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

Abstract

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.

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells

“…This is in conformance with other EDX analyses, showing spillover in the opposite direction, i.e., into cilia during secretion stimulation [45], and with the occurrence of ciliary reversal in d4-500r cells during exocytosis stimulation [45,46]. Considering this rapid spread of Ca 2+ in either direction we have averaged in our figures all data points recorded along ciliary shafts.…”

“…In this case, one has to consider that [Ca] increases may be higher than in electrophysiological recordings. In fact, chemical depolarisation over longer times combined with 45 Ca 2+ flux measurements shows much higher Ca 2+ uptake since Ca 2+ flux into cells (unstimulated background subtracted) lasts much longer than recorded during electrical stimulation [22]. This was explained by masked Ca 2+ flux during ongoing depolarisation [13].…”

“…This was explained by masked Ca 2+ flux during ongoing depolarisation [13]. Concomitantly, pilot calculations from 45 Ca 2+ flux data published with chemical depolarisation [22] would result in [Ca]-values approaching those we see by EDX.…”

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources

“…To us, involvement of some somatic Ca 2+ channels appears appealing, not only because we see a Ca 2+ influx component (Fig. 3 column 1a versus column 3a, and column 1b versus column 3b), but also because in another context we have found that spill-over of Ca 2+ from somatic domains into cilia can easily take place, but not in the opposite direction [18]. One has to recall that we record f/f 0 changes not directly in cilia, but in the nearby cell cortex, as frequently done in work with cilia [5,9,48].…”

“…A reversal reaction is easily observed during depolarization-induced activation of voltage-dependent Ca 2+ channels in the ciliary membrane [15,16]. But spill-over from cortical regions during exocytosis stimulation can also produce ciliary reversal [17,18].…”