Imaging of Ca²⁺ transients induced in Paramecium cells by a polyamine secretagogue

Since the development of Ca²⁺-sensitive fluorochromes (Tsien, 1988) it has been documented with many cells that a transient increase in free Ca²⁺ concentration, [Ca²⁺]_i, in the cell cortex accompanies stimulated exocytosis. Ca²⁺ may, thus, be an important regulator of exocytosis (Tsien and Tsien, 1990; Burgoyne and Morgan, 1993; Cheek and Barry, 1993; Bootman and Berridge, 1995; Clapham, 1995).

Although the Paramecium cell is an established model system to analyse several aspects of triggered exocytosis (Plattner et al., 1991), also with the implication of Ca²⁺ as a regulator, [Ca²⁺]_i transients have not been documented so far because of a variety of severe technical problems. (i) As a ciliated protozoan this cell is highly mobile. (ii) During vigorous trichocyst discharge cells can be locally deformed and displaced. (iii) They do not readily take up fluorochrome esters. (iv) Once incorporated, fluorochromes may be sequestered into digesting vacuoles.

We now have immobilized cells for microinjection with different fluorochromes, not only for conventional but also for confocal laser scanning microscopy (CLSM) with high time resolution. For the technical reasons mentioned we have included non-secretory strains in our analyses. Thus, we can show for the first time calibrated resting values, [Ca²⁺]_i^rest, and rapid local transients during activation, [Ca²⁺]_i^act, by the secretagogue aminoethyldextran (AED). Within the cell cortex, [Ca²⁺]_i^act may culminate very rapidly after stimulation, i.e. at ≤2 seconds by conventional analyses and even much faster according to CLSM. Then rapid spillover into deeper cell layers occurs, followed by slow recovery. In wild-type cells a cortical [Ca²⁺]_i^act transient rise to >1 to 10 μM is accompanied by fast synchronous trichocyst exocytosis. Experiments with some exocytosis-incompetent mutants show similar Ca²⁺ signals. Microinjected chelators of different Ca²⁺-affinity indicate that [Ca²⁺]_i may have to rise locally well beyond 1 μM for exocytosis to occur.

Cultures of Paramecium tetraurelia, wild-type (strain 7S) and trichocyst-free mutant cells (trichless, tl; Pollack, 1974) were grown at 25°C; strain nd9 with docked but non-dischargeable trichocysts (Beisson et al., 1976) was grown at non-permissive 28°C, all in dried lettuce medium monoxenically inoculated with Enterobacter aerogenes containing [Ca²⁺] = 0.5×10⁻⁴ M. Mutants were grown to early stationary phase, 7S cells to mid-logarithmic phase and concentrated before use to 10⁵ cells/ml by low-speed centrifugation.

Individual cells were transferred under a dissecting microscope into 1 μl droplets of culture medium on a siliconized glass slide and covered with paraffin oil to prevent evaporation. In an inverted microscope (ICM 405, Zeiss, Oberkochen, Germany) a sucking capillary of ∼10 μm inner diameter was used to aspirate excess fluid and, thus, to immobilize isolated cells for microinjection. Ca²⁺-sensitive fluor-ochromes, dissolved in 10 mM Tris-HCl, pH 7.2, were microinjected as indicated by Kersken et al. (1986), to yield a final concentration of 50 (Fura Red) to 100 μM (Fura-2, Fluo-3). The volume injected was ∼10⁻¹¹ l, i.e. ∼10% of cell volume. All intracellular concentrations indicated are estimated final concentrations. In controls, equal distribution throughout the cell body was ascertained, e.g. after 0.5 to 2 minutes, by confocal laser scanning microscopy, CLSM, as described below. A solution of 1% AED was pressure ejected (0.1 seconds) from a second capillary and the dilution factor, yielding a EC100 = 0.05% (Plattner et al., 1984, 1985), was estimated by free fluorescein added. Usually final [Ca²⁺]e was 50 μM in trigger experiments.

