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

Abstract

We analyzed preparative and analytical aspects of the dynamic localization of Ca 2 + 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 EDXlstructure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (~lOO-nm thickness) or cilia (-250-nm diameter). We used widely different standards to calibrate the ratio of CaK" net counts in relation to actual [Cal. Calibration curves showed a linear relationship and a detection limit of rCal = 2 mM, while rCal in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr 2 + can rapidly be substituted for Ca 2 + in the medium before and during stimulation, thus allowing one to determine Me 2 + fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca 2 + mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.1999 Academic Press

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction

“…For comparison, synchronous trichocyst exocytosis occurs within 80 ms [40], whereas one ciliary beat requires ∼50 ms (see "Section 3"). While we register considerable changes in Ca K␣ EDX signals upon exocytosis stimulation [38,39], we find no evidence of any artificial redistribution during mock stimulation. Exocytosis stimulation involves depletion of cortical Ca stores (alveolar sacs) superimposed by a Ca 2+ influx during exocytosis stimulation, i.e.…”

“…Briefly, the methodology developed in our lab was used for quenched-flow [40] and for freeze-substitution under conditions allowing to retain Ca 2+ [38,41] …”

“…bound calcium and free Ca 2+ [38,39]. The preparative method we apply is quenched-flow/fast freezing [40] combined with freeze-substitution at 193 K in presence of fluoride as a calcium precipitating ion of very low solubility [38,41], based on previous work by Poenie and Epel [42].…”

Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx

“…Also [Ca] values in stores before .imulation are quite similar in the two systems, i.e. 43 mM i; ASs [38] and 33 mM in skeletal muscle SR [54], both estimated on the basis of wet weight. However, refilling times evidently are widely different between the SR of skeletal muscle and our system.…”

“…For EDX, cells contained in their medium with [Ca2+]o = I mM were stimulated for different times with an equal part of 2 fLM AED (removed in long-time stimulation experiments) in the quenched-flow device [20,23] for cryofixation in melting propane and subsequent freeze-substitution under conditions appropriate to retain Ca 2 + in place according to the method of Poenie and Epel [37], modified as previously described [21,26,29,38]. Then, cells were embedded in Spurr's resin and semithin sections of 0.5 fLm were ana-Iyzed in an analytical EM, type ZeisslLeo EM912 Omega operated in the STEM mode.…”