We asked to what extent Ca 2+ signals in two different domains of Paramecium cells remain separated during different stimulations. Wild-type (7S) and pawn cells (strain d4-500r, without ciliary voltage-dependent Ca 2+ -channels) were stimulated for trichocyst exocytosis within 80 ms by quenched-flow preparation and analysed by energy-dispersive X-ray microanalysis (EDX), paralleled by fast confocal fluorochrome analysis. We also analysed depolarisation-dependent calcium signalling during ciliary beat rerversal, also by EDX, after 80-ms stimulation in the quenched-flow mode. EDX and fluorochrome analysis enable to register total and free intracellular calcium concentrations, [Ca] and [Ca 2+ ], respectively. After exocytosis stimulation we find by both methods that the calcium signal sweeps into the basis of cilia, not only in 7S but also in pawn cells which then also perform ciliary reversal. After depolarisation we see an increase of [Ca] along cilia selectively in 7S, but not in pawn cells. Opposite to exocytosis stimulation, during depolarisation no calcium spill-over into the nearby cytosol and no exocytosis occurs. In sum, we conclude that cilia must contain a very potent Ca 2+ buffering system and that ciliary reversal induction, much more than exocytosis stimulation, involves strict microdomain regulation of Ca 2+ signals.

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ("trichocysts") exocytosis in Paramecium involves a Ca2+ -influx" superimposed to Ca2+ -release from cortical stores ("alveolar sacs" (ASs). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca2+]o in the medium, from~20-25% at [Ca 2 +]0 :::0.25 j.LM to 100% at [Ca 2 +]0 between 2 and 10 j.LM, i.e. close to the range of estimated local intracellular [Ca2+] during membrane fusion. Next, we analyzed Ca2+ -specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca2+ -signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca2+ -signal (due solely toCa2+ released from cortical stores) recovered with 11/2~65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca2+0),11/2 for store refilling was similar,~60 min. These values are similar to previously measured 45Ca2+ -uptake by isolated ASs. In sum we find, (i) exogenous Ca2+ increases exocytosis/membrane fusion performance with EC50 = 0.7 j.LM, (ii) Ca 2 +-signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.

As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca 2+ influx selectively via ciliary voltage-dependent Ca 2+ -channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca 2+ has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (≥30 ms) smaller than one ciliary beat (∼50 ms) and beyond. Particularly under overstimulation conditions (∼10 −5 M Ca 2+ before, 0.5 mM Ca 2+ during stimulation) we find in cilia a [Ca] peak at ∼80 ms and its decay to near-basal levels within 110 ms (90%) to 170 ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at ∼100 ms, with a decay to original levels within 170 ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca 2+ through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells.