Studies on the mechanism of cell elongation in Blepharisma japonicum I. A physiological mechanism how light stimulation evokes cell elongation

Europ.J.Protistol. 25, 182-186 (1989) October 27, 1989

European Journal of

PROTISTOLOGY

Studies on the Mechanism of Cell Elongation in Blepharisma japonicum I. A Physiological Mechanism How Light Stimulation Evokes Cell Elongation Masaki Ishida and Yoshinobu Shigenaka Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan

Koji Taneda Department of Biology, Kochi University, Kochi, Japan

SUMMARY In a heterotrichous ciliate, Blepharisma japonicum, longitudinal elongation of the cell bodyis induced by light stimulation (1000 to 3000 lux). This light-induced response was inhibited under the existence of cyclic mononucleotide phosphodiesterase (PDE) antagonists such as papaverine (10--4 M), theophylline (10-3 M), dibutyril-cAMP (10--4 M), 3-isobutyl-1-methylxanthine (10--4 M) and dibutyril-cGMP (10--4 M). Microinjection of cyclic mononucleotides, especially cGMP, inhibited cell elongation. These observations suggest that the cell elongation was mediated by intracellular cyclic mononucleotide. K+ specific ionophore valinomycin (10-L 10-7 M) enhanced light-induced cell elongation. Thiseffect of valinomycin became more remarkable when valinomycin coexisted with PDE antagonists, while it was diminished under high K+ conditions. Moreover, the K+ channel blockers tetraethylammonium (TEA) and CsCI inhibited cell elongation. These observations suggest that the cell elongation isalsomediated by K+ hyperpolarization, and that this electrical change is probably elicited afterthe intracellular concentration of cyclic mononucleotide decreased.

Introduction In some heterotrichous ciliates such as Spirostomum, Stentor and Condylostoma, contraction of the cell body has been investigated mainly from morphological viewpoints [4, 6, 11, 13, 14]. These studies have demonstrated that such ciliates have a certain fibrillar system, termed myoneme, which is regarded as a force-generating structure for cell contraction [10-13]. Similarly in peritrichous ciliates such as Vorticella, Carchesium and Zoothamnium, so-called stalk contraction is attributed to the conformation change of an elastic polymer, termed spasmoneme [1]. Although there have been many investigations on the 0932-4739/89/0025-0182$3.50/0

fibrillar systems correlated with the motive force for cell contraction, only a few investigations have been carried out as to the mechanism of re-elongation of the cell, which takes place during the recovery process from cell contraction. In Stentor, the presence of a microtubular system has been suggested as indispensable for cell elongation [2]. Huang and Pitelka [5] also reported that the motive force for cell elongation of Stentor may be generated by active sliding between adjacent microtubules in Km-fibers. In another heterotrichous ciliate, Blepharisma, cell elongation can be induced by light stimulation [8, 9]. Recently, Matsuoka and Shigenaka [9] have examined the process of © 1989 by Gustav Fischer Verlag, Stuttgart

Mechanism of Cell Elongation in Blepharisma . 183

cell elongation of Blepharisma by electron microscopy and reported that this movement may result from active sliding of vacuole associated microtubules (VAMs) in addition to the sliding of post-ciliary microtubular sheets (Km-fiber). However, the physiological mechanism how light stimulation induces cell elongation still remains unknown. The present study on Blepharisma was therefore undertaken to elucidate the signal transduction mechanism from light stimulation to mechanical cell elongation by means of light microscopy, especially by using drugs such as channel blockers, ionophores and cyclic mononucleotide phosphodiesterase inhibitors. As a result, we suggest that the whole process of cell elongation involves the following mediating steps; light reception - decrease in cyclic mononucleotide concentration - change of membrane conductance for K+-membrane hyperpolarization - cell elongation. Material and Methods A heterotrichous ciliate, Blepharisma [aponicum strain R-13, was cultured at 23 ± 1 °C in a lettuce infusion containing a small amount of CaC0 3• The culture was always kept in darkness. Prior to each experiment, cells were washed in a test solution (1 mM CaCh, 1 mM KCl, 1 mM Tris-HCl buffer at pH 7.2), collected by using low speed centrifugation, and kept for 1 hour in a small test tube (10 rnl) for adaptation to the test solution. At that time, the cell density was adjusted to be 100-200 cells/m!. The cell suspension was then concentrated up to 1000-2000 cells/ml and the cells were transferred into a small observation chamber (2 ml) attached to a light microscope (Nikon DIAPHOTO). The chamber was thermo-controlled (24°C) with a water circulation system. A halogen lamp was used for light stimulation (1000 or 3000 lux) and the light intensity was measured with a photoelectric illuminometer (Tokyo Photo-electric, ANA-999). An infrared-absorbing filter was placed between the lamp and the cell suspension to eliminate the effect of heat ray.

