EFFECTS OF Ca++ AND K+ ON MOTILITY AND PHOTOMOTILITY OF THE MARINE CILIATE FABREA SALINA



Spartaco Puntoni, Roberto Marangoni, Domenico Gioffrè and Giuliano Colombetti


Istituto di Biofisica del C.N.R., Via S. Lorenzo 26, 56127 Pisa, Italy



Abstract


Fabrea salina is a motile ciliate marine protozoan belonging to the order Heterotrichida, that shows photomotile reactions. We report here the results of a study on motility and phototaxis of F. salina exposed to different Ca++ and K+ concentrations. Our findings show that concentrations of potassium ions greater than that of the growth medium and concentrations of calcium ions lower than that of the growth medium cause a significant increase in phototactic response, while the cell speed is not significantly affected. A more thorough analysis shows that the absolute concentrations of Ca++ or K+ are not important and that the phototactic responsiveness depends mainly on the ratio of the K+ concentration to the square root of the Ca++ (the so called Ja value), in agreement with the results obtained in other protozoa. Our results are tentatively discussed in terms of altered membrane potential or ion channel conductances.

1. Introduction


The motile behavior of ciliated protozoa is strongly influenced by membrane potential and this, in its turn, depends on the amount of monovalent and divalent cations in the suspension medium [1,2,3,4,5]. For instance, when Paramecium is transferred from its adaptation medium to higher K+ concentrations it shows a period of ciliary reversal [6,7,8,9], the duration of which depends on the Ja value, defined as the ratio of K concentration to the square root of Ca concentration [7,8,9,10,11,12,13]. Iwatsuki and Song [14] and Iwatsuki and Kobayashi [15] have shown that this is true also in Stentor coeruleus. These authors have shown, furthermore, that the latency of the step-up response in this ciliate is determined by the Ja value [15], as are the phototactic orientation (which increases by increasing Ja values) and the percentage of cells showing photophobic response (which increases by decreasing Ja values) [14]. Fabrea salina Henneguy (1889) is a light-sensitive marine ciliate, which belongs to the order Heterotrichida, like Blepharisma japonicum and Stentor coeruleus. Like these ciliates, it shows photomotile responses, but, whilst Blepharisma japonicum only shows step-up photophobic responses [16] and Stentor coeruleus negative phototaxis and step-up photophobic responses, Fabrea salina exhibits positive phototaxis and photophobic step-down reactions [17,18]. The experimental evidence available so far suggests that membrane potential may play an important role in the mechanism of photomotile reactions of Stentor coeruleus and Blepharisma japonicum [15,19,20]. We have therefore investigated the phototactic response of Fabrea salina as a function of the ionic concentrations to which the cells are exposed, with attention given to the role of K and Ca ions. Our results indicate that the phototactic response does not depend on the absolute concentrations of K and Ca, but that the parameter that better correlates with the alteration of phototactic sensitivity is the Ja value, as defined above.

2. Materials and methods


Fabrea salina were grown in artificial sea water (density 1.022) at a constant temperature of 22 C in a light-dark cycle (15 h light-9 h dark). F. salina were fed the green marine alga Dunaliella salina, which was grown in the growth medium suggested by Johnson [21] and on the same light-dark cycle. D. salina used to feed F. salina were centrifuged and washed three times in the ciliate growth medium. F. salina cells were starved for four days before being used for the experiments. F. salina has a pear-shaped body, the dimensions of which range from 100 to 300 mm for the long axis and from 50 to 200 mm for the short axis. The cell surface is very densely covered with cilia: somatic and oral cilia are clearly distinguishable. The adoral zone is characterized by a deep spiral-shaped invagination, on the edge of which adoral membranelles are visible. F. salina swims along a left-handed helix with an average speed of 200-300 mm/s (at 24 C) and shows two kinds of responses to light stimuli: positive phototaxis (cell displacement towards the light source) and a photophobic step-down reaction (upon a sudden decrease in light fluence rate the cells stop, turn and start swimming again in a new direction) [17,18]. Fabrea salina cells were collected and concentrated from cultures starved for four days; part of the cells, used as control, was resuspended in a medium with the following composition: NaCl 300 mM, MgCl2 67 mM, CaCl2 14 mM, KCl 9 mM, Na2SO4 31 mM, NaHCO3 2 mM and the pH adjusted to 8 with approximately 5 mM Tris- HCL; the cells to be the tested were resuspended in media with different calcium and/or potassium ions concentrations. The range of ionic concentrations investigated varied from 9 mM to 90 mM for potassium and from 1.75 to 56 mM for calcium. Ca-concentrations lower than 1.75 mM and higher than 60 mM resulted in a complete arrest of motility, followed by cell death. The same was true for K concentrations higher than 100 mM. Immediately after transfer to the new medium, the cells usually showed a prolonged period of tumbling and backward swimming, which lasted for some hours, depending on the different ionic composition of the resuspension media. In order to allow the cells to completely adapt to the new ionic conditions, we left them overnight in the new media; the next day we examined the cells and considered them adapted and suitable for the experiments only when at least 70% of them were motile and their speed was not significantly different from that of the control samples (about 10% of the control value). In each experiment about 500 ml of cell suspension containing 150-200 cells, the maximum number that we can simultaneously track, were placed in the experimental chamber, the dimensions of which are 15mm*10mm*4mm. Cell motion parameters (average speed, frequency of directional changes and frequency distribution of the angles of directional changes) and phototactic reaction were measured in control and treated samples. The measurements were performed by means of a laboratory developed automatic tracking system, described in detail in a previous paper (Marangoni et al., 1996). Phototaxis stimulating light was provided by means of a 150W Xenophot HLX lamp; its wavelength was selected by means of a B579 filter (Balzers, Germany) with maximum transmission at 579 nm and band width at half maximum of about 10 nm; the light stimulus was kept on for three min at a fluence rate of 9 W/m2 . These conditions induce a significant phototactic response in non- treated samples, as described in detail in a previous paper [22]. The phototactic coefficient thus measured was corrected for variations in cell speed [23] and normalized with respect to the phototactic responsiveness of the control samples, taken arbitrarily equal to 1. Each experiment was repeated four times and the data shown are the average of the values obtained together with their standard deviation.

3. Results


Fig 1 reports the results of a series of experiments in which we have varied the K-concentration at different Ca concentrations.


Fig. 1 The phototactic response (normalized with respect to the control sample as described in Materials and methods) at fixed calcium concentrations. Abscissa:  potassium concentrations. Ordinate: phototactic response in arbitrary units. The error bars indicate the standard deviation.
R vs k at Ca= 1.75 mM, JPEG, (9339 bytes) R vs k at Ca= 3.5 mM, JPEG, (9134 bytes)
R vs k at Ca= 7 mM, JPEG, (8977 bytes) R vs k at Ca= 14 mM, JPEG, (9315 bytes)
R vs k at Ca= 28 mM, JPEG, (8676 bytes) R vs k at Ca= 56 mM, JPEG,  (9010 bytes)


A general feature of these results is that at each Ca concentration an increase in [K] brings about a meaningful increase in phototactic response. This increase is, however, also dependent on the Ca-concentration and is stronger at the lowest Ca concentration used. In this case, moreover, at high K potassium concentration the phototactic response shows a net decrease. Within the general trend of an increase in phototaxis with increasing [K], we can, moreover, observe a general slight decrease for [K] = 36 mM.

The effect of an increase in [K] on cell speed is less clear and, if any, it seems to depend also on the amount Ca present, as shown in Fig. 2.


Fig. 2 The average cell population speed at the various ionic compositions used. The different curves refer to the different Calcium concentrations used, indicated by the box inside.  Abscissa: potassium concentration (mM). Ordinate: average sample speed relative to that of the control sample.

Speed vs. K and Ca, JPEG, (15075 bytes)


Figure 3 shows the trend of the phototactic response at fixed potassium concentration as a function of [Ca]. There is a general decrease of phototaxis, which is more marked when the phototactic responsiveness is higher, as, for example, at [K] = 54 mM..


Fig. 3 The phototactic response (normalized as described above) at fixed potassium concentrations. Abscissa:  different calcium concentrations used. Ordinate:  phototactic response in arbitrary units. The error bars indicate the standard deviation.
R vs Ca at K= 18 mM, JPEG, (7732 bytes) R vs Ca at K= 36 mM, JPEG,  (7816 bytes)
R vs Ca at K= 54 mM, JPEG, (7935 bytes) R vs Ca at K= 72 mM, JPEG, (8002 bytes)
R vs Ca at K= 90 mM, JPEG,  (8089 bytes)


Figure 4 reports a 3D graph, which summarizes the behavior of phototaxis as a function of both [K] and [Ca] concentrations. In control experiments we have varied the ionic strength of the medium but kept [K] and [C] constant. In these conditions there was no measurable effect on the phototactic response of F. salina (data not shown).


