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S Bolsover

Publications and source records attributed to S Bolsover.

9 recordsLinked to original sources
PubMed2001

Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions.

We have studied the degree to which fluorescent Ca(2+) indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca(2+) indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca(2+) indicator Fura Red; EGFP can be used with the Ca(2+) indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca(2+) signals in cells transfected with fluorescent proteins.

Animals↗
PubMed1999

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.

Annexin A6↗
PubMed1998

Calcium buffering capacity of neuronal cell cytosol measured by flash photolysis of calcium buffer NP-EGTA.

N1E-115 mouse neuroblastoma cells were injected with a calcium buffer/indicator solution to allow both ratiometric measurement of free calcium concentration and the release of calcium ions upon UV flash. The solution contained sulforhodamine, a marker dye used to estimate the volume injected; fluo-3, a calcium indicator, and NP-EGTA, a high affinity calcium-selective buffer that is converted by UV flash to products with negligible calcium affinity. The calcium increase recorded upon UV irradiation (Delta[Ca2+]i) was small for small injection volumes, increased with larger injection volumes, but approached a plateau at the largest injection volumes. From this relation we estimate the buffering capacity of the cytosol as 1700 ions bound per ion free.

Animals↗
PubMed1998

Real-time measurements of calcium dynamics in neurons developing in situ within zebrafish embryos.

We have developed a non-invasive technique to measure intracellular calcium ([Ca2+]i) in neurons growing within intact embryos of the zebrafish (Danio rerio). A single blastomere was injected with a calcium-sensitive fluorescent dye (Calcium Green dextran) between the 32- and 128-cell stage and the embryo imaged between 16 h and 20 h postfertilisation using laser scanning confocal microscopy. Labelled nerve cells from embryos preinjected with dye and dissociated at 16 h showed a fluorescence increase (66+/-22%; n=11) in response to depolarisation with KCl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca2+]i changes in vivo. Imaging of dye loaded cells over 30-min periods in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescence changes distinguishable from noise. However, a subset of neurons within the ventral spinal cord exhibited spontaneous, repetitive [Ca2+]i oscillations which may have a functional significance during neuronal development.

Animals↗
PubMed1998

Nuclear and cytosolic calcium changes in osteoclasts stimulated with ATP and integrin-binding peptide.

Cytosolic calcium modulates the activity of osteoclasts, large multinucleate cells that resorb bone. Nuclear events, such as gene transcription, are also calcium-regulated in these cells, and fluorescence imaging has suggested that calcium signals produced by some stimuli are specifically targeted to, or amplified within, osteoclast nuclei. We used two alternative techniques of dye loading to examine the changes of intracellular calcium induced in rat osteoclasts by three stimuli. Osteoclasts loaded with the calcium indicator Fura-2 by the acetoxymethyl (AM) ester technique appeared to display marked nuclear calcium amplification. During stimulation with integrin-binding peptides, ATP, or high extracellular calcium, fluorescence ratios recorded from the nuclei rose higher than did ratios recorded from extranuclear regions. In contrast, nuclear calcium amplification was not observed after AM loading in the presence of the anion transport inhibitor sulfinpyrazone, nor in osteoclasts injected with Fura-2 conjugated to a high MW dextran. In these cells, nuclear fluorescence ratios were equal to the extranuclear values at all times: upon stimulation by an agonist, the nuclear and cytosolic calcium concentrations increased by the same amount. The calcium changes seen in stimulated osteoclasts can no longer be taken as evidence for the general validity of the phenomenon of nuclear calcium amplification.

Adenosine Triphosphate↗
PubMed1996

Spatial organization of calcium dynamics in growth cones of sensory neurones.

The concentration of calcium ions in the cytosol ([Ca2+]i) has a dominant influence on neuronal development. A [Ca2+]i rise can, depending on the amplitude and location, promote outgrowth or dramatically inhibit it. We have used the fluorescent calcium indicators Fura-2 and Fura-2 dextran to measure [Ca2+]i dynamics in sensory neurones from the adult rat. [Ca2+]i was low and uniform in advancing growth cones, even during specific behaviours such as protrusion, filling and consolidation. A brief train of action potentials caused [Ca2+]i to rise at the extreme leading edge of the growth cone. [Ca2+]i changes in more proximal regions of the growth cone were much smaller. This spatially organized [Ca2+]i change, which may result from a concentration of calcium channels at the growth cone leading edge, is likely to function in spontaneously active regenerating axons in vivo to specifically activate calcium-dependent processes at the growth cone tip.

Animals↗