As an index for cell elongation, the elongation rate (ER) was employed here, which is represented by the following formula: ER = ((La - Lb)/Lb) x 100 (%), where La and Lb stand for the cell body lengths after and before the stimulation was given, respectively. Microinjection was carried out with a single micropipette attached to a micro-manipulator (Narishige, MO-202). The micro-pipette was connected by vinyl or polyethylene tubing to a 1 ml syringe, which was controlled by a microinjector (Narishige, IM-SB). The whole system was filled with liquid paraffin. Micropipettes were drawn out by a micro-electrode puller (Narishige, PD-S) from Pyrex tubing (1 mm in inner diameter), and the tip was bent at an angle of 90° by hearing with a small frame of a glass-made burner. The tips were then broken off to the desired diameter (2-3 urn) by pushing them against a solid surface under the light microscope. A short column of oil was sucked up into the micropipette and its length was measured with an ocular micrometer. The liquid paraffin column was then expelled into a drop of water and the volume of the resultant spherical oil droplet was calculated from its diameter. Thus, by knowing the length of the column, we could inject a desired volume. About 7-8 x 104 flm3 of the fluids, about 10% of the cell volume, was injected into each Blepharisma. Mononucleotides such as cAMP and cGMP, dissolved in deionized distilled water (DDW), were injected into the cel!. As the control for this, only DDW was injected.

Results Under the normal cultural condition in darkness, Blepharisma japonicum is always rice-shaped (Fig. 1a). When the light stimulation (1000 or 3000 lux) was applied to the ciliate, it elongated for 5-15 minutes after the stimulation was given (Fig. 1b).

1a

Fig. 1. Light micrographs of a single specimen of Blepharisma japonicum at normal (a) and elongated (b) states. The specimen was stimulated by 10 min exposure to 1000 lux light in the standard test solution. Scale bar indicates 100 urn,

1b

184 . M. Ishida, Y. Shigenaka and K. Taneda

Effect of Valinomycin, TEA and CsCI The effect of valinomycin on the cell elongation is shown in Figs. 2-4. When the cells were stimulated by light (1000 lux), valinomycin was found to enhance the cell elongation in the concentration range of 10-8 M to 10-7 M (Fig. 2). Moreover, cell elongation was induced by valinomycin even in darkness in the concentration range of 10-7 M to 10-6 M (Figs. 2 and 3). The concentration effect of K+ ions (0.1 to 30 mM) on the valinomycin-induced cell elongation in darkness is shown in Fig. 4. Valinomycin (10-6 M) induced cell elongation only when K+ concentrations were below 1 mM. Potassium channel blockers, TEA (tetraethylammonium chloride, 20 mM) and CsCi (20 mM), had inhibitory effects on the cell elongation (Table 1). The effect of CsCI was more remarkable (two-tailed t-test, P < 0.05) than that of TEA (0.05 < P < 0.1).

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Fig. 4. Concentration effect of K+ on the cell elongation in darkness. Every test solution contains 10- 6 M valinomycin in 0.1 % DMSO. Each point and bar stand for mean value and SE from 40 specimens. 0, valinomycin; e, valinomycin-free control.

Mechanism of Cell Elongation in Blepharisma . 185

the following compounds were used as PDE-antagonists; theophylline (10- 3 M), papaverine (10-4 M), dibutyrilcAMP (10-4 M), 3-isobutyl-1-methylxanthine (10-4 M) and dibutyril-cGMP (10-4 M). These antagonists showed about the same degree of inhibitory effect among them on the cell elongation. As shown in Table 2, statistically significant differences could be detected except for papaverine and dibutyril-cAMP. These inhibitory effects were found to disappear by the coexistence of valinomycin.

Table 1. Effect of potassium channel blockers on the cell elongation in standard test solution. Mean values of elongation rate and SEs from 40 specimens are presented. Cells were adapted to the solution containing blockers for 10 min and then stimulated by 15 min exposure to 1000 lux light. CONCENTRATION (M)

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5.70

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1.13

Effect of Microinjection of Cyclic Mononucleotides into the Cell The effect of microinjection of cAMP, cGMP and DDW on the cell elongation is shown in Fig. 5. Among these two mononucleotides, cGMP showed stronger inhibition to the cell elongation. This inhