Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale : preparative and analytical aspects as exemplified with Paramecium Cells

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1999
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Hardt, Martin
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Funktionell-ultrastrukturelle Analyse des Exocytose-Prozesses (Sekretausschleusung)
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Journal of Structural Biology. 1999, 128(2), pp. 187-199. ISSN 1047-8477. Available under: doi: 10.1006/jsbi.1999.4188
Zusammenfassung

We analyzed preparative and analytical aspects of the dynamic localization of Ca2+ during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (~100-nm thickness) or cilia (~250-nm diameter). We used widely different standards to calibrate the ratio of CaK net counts in relation to actual [Ca]. Calibration curves showed a linear relationship and a detection limit of [Ca] = 2 mM, while [Ca] in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr2+ can rapidly be substituted for Ca2+ in the medium before and during stimulation, thus allowing one to determine Me2+ fluxes. With our ''model'' cell, Paramecium, we showed that, upon stimulation (causing rapid Ca2+ mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

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570 Biowissenschaften, Biologie
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calcium, cilia, EDX, electron microscopy, exocytos, Paramecium, quenched-flow, secretion
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ISO 690HARDT, Martin, Helmut PLATTNER, 1999. Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale : preparative and analytical aspects as exemplified with Paramecium Cells. In: Journal of Structural Biology. 1999, 128(2), pp. 187-199. ISSN 1047-8477. Available under: doi: 10.1006/jsbi.1999.4188
BibTex
@article{Hardt1999Quant-8546,
  year={1999},
  doi={10.1006/jsbi.1999.4188},
  title={Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale : preparative and analytical aspects as exemplified with Paramecium Cells},
  number={2},
  volume={128},
  issn={1047-8477},
  journal={Journal of Structural Biology},
  pages={187--199},
  author={Hardt, Martin and Plattner, Helmut}
}
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