Fig. 4 3D diagram of the phototactic response vs. [K] and [Ca] concentrations

3D histogram (30127 bytes)


In figure 5 we report our results as a function of the Ja-value, defined as the ratio between [K] and the square root of [Ca].


Fig. 5 The phototactic response vs. the Ja-value. The r^2 is about 0.84, then p is less than 0.05.
Ja value, JPEG, (18670 bytes)

4. Discussion


All the available experimental evidence strongly indicate a close coupling between membrane potential and motile responses mediated by the cilia. It is, therefore, not surprising that it is possible to influence the photomotile reactions by changing the ionic environment in which the cells are suspended. Iwatsuki and Song [14] have found results similar to ours in the case of Stentor coeruleus. In this protozoan an increase in [K] at a certain Ca concentration brings about a meaningful increase in the phototactic response, whilst an increase in [Ca] at fixed [K] causes a significant decrease in phototaxis. These authors suggest that the behavioral response to the light stimulus might be linked not to the absolute [K] and/or [Ca], but rather be affected by the so-called Ja-value. However, these authors weaken their hypothesis, by suggesting that an increase in [Ca] can diminish the phototactic response of Stentor, for a determined Ja value. In fact their conclusion goes beyond their experimental evidence, which only shows that a simultaneous increase in [K] and [Ca] (necessary in order to keep the Ja value constant) is accompanied by a decrease in phototaxis. The results shown in Fig. 1 indicate that by keeping [Ca] constant and increasing [K] we can increase the phototactic response of Fabrea salina, whereas an increase in [Ca] at constant [K] brings about a strong decrease in phototactic sensitivity of the cells. We have then asked the question whether Ca and K were able to affect the phototactic response t independently of each other, or if there was a sort of competition between the two ions, as suggested, for example, by Colombetti et. al [24] in Stentor. These authors had suggested that the relative ratio [K]/Sqrt([Ca]) might be involved in the regulation of the capability of Stentor to respond to light stimuli, but were unable to establish a more precise relationship between the ionic concentrations and the photomotile response. Our results, shown in Fig. 5, indicate that there is a meaningful correlation between Ja value and phototaxis in F. salina. The r2 value calculated from our data is meaningful at the confidence level; this indicates that there is a strong dependence of phototaxis on the combined action of [Ca] and [K], which assumes the character of an almost functional relation. What remains to be understood is the step (or are the steps) of the photosensory transduction chain of Fabrea salina where high Ja values can induce an amplification, that is reflected in a the amplification of the phototactic response (which can be as high as a factor of 4). There is little doubt that this effect cannot be at the level of light absorption itself. It is difficult to imagine that a rhodopsin, which has been suggested as a possible photoreceptor in F. salina [25], may change its optical absorption properties simply by changing the relative ratio of K to the square root of Ca in the suspension medium. In the same manner, if we assume that phototaxis in Fabrea salina relies on a shading mechanisms, it is hard to think of how changing the ionic composition of the medium could render the shading mechanism more efficient. One could possibly imagine that, if all the molecules involved in the transduction chain are located on the ciliary membrane, as is the case for the rods, by changing the relative ratio of K to the square root of Ca one could make one or more of the amplification steps more efficient for a surface charge effect. Probably it is more reasonable to consider the final steps of the sensory transduction chains, where usually the opening or closing of ion channels are involved. These events might be affected by a different ionic environment, with a competition between K and Ca for cell membrane binding sites and a possible, subsequent activation (or deactivation) of membrane channels, for instance. With regard to this it may be interesting to recall that in Stentor high Ja values inhibit the step-up photophobic reaction, which has been shown to be linked to a membrane depolarization followed by the opening of Ca channels in the cell membrane and a subsequent Ca action potential, leading to ciliary reversal. In this case high Ja values could bring about block the opening of the channels responsible for depolarization. Similar behavioral results have been observed (Colombetti et al., unpublished) for the step- down reaction of F. salina, which appears to be inhibited at high Ja values. Let us now assume that a "good" phototaxis requires a prolonged swimming towards the light source, which implies that the cells should avoid random turns during their motion. A random turn in a ciliated protozoa can take place whenever there is a depolarization of the membrane potential, in the limit when there is a Ca action potential. If it is true that high Ja values can inhibit or make more difficult these action potentials, as seems to be suggested by the results for the step-up in Stentor or the step-down in Fabrea salina, we have a mechanism that can explain the enhanced phototactic reaction in both systems. A direct proof of this hypothesis requires a measurement of the random membrane fluctuations in these ciliates under different Ja values.

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