Three-dimensional intracellular calcium gradients in single human burst-forming units-erythroid-derived erythroblasts induced by erythropoietin.

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Abstract
We have previously shown that the intracellular free Ca2+ increase induced by erythropoietin is likely related to differentiation rather than proliferation in human BFU-E-derived erythroblasts (1989. Blood. 73:1188-1194). Since cell differentiation involves transcription of specific regions of the genome, and since nuclear endonucleases responsible for single strand DNA breaks observed in cells undergoing differentiation are Ca2+ dependent, we investigated whether the erythropoietin-induced calcium signal is transmitted from cytosol to nucleus in this study. To elucidate subcellular Ca2+ gradients, the technique of optical sectioning microscopy was used. After determining the empirical three-dimensional point spread function of the video imaging system, contaminating light signals from optical planes above and below the focal plane of interest were removed by deconvolution using the nearest neighboring approach. Processed images did not reveal any discernible subcellular Ca2+ gradients in unstimulated erythroblasts. By contrast, with erythropoietin stimulation, there was a two- to threefold higher Ca2+ concentration in the nucleus compared to the surrounding cytoplasm. We suggest that the rise in nuclear Ca2+ may activate Ca2(+)-dependent endonucleases and initiate differentiation. The approach described here offers the opportunity to follow subcellular Ca2+ changes in response to a wide range of stimuli, allowing new insights into the role of regional Ca2+ changes in regulation of cell function.