Determining calcium concentration in heterogeneous model systems using multiple indicators

Hyrc, Krzysztof L.; Rzeszotnik, Ziemowit; Kennedy, B.R.; Goldberg, Mark P.

doi:10.1016/j.ceca.2007.02.002

Cited by 10 publications

(6 citation statements)

References 51 publications

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“…[70–72]), a difference often masked by excessive Ca 2+ buffering by an indicator [71, 73, 66] and/or presence of optically unresolved Ca 2+ domains [74]. In either case, the low affinity indicators are far more likely to report actual [Ca 2+ ] i than their high affinity counterparts [71, 73, 74]. The fact that ACR-1-LA, but not ACR-1, detects this difference shows that the indicators maintain their Ca 2+ affinity status in situ , although their apparent dissociation constants (K d ) might vary from those determined in vitro (e.g.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Hyrc

Minta²,

Escamilla³

et al. 2013

Cell Calcium

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Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (~780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd=0.49±0.07 μM; ACR-1) or low affinity (Kd=6.65±0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd =0.38±0.02 nM) or Mg2+ (Kd ~5 mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=6.31±0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

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Section: Resultsmentioning

confidence: 99%

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Hyrc

Minta²,

Escamilla³

et al. 2013

Cell Calcium

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“…Interpreting these signals can be complicated if the region contains a non-uniform distribution of Ca 2+ ions, an issue that should be considered when inferring [Ca 2+ ] i activity from calcium indicator fluorescence intensity (Hyrc et al . 2007 ).…”

Section: Discussionmentioning

confidence: 99%

Fine spatiotemporal activity in contracting myometrium revealed by motion‐corrected calcium imaging

Loftus

Shmygol

Richardson

2014

The Journal of Physiology

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Successful childbirth depends on the occurrence of precisely coordinated uterine contractions during labour. Calcium indicator fluorescence imaging is one of the main techniques for investigating the mechanisms governing this physiological process and its pathologies. The effective spatiotemporal resolution of calcium signals is, however, limited by the motion of contracting tissue: structures of interest in the order of microns can move over a hundred times their width during a contraction. The simultaneous changes in local intensity and tissue configuration make motion tracking a non-trivial problem in image analysis and confound many of the standard techniques. This paper presents a method that tracks local motion throughout the tissue and allows for the almost complete removal of motion artefacts. This provides a stabilized calcium signal down to a pixel resolution, which, for the data examined, is in the order of a few microns. As a byproduct of image stabilization, a complete kinematic description of the contraction–relaxation cycle is also obtained. This contains novel information about the mechanical response of the tissue, such as the identification of a characteristic length scale, in the order of 40–50 μm, below which tissue motion is homogeneous. Applied to our data, we illustrate that the method allows for analyses of calcium dynamics in contracting myometrium in unprecedented spatiotemporal detail. Additionally, we use the kinematics of tissue motion to compare calcium signals at the subcellular level and local contractile motion. The computer code used is provided in a freely modifiable form and has potential applicability to in vivo calcium imaging of neural tissue, as well as other smooth muscle tissue.

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“…ER) and those with lower Ca 2+ (e.g. cytosol) so this technique may underestimate [Ca 2+ ] ER (Hyrc et al. 2007), but such averaging is unlikely to account for the observed decrease in [Ca 2+ ] ER in proteasome inhibitor‐treated cultures.…”

Section: Discussionmentioning

confidence: 99%

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Hyrc

Moulder

et al. 2009

Journal of Neurochemistry

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Cytosolic Ca2+ concentration ([Ca2+]i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage-gated Ca2+ channels restores cytosolic Ca2+ levels and reduces this neuronal death (Snider et al. 2002). We now show that this reduction in [Ca2+]i is transient and occurs early in the cell death process, before activation of caspase-3. Agents that increase Ca2+ influx such as activation of voltage-gated Ca2+ channels or stimulation of Ca2+ entry via the plasma membrane Na-Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage-gated Ca2+ channels and a less robust increase in [Ca2+]i after depolarization. Levels of endoplasmic reticulum (ER) Ca2+ were reduced and capacitative Ca2+ entry was impaired early in the cell death process. Mitochondrial Ca2+ was slightly increased. Preventing the transfer of Ca2+ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca2+ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum (ER) Ca2+, possibly mediated by dysfunction of voltage-gated Ca2+ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.

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Determining calcium concentration in heterogeneous model systems using multiple indicators

Cited by 10 publications

References 51 publications

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Fine spatiotemporal activity in contracting myometrium revealed by motion‐corrected calcium imaging

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

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