scholarly journals Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin.

1996 ◽  
Vol 135 (1) ◽  
pp. 181-190 ◽  
Author(s):  
A Muto ◽  
S Kume ◽  
T Inoue ◽  
H Okano ◽  
K Mikoshiba
Keyword(s):  
Calcium Transients ◽  
Free Calcium ◽  
Calcium Waves ◽  
Cleavage Furrow ◽  
Cleavage Stage ◽  
Xenopus Embryos ◽  
Calcium Indicator ◽  
Spatio Temporal ◽  
Indicator Dyes ◽  
Insp3 Receptor

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

Measurement of [Ca] in single cells and pH in single organelles by fluorescence microscopy

1988 ◽  
Vol 46 ◽  
pp. 46-47
Author(s):  
F.R. Maxfield ◽  
M.L. Shelanski
Keyword(s):  
Single Cells ◽  
Living Cells ◽  
Free Calcium ◽  
Ion Concentrations ◽  
Calcium Indicator ◽  
Controlled Illumination ◽  
Computer Controlled ◽  
Temporal And Spatial ◽  
Intracellular Sorting ◽  
Indicator Dyes

Microscope spectrofluorometry and digital image processing provide the ability to study changes in ion concentrations in living cells with high temporal and spatial resolution. We have used fluorescein labeled macromolecules to measure the pH of specific endosomal compartments (1-3). The ratio of fluorescence intensities at 450 nm and 490 nm excitation provides a measure of the pH (4). The acidification of endosomes detected by this technique provide an explanation for endosome functions including intracellular sorting of ligands and receptors, release of iron from transferrin, and penetration of viruses and toxins into the cytosol (3).Using the tetracarboxylate calcium indicator dyes synthesized by R. Tsien and his colleagues (5), the same instruments can be used for measuring intracellular free calcium, [Ca2+]i, in single cells. We have used the system to measure [Ca2+]i changes associated with cell motility (6-9).Cells are examined using a Leitz Diavert microscope with a computer-controlled illumination and photometry system.

IMAGING CALCIUM WAVES IN EGGS AND EMBRYOS

1993 ◽  
Vol 184 (1) ◽  
pp. 213-219 ◽  
Author(s):  
I Gillot ◽  
M Whitaker
Keyword(s):  
Latent Period ◽  
Calcium Concentration ◽  
Calcium Influx ◽  
Embryonic Cell ◽  
Calcium Wave ◽  
Free Calcium ◽  
Calcium Indicator ◽  
Free Calcium Concentration ◽  
Indicator Dyes ◽  
Indicator Dye

Sea urchin eggs and those of most other deuterostomes are activated at fertilization by an increase in cytoplasmic free calcium concentration ([Ca2+]i) that triggers the onset of the embryonic cell division cycles. We can image the calcium wave using fluorescent calcium indicator dyes and confocal microscopy. There are two components to the [Ca2+]i increase at fertilization. The first is due to a rapid calcium influx caused by a calcium action potential; this leads to a small increase in [Ca2+]i just beneath the plasma membrane with spherical symmetry. After a latent period of some 15 s, there is a second large and rapid increase in [Ca2+]i localized to the region of sperm-egg contact: during the latent period [Ca2+]i does not change but within 1 s of the end of the latent period [Ca2+]i reaches 2 micromolar. The calcium wave then spreads across the egg with a velocity of 5 micrometre s-1. Behind the advancing wavefront, the calcium concentration is uniformly high, even within the egg nucleus, though there are no indications that intranuclear calcium concentration differs from [Ca2+]i. [Ca2+]i falls uniformly towards resting levels over the next 500 s. In cases where there is an apparent inhomogeneity in [Ca2+]i in either the cortex or the nucleus, we find that the calcium indicator dye is inhomogeneously distributed. This appears to be due to uptake of the indicator dye (Fluo-3), probably into mitochondria. The artefact can be avoided by using a dextran-conjugated dye.

scholarly journals Calcium spikes accompany the cleavage furrow ingression and cell separation during fission yeast cytokinesis

2020 ◽  
Author(s):  
Abhishek Poddar ◽  
Oumou Sidibe ◽  
Aniruddha Ray ◽  
Qian Chen
Keyword(s):  
Fission Yeast ◽  
Embryonic Cell ◽  
Calcium Transients ◽  
Cleavage Furrow ◽  
Cell Integrity ◽  
Calcium Spikes ◽  
Division Plane ◽  
Calcium Indicator ◽  
Intracellular Calcium Level ◽  
Ring Constriction

AbstractThe role of calcium signaling during cytokinesis has long remained ambiguous. The studies of embryonic cell division discovered that calcium concentration increases transiently at the division plane just before the cleavage furrow ingression, leading to the hypothesis that these calcium transients trigger the contractile ring constriction. However, such calcium transients have only been found in animal embryos and their function remains controversial. Here we explored cytokinetic calcium transients in the model organism fission yeast. We adopted GCaMP, a genetically encoded calcium indicator, to determine the intracellular calcium level. We validated GCaMP as a highly sensitive calcium indicator which allowed us to capture the calcium transients stimulated by osmotic shocks. To identify calcium transients during cytokinesis, we first identified a correlation between the intracellular calcium level and cell division. Next, we discovered calcium spikes at the start of the cleavage furrow ingression and the end of the cell separation using time-lapse microscopy to. Inhibition of these calcium spikes slowed down the furrow ingression and led to frequent lysis of the daughter cells. We conclude that like the larger animal embryos fission yeast triggers cytokinetic calcium transients which promote the ring constriction and daughter cell integrity (194).Highlight summary for TOCCalcium rises transiently at the division plane during embryonic cell cytokinesis, but the conservation and function of such calcium transients remain unclear. We identified similar calcium spikes during fission yeast cytokinesis and demonstrated that these spikes promote the contractile ring constriction and the daughter cell integrity (257).

Measurement of calcium transients in frog muscle by the use of arsenazo III

1977 ◽  
Vol 198 (1131) ◽  
pp. 201-210 ◽  
Keyword(s):  
Calcium Release ◽  
Calcium Transients ◽  
Arsenazo Iii ◽  
Free Calcium ◽  
Muscle Fibres ◽  
Bathing Solution ◽  
Intracellular Injection ◽  
Calcium Indicator ◽  
Skeletal Muscle Fibres ◽  
Indicator Dye

Intracellular injection of the calcium indicator dye arsenazo III was used to measure the calcium transients occurring during depolarization of frog skeletal muscle fibres. A quantitative estimate of the rise in intracellular free calcium during a twitch was made, and the relation between membrane potential and calcium release was examined. The results also indicate that calcium in the bathing solution plays no important part in the generation of calcium transients during single twitches.

scholarly journals Timing of Ca2+ Release from Intracellular Stores and the Electrical Response of Limulus Ventral Photoreceptors to Dim Flashes

2000 ◽  
Vol 115 (6) ◽  
pp. 735-748 ◽  
Author(s):  
Richard Payne ◽  
James Demas
Keyword(s):  
Latent Period ◽  
Electrical Response ◽  
Cyclopiazonic Acid ◽  
Receptor Potential ◽  
Ion Concentration ◽  
Free Calcium ◽  
Flash Intensity ◽  
Stochastic Variation ◽  
Highly Correlated ◽  
Indicator Dyes

Light-induced release of Ca2+ from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca2+ indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 μm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Cai) after flashes delivering between 10 and 5 × 105 effective photons. Peak Cai increased with flash intensity to 138 ± 76 μM after flashes delivering ∼104 effective photons, while the latent period of the elevation of Cai fell from ∼140 to 21 ms. The onset of the light-induced elevation of Cai was always highly correlated with that of the receptor potential. The time for Cai to exceed 2 μM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 ± 6.4 ms). Cai was also measured during steps of light delivering ∼105 effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Cai were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Cai and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Cai resulted from Ca2+ release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca2+ pumps, but not by removal of extracellular Ca2+ ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca2+ ions that appears necessary for a highly amplified electrical response to dim flashes.

scholarly journals Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos

2000 ◽  
Vol 113 (19) ◽  
pp. 3519-3529 ◽  
Author(s):  
C. Leclerc ◽  
S.E. Webb ◽  
C. Daguzan ◽  
M. Moreau ◽  
A.L. Miller
Keyword(s):  
Xenopus Laevis ◽  
Calcium Signalling ◽  
Neural Induction ◽  
Calcium Transients ◽  
Free Calcium ◽  
Treated Animal ◽  
Cell Stage ◽  
Subsequent Formation ◽  
Xenopus Laevis Embryos

Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.

scholarly journals Intracellular free calcium oscillates during cell division of Xenopus embryos.

1991 ◽  
Vol 112 (4) ◽  
pp. 711-718 ◽  
Author(s):  
N Grandin ◽  
M Charbonneau
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Volume ◽  
Intracellular Ph ◽  
Calcium Ions ◽  
Volume Ratio ◽  
Mean Amplitude ◽  
Free Calcium ◽  
Periodic Oscillations ◽  
Xenopus Embryos

In Xenopus embryos, previous results failed to detect changes in the activity of free calcium ions (Ca2+i) during cell division using Ca2(+)-selective microelectrodes, while experiments with aequorin yielded uncertain results complicated by the variation during cell division of the aequorin concentration to cell volume ratio. We now report, using Ca2(+)-selective microelectrodes, that cell division in Xenopus embryos is accompanied by periodic oscillations of the Ca2+i level, which occur with a periodicity of 30 min, equal to that of the cell cycle. These Ca2+i oscillations were detected in 24 out of 35 experiments, and had a mean amplitude of 70 nM, around a basal Ca2+i level of 0.40 microM. Ca2+i oscillations did not take place in the absence of cell division, either in artificially activated eggs or in cleavage-blocked embryos. Therefore, Ca2+i oscillations do not represent, unlike intracellular pH oscillations (Grandin, N., and M. Charbonneau. J. Cell Biol. 111:523-532. 1990), a component of the basic cell cycle ("cytoplasmic clock" or "master oscillator"), but appear to be more likely related to some events of mitosis.

scholarly journals Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 231-233 ◽  
Author(s):  
PD Lew ◽  
C Wollheim ◽  
RA Seger ◽  
T Pozzan
Keyword(s):  
Chronic Granulomatous Disease ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Granulomatous Disease ◽  
Chemotactic Peptide ◽  
Intact Cells ◽  
Calcium Indicator ◽  
Free Calcium Concentration

Abstract Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

Measurement and Interpretation of Cytoplasmic [Ca2+] Signals From Calcium-Indicator Dyes

Physiology ◽  
2000 ◽  
Vol 15 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Stephen M. Baylor ◽  
Stephen Hollingworth
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Skeletal Muscle Fibers ◽  
Calcium Indicator ◽  
Indicator Dyes

Ca2+-indicator dyes are widely used in biology yet difficult to characterize inside cells. Studies in skeletal muscle fibers provide important information about indicator behavior and about Ca2+ signaling within the cytoplasm.

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