Jiang Martin D. Haustein Michael V. Sofroniew Baljit S. Khakh 《Journal of visualized experiments : JoVE》2014,(93)
Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry. 相似文献
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Ágota Apáti Katalin Pászty Luca Hegedűs Orsolya Kolacsek Tamás I. Orbán Zsuzsa Erdei Kornélia Szebényi Adrienn Péntek Ágnes Enyedi Balázs Sarkadi 《Cellular signalling》2013,25(4):752-759
Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca2 + indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca2 + oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca2 + signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging. 相似文献
19.
B. Walz B. Zimmermann S. Seidl 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1994,174(4):421-431
We have measured Cai at rest and upon light stimulation in the photoreceptors of the honeybee drone microfluorometrically with the fluorescent Ca2+ indicator dyes fura-2, fluo-3 and Ca-green 5N.In darkness, Cai was 90 nM after 5 min of dark adaptation. A saturating light step caused Cai to rise in the bulk cytoplasm to 750 nM within 1 s. Our measurements with the low affinity dye Ca-green 5N showed that bright 1-s light flashes cause a rapid increase in Cai which was graded with stimulus intensity. Ca-green 5N fluorescence reached a peak in about 200 ms, and then decayed to a slightly lower sustained plateau. The fluorescence signal peaked, when the receptor potential was repolarizing from its peak to the plateau. This observation is in agreement with the proposal that the peak-to-plateau transition of the receptor potential is caused by the rise in Cai
From our Fluo-3 measurements it appears that the latency of the Ca2+ increase is by 3–4 ms longer than the latency of the receptor potential elicited by bright 100-ms light flashes. This result provides no support for the proposal that Ca2+ mediates the opening of those membrane channels responsible for the upstroke of the receptor potential.Abbreviations ER
endoplasmic reticulum
- IP3
Inositol 1,4,5-trisphosphate
- SMC
submicrovillar cisternae 相似文献
20.
Jansen MA Badlou BA van Echteld CJ Ruigrok TJ 《Molecular and cellular biochemistry》2000,203(1-2):169-175
Reperfusion of isolated mammalian hearts with a Ca2+-containing solution after a short Ca2+-free period at 37°:C results in massive influx of Ca2+ into the cells and irreversible cell damage: the Ca2+paradox. Information about the free intracellular, cytosolic [Ca2+] ([Ca2+]i) during Ca2+ depletion is essential to assess the possibility of Ca2+ influx through reversed Na+/Ca2+ exchange upon Ca2+ repletion. Furthermore, the increase in end-diastolic pressure often seen during Ca2+-free perfusion of intact hearts may be similar to that seen during ischemia and caused by liberation of Ca2+ from intracellular stores. Therefore, in this study, we measured [Ca2+]i during Ca2+- free perfusion of isolated rat hearts. To this end, the fluorescent indicator Indo-1 was loaded into isolated Langendorff-perfused hearts and Ca2+-transients were recorded. Ca2+-transients disappeared within 1 min of Ca2+ depletion. Systolic [Ca2+]i during control perfusion was 268±54 nM. Diastolic [Ca2+]i during control perfusion was 114±34 nM and decreased to 53±19 nM after 10 min of Ca2+ depletion. Left ventricular end-diastolic pressure (LVEDP) significantly increased from 13±4 mmHg during control perfusion after Indo-1 AM loading to 31±5 mmHg after 10 min Ca2+ depletion. Left ventricular developed pressure did not recover during Ca2+ repletion, indicating a full Ca2+ paradox. These results show that LVEDP increased during Ca2+ depletion despite a decrease in [Ca2+]i, and is therefore not comparable to the contracture seen during ischemia. Furthermore, calculation of the driving force for the Na+/Ca2+ exchanger showed that reversed Na+/Ca2+ exchange during Ca2+ repletion is not able to increase [Ca2+]i to cytotoxic levels. 相似文献