[Ca²⁺]i increase during AED stimulation was eventually manipulated by injection of BAPTA or 4,4′-difluoro-BAPTA (Molecular Probes, Eugene, OR). Alternatively, we have injected CaCl2 after loading cells with Fura Red (for details, see Results).

Fluorochromes used, Fura-2, Fura Red and Fluo-3, were all from Molecular Probes and were all microinjected. Fura-2 and Fura Red were detected in the ICM 405 microscope (Zeiss) as follows. Excitation light was selected from a 50 W mercury lamp (HBO 50, Osram) by a 10 nm bandpass interference filter (360 and 380 nm for Fura-2, 440 and 490 nm for Fura Red; from L.O.T., Darmstadt, Germany) mounted on a home-made filter wheel (mechanically separated from the microscope) and reflected into the objective (63/1.3 oil, Zeiss) using a dichroic mirror (FT 460 for Fura-2, FT 510 nm for Fura Red, Zeiss). Emission was collected by the objective and passed the dichroic and barrier filter (LP 470 nm for Fura-2, LP 590 nm for Fura Red, Zeiss) before reaching the image intensifier of a moonlight camera from Panasonic (AVT Horn, Aalen, Germany). Diffusion of Fura Red into outermost cell layers was analysed by zseries in a CLSM operated at 488 nm excitation (50 mW Ar-laser output) and a longpass filter of 510 nm.

Fluorescence was excited for 250 milliseconds at each wavelength and, during turning of the filter wheel, phase contrast images were eventually taken to control cell behaviour. One cycle consisting of two fluorescence and one phase contrast image, thus, lasted 2 seconds.

Conventional fluorescence images were digitized on a 486 PC equipped with a framegrabber and ratio-imaging was performed with Image-1 software (Universal Image Cooperation, West-Chester, PA, USA) resulting in a false-colour picture of the cell. Alternatively average fluorescence intensities were measured at both excitation wavelengths and for each situation the ratio was calculated (Sigma Plot, Jandel Scientific Software, Corte Madera, CA, USA) and converted to [Ca²⁺] according to calibration curves for [Ca²⁺]. These were obtained by adding different amounts of CaCl2 to a solution containing 10 mM Na2Pipes, 50 mM KCl, 5 mM NaCl and 10 mM EGTA, pH 7.0. [Ca²⁺] was calculated using a software program (Föhr et al., 1993) for conditions closely resembling ionic conditions in Para-mecium cells (Lumpert et al., 1990; see Results). To mimic optical path conditions, calibration solutions with fluorochromes were filled in rectangular microcapillaries of 50 μm lumen (thickness of a Paramecium cell) for [Ca²⁺] calibration in the fluorescence microscope.

Semi-quantitative Ca²⁺ measurements were done with a confocal laser scanning system (Odyssey, Noran Instruments, Middleton, WI, and Bruchsal, Germany), with 33 frames/second acquisition rate by optoacustic beam deflection. This was mounted on an inverted microscope (Axiovert form Zeiss) equipped with an oil immersion objective (63/1.4, Zeiss). In Fluo-3 injected cells a rise of [Ca²⁺] was shown by an increase in fluorescence intensity at ≥514 nm referred to the fluorescence before stimulation (f/fo), with excitation at 488 nm, as used in other CLSM studies (Bacskai et al., 1995). In some experiments alternate acquisition of fluorescence images and images generated by the laser light passing the cell was used to correlate Ca²⁺ signals with exocytosis.

Attempts to incubate cell suspensions with fluorochrome esters were unsuccessful because of lack of uptake. Injection of fluorochrome salt into immobilized cells was achieved by isolation in a small droplet and cautious reduction of the volume, to mechanically stop swimming activity, as described in Materials and Methods. Such cells could be locally triggered with AED through a closely apposed capillary by pressure ejection and superfusion. When mimicked by buffer application only these procedures did not cause any important changes in global [Ca²⁺]_i (Table 1-B) with different fluorochromes.

Because of lack of any information with our system and since we had to compare conventional double wavelength with single wavelength CLSM recording, we had to try out different fluorochromes. Calibration of Fura Red revealed suitability for our cells in a linear range between ≥20 nM and ≤5 μM, i.e. close to the manufacturer’s specifications, and a similar curve was obtained for Fura-2 (not shown). For CLSM analysis a fluorochrome with lower Ca²⁺ affinity was advised since within short periods relatively high [Ca²⁺]_i^act had to be expected. Fluo3 is reported as suitable for 30 nM-4 μM (Minta et al., 1989).

K_d values under ionic conditions closely resembling our cells (Lumpert et al., 1990) are 130 nM for Fura Red (manufac-turer’s data), 170 nM for Fura-2 (Pethig et al., 1989) and 400 nM for Fluo-3 (Minta et al., 1989). An important prerequisite is even distribution of fluorochromes throughout the interior of the cells, up to the outermost layers. This is shown specifically for Fura Red by CLSM single wavelength recording (Fig. 1). While most fluorochromes used are excluded from pulsatile and digesting vacuoles as well as from trichocysts (Fig. 1), this is not true for Fluo-3 (see below). Sequestration into some of the digesting vacuoles is finished within ∼1 minute after injection and does not change subsequently, i.e. when [Ca²⁺]_i analysis is performed.

Fura-2 registrations showed [Ca²⁺]_i^rest = 80 nM in 7S cells, but Fura-2 proved problematic because of notorious photoactivation (Grapengiesser, 1993) resulting in exocytosis at a global threshold of [Ca²⁺]_i ≥400 nM (not shown). We then tested Fura Red and Fluo-3 for suitability. In the three strains analysed [Ca²⁺]_i^rest remains rather constant around 50 nM over 20 seconds (Table 1-A), i.e. the usual [Ca²⁺] transient recording time, and even beyond 1 minute. This largely excludes cell impairment. Using Fluo-3 with CLSM in 7S cells, only a small fluorescence change was observed during continuous laser illumination over 20 seconds (Table 1-A) or more.

Mechanical restraints were required for analysing 7S cells since they move quickly particularly during exocytosis (see Introduction). Therefore, we started with mutants for controls. Both, nd9-28°C and tl cells show rapid [Ca²⁺]_i increase at the site of AED application (Figs 2 and 3 top). As AED spreads around a cell, a cortical [Ca²⁺]_i transient also spreads over increasing cell surface areas. Simultaneously Ca²⁺ sweeps the cell interior. In the examples shown [Ca²⁺]_i^rest may be re-established within a short time, but this may mostly last ≥20 seconds. Mock stimulation by buffer application causes no remarkable [Ca²⁺]_i change (Figs 2 and 3 bottom; Table 1-B).

[Ca²⁺]_i transients are evaluated quantitatively in Fig. 4 for nd9-28°C and in Fig. 5 for tl cells, respectively. Analysis of global changes (Table 2) blurrs actual dynamics, although it facilitates estimation of the total Ca²⁺ load in cells during stimulation (see Discussion). Whereas global values culminate at 4 seconds, about 3 to 5 times the basal [Ca²⁺]_i values, spatially resolved evaluation shows that [Ca²⁺]_i^act peaks already 2 seconds after AED application in nd9-28°C cells (Fig. 4). This is at the time resolution limited by filter change. Surprisingly data obtained with tl cells are much more variable (Fig. 5). With both strains analysed, more or less pronounced Ca²⁺ transients reach deeper cell regions and [Ca²⁺]_i^rest may not yet be fully re-established after 20 seconds (Table 2). Time required for filter change did not allow similar analyses with fast moving 7S cells.

Therefore, we switched to high time resolution, using Fluo-3 injection in conjunction with CLSM. First we analysed nd9-28°C cells for comparison with previous double wavelength recordings and then Fluo-3 loaded 7S cells to visualize Ca²⁺ transients in spite of cell displacement and deformation during trichocyst discharge (see Introduction). Semi-quantitative evaluation of signals was by the ratio f/f_o, i.e. fluorescence after stimulation vs values at rest, when images were taken in minimal intervals of 33 milliseconds (see Materials and Methods).

