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

Abstract

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.

A broad spectrum of actin paralogs inParamecium tetraureliacells display differential localization and function

“…A similar structure/function effect is discussed for mammalian cells whose cortical Ca 2+ stores may be positioned by F-actin (Rosado and Sage, 2000;Wang et al, 2002;Roderick and Bootman, 2003;Turvey et al, 2005). Although we could not show such an effect in response to cytochalasin in Paramecium (Mohamed et al, 2003), this may be due to the involvement of drug-insensitive isoforms. Any disturbance, e.g.…”

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

“…A quite similar phenomenon occurs during synchronous exocytosis, also analysed by EDX [30]. Considering the slow kinetics of the SERCA-type Ca 2+ -pump in general [4] and in Paramecium in particular [14,28,49] this can impossibly explain the rapid Ca 2+ -uptake and release during overstimulation of ciliary reversal.…”

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

“…Following this, these cells have been stimulated with GTP. The time elapsed between the two stimuli was only ≤3 min, which is much too short to allow for any significant refilling [51]. Under these conditions, during GTP stimulation, we observe oscillating f/f 0 signals with a large first peak (Figs.…”

“…During stimulation of synchronous exocytosis by AED, [Ca] in alveolar sacs is reduced to ∼20% of its original value within 1 s [44]. We also know that refilling is unexpectedly slow, with a half-time of ∼60 min [51]. Although [Ca] may be less high in the ER, it deserves interest in the context of GTP-mediated Ca 2+ signalling because it approaches ciliary bases where no alveolar sacs occur [52].…”

“…No Ca 2+ release from alveolar sacs is achieved, neither with ryanodine, nor with InsP 3 [54], but with the polyamine secretagogue AED [44,53]. These data, together with the unexpectedly slow refilling [51], shed some doubt on conclusions derived from experiments with SERCA inhibitors, assuming a role for alveolar sacs in Ca 2+ release and uptake cycles during GTP stimulation [24]. Considering the endogenous Ca 2+ signalling component which we assume for GTP activation, the ER is a more likely candidate than alveolar sacs (see below), unless one would concede to alveolar sacs an unexpected hybrid character.…”