Fig. 6 shows [Ca²⁺]_i increase in a nd9-28°C cell, again starting from the site of AED application, from where the signal spreads laterally and inside the cell. Cortical signal increases within 1 second to about 9 times basal values. We attribute this faster increase, though to relative values approaching those reported in Fig. 4, to improved resolution with CLSM. Concomitantly the line scan evaluation in Fig. 6 (bottom) shows continuous spillover into deeper cell regions. From this we could expect realistic data for 7S cells.

To obtain methodically unimpaired semi-quantitative data for 7S cells we used different approaches. In a first approach we applied only a brief AED stimulus (which remained locally restricted according to extracellular emission from fluorescein added), thus reducing cell dislocation (Fig. 7). Under these conditions cortical [Ca²⁺]_i^act rose, though only locally, considerably above basal levels and the Ca²⁺-signal was paralleled by local trichocyst exocytosis.

We then mimicked conditions of mutant cells by producing tl phenocopies from 7S cells. We depleted Fluo-3 loaded 7S cells of their trichocysts by an AED stimulus, before exposure to a second AED stimulus 10 minutes later. By this time interval Ca²⁺ homeostasis can be reasonably assumed to be re-established. The second stimulus also results in considerable cortical [Ca²⁺]_i increase by a factor of 3 (Figs 8 and 9). From [Ca²⁺]_i^rest = 68 nM determined conventionally (Table 2) one may derive a cortical peak value of [Ca²⁺]_i^act = 210 nM in 7S cells. (This smaller response may be due to only partial store refilling). These double trigger experiments also showed Ca²⁺ spillover into central cell regions, just as with mutants.

Can Ca²⁺ injection induce exocytosis and can exocytosis be suppressed by injected Ca²⁺ chelators? In Fig. 10 the Ca²⁺ injected would amount to 500 μM, disregarding cytosolic binding and pumping activities. Actual [Ca²⁺]_i values recorded in this typical experiment, however, are only 1.03 μM (at 2 seconds around injection needle) and 685 or 900 nM in cell cortex after 2 or 4 seconds, respectively, where it entails trichocyst exocytosis. This heavy Ca²⁺ load is only slowly down-regulated to resting level (50 nM at t_o) since global concentration is still 240 nM after 20 seconds. These experiments showed that: (i) the Paramecium cytoplasm has a very high Ca²⁺ buffering and/or sequestration capacity; (ii) that trichocyst release may require ≥1 μM Ca²⁺, possibly up to 10 μM; and finally (iii) that micromolar [Ca²⁺]_i can cause local cell contraction. A similar threshold [Ca²⁺]_i^act value of >1 μM was estimated by the effect of AED application after injection of Ca²⁺ buffers (Table 3). While BAPTA incompletely inhibited exocytosis, the lower affinity derivative, 4,4′-F₂-BAPTA blocked exocytosis in 5 out of a total of 8 cells.

Up to now serious methodical problems have impeded imaging of [Ca²⁺]_i transients during exocytosis in Paramecium cells, e.g. cell movement, deformation and dislocation during trichocyst release, lack of penetration of fluorochromes, and their intracellular sequestration. We now have largely overcome these problems, however, some blurring of cortical [Ca²⁺] still may impair our measurements, even with fast CLSM. As a semi-quantitative method this also required, in parallel, quan-titative double wavelength measurements to make data more reliable. During AED application this could be achieved best with exocytosis incompetent strains.

Fluorochromes were found to rapidly distribute all over the cell, although we can neither establish their availability in the narrow subplasmalemmal space nor can we record signals selectively from this critical region. Measurements with Fura-2 support values for [Ca²⁺]_i^rest obtained with Fura Red, but it caused photoactivation and exocytosis at [Ca²⁺]_i ≥400 nM. After AED-stimulation similar activation values were measured with Fura Red (see Results). Hence, Fura-2 had to be abandoned. Fluo-3 was most appropriate for CLSM, but it was partially sequestered into vacuoles. Since trapping occurred before activation analyses, it should not have affected our recordings.

As discussed below, the data we obtained for Ca²⁺-signalling during secretagogue application are well comparable with other systems (for review, see Bootman and Berridge, 1995).

Ironically Paramecium was among the first cells whose Ca channels could be well defined by electrophysiology (voltagedependent channels in ciliary membranes; Eckert and Brehm, 1979), while Ca²⁺ imaging had to wait up to now. Still we do not yet know the molecular identity of Ca channels involved in exocytosis. Yet we can exclude ciliary Ca channels (Plattner et al., 1984) and, thus, conclude that any Ca²⁺ influx, in parallel to store activation, takes place along the somatic cell membrane (Erxleben and Plattner, 1994).

Estimations of [Ca²⁺]_i^rest in Paramecium by electrophysiology (Naitoh and Kaneko, 1972; Nakaoka et al., 1984; Machemer, 1989) are slightly above our values. This explains the occurrence of a Ca²⁺ influx even at [Ca²⁺]_e = 40 μM in response to AED (Kerboeuf and Cohen, 1990), i.e. close to the conditions we normally work with (50 μM). We now show a rapid increase in cortical [Ca²⁺]_i, in parallel to AED stimulated trichocyst exocytosis, though the time correlation we can achieve, even with CLSM, is just at the limits of AED induced exocytosis. According to our previous quenched-flow/ultrastructural analyses trichocyst release is completed within 80 milliseconds (Knoll et al., 1991).

As discussed below, we have to envisage two sources of Ca²⁺: (i) influx from the medium; and (ii) mobilisation from the vast subplasmalemmal pools, i.e. the alveolar sacs (Stelly et al., 1991, 1995). How could these two components account for the Ca²⁺ transients observed?

During AED stimulation, ⁴⁵Ca²⁺ flux measurements showed uptake of 5 fMoles Ca²⁺ per cell (Knoll et al., 1992). Based on analytical microscopic methods, about half of the Ca stored in alveolar sacs, in a concentration of ∼5 mM, may be mobilised during AED stimulation (Stelly et al., 1995). Ca²⁺ influx would necessarily increase cortical [Ca²⁺]_i primarily in the ∼15 nm narrow subcortical space between cell membrane and alveolar sacs (Plattner et al., 1991). According to morphometric evalu-ations this subplasmalemmal space has a volume of 160 μm³ (Erxleben et al., 1997). Possibly Ca²⁺ released from alveolar sacs could also flow selectively into this space. If one would disregard Ca²⁺ diffusion and binding, as well as sequestration and extrusion, Ca²⁺ influx and mobilisation would result in concentrations in the subplasmalemmal compartment ∼10⁵ times above measured values (Erxleben et al., 1997, and this paper), thus by far reversing gradients. This is rapidly counteracted and requires further explanation. [Ca²⁺] transients might be too rapid to be detected even by fast CLSM with a ‘fast’ fluorochrome. Although time points achieved with Fluo-3/CLSM are within the range of synchrony of AED induced exocytosis (Knoll et al., 1991), signal build cannot compete with recordings of Ca²⁺-activated currents which accompany individual AED stimulated exocytotic events (Erxleben and Plattner, 1994), with a rise time of 7 milliseconds and a half width of 21 milliseconds (Erxleben et al., 1997). As found with motor nerve endings by electrophysiology actual [Ca²⁺]_i^act values precisely at exocytosis sites may be as high as several 100 μM for milliseconds (Llinás et al., 1995; Zucker, 1993). Although similar values would appear possible (see above), fluorescence data obtained with our system show a greater resemblance to chromaffin cells with ≤10 μM [Ca²⁺]_i^act within 30 milliseconds (Chow et al., 1994), Purkinje cells with ≤3 μM within 100 milliseconds (Eilers et al., 1995) or cerebellar granule cells with several 100 nM [Ca²⁺]_i^act during a similar time range (Regehr and Atluri, 1995). Additionally any buildup of subplasmalemmal [Ca²⁺] may be quickly counterregulated by the mechanisms mentioned and, thus, restrict the lifetime of current signals. With this regard the spillover into the cell body which we observe may be of particular functional interest (see below).

All these restrictions in interpreting actual [Ca²⁺]_i^act values at exocytosis sites, however, concern any Ca²⁺ imaging study (Morgan, 1993; Ross, 1993) and, to some extent, even electrophysiological measurements (Augustine and Neher, 1992; Bootman and Berridge, 1995).

To compensate for these problems one can inject Ca²⁺ buffers of different sensitivities and kinetics before stimulation. For instance, BAPTA has a lower K_d than its low affinity difluoro-derivative (Table 3). Due to very rapid Ca²⁺ binding kinetics this allows us to estimate local [Ca²⁺]_i^act well within <1 milliseconds in a region <1 μm around a Ca²⁺ source, as cal-culated for BAPTA (Adler et al., 1991; Stern, 1992) or for 4,4′-F₂-BAPTA (Speksnijder et al., 1989). Data in Table 3 give pilot values from which a requirement of [Ca²⁺]_i^act of 1 μM or somewhat more can be derived. This is compatible (i) with our [Ca²⁺] imaging values, and (ii) with the threshold free Ca²⁺-concentration causing trichocyst exocytosis after Ca²⁺ injection. None of these data can exclude the possibility, however, that a very locally higher [Ca²⁺]_i^act may occur, particularly since Ca²⁺ buffers have recently been shown to entail some uncertainties on their own (Borst and Sackmann, 1996; Neher, 1996).

From the volume of a Paramecium cell (0.7×10⁻¹⁰ l; Erxleben et al., 1997) and the data for Ca²⁺ influx and pool mobilisation (see above), a global [Ca²⁺]_i^act = 2 mM would result. This is ∼10⁴ times above global peak values observed with nd9-28°C or tl cells, respectively. Injection of 500 μM Ca²⁺ induced a global rise of free [Ca²⁺]_i to ∼500 nM, indicating a Ca²⁺ binding ratio of ∼100 for Paramecium cytoplasm, a value well comparable to the Ca²⁺-buffer capacity of other systems (Neher, 1995). We assumed that ∼99% of Ca²⁺ would be rapidly bound to cytosolic proteins (Neher and Augustine, 1992; Helmchen et al., 1996), ∼99% of the remaining Ca²⁺ would have to be removed in another way (see Conclusions, below). Evidently after AED stimulation the different strains operate with different kinetics to re-establish [Ca²⁺] homeostasis, whereby the state of trichocyst docking sites might play a role.

Despite some uncertainties (which are not unique to our own work) we can derive from our analyses several important con-clusions. (i) Trichocyst exocytosis is accompanied by a cortical [Ca²⁺] increase in the subsecond time range. (ii) This increase occurs also with mutants with defective or missing targets. (iii) As to the ensuing [Ca²⁺] increase in deeper cell regions we want to speculate along the following lines. The cell body serves as a sink for the tremendous excess of Ca²⁺, from influx and store mobilisation, which is required for a successful exoendocytosis cycle (Plattner et al., 1997a). This spillover accounts for dilution by diffusion and favours binding to endogenous buffers throughout the cell body. Sequestration into endoplasmic reticulum, occurring throughout the cell body and endowed with calreticulin-like protein (Plattner et al., 1997b), will also facilitate re-establishment of Ca²⁺ homeostasis, within >20 seconds, after AED stimulation.

We thank Dr J. Beisson (CNRS, Gif-sur-Yvette) for providing the nd9 mutant, Dr J. Hentschel for setting up CLSM technology, Mrs R. Schmidt, D. Schmid and W. Schweinbeck for the filter wheel design. This study was supported by DFG grant Pl78/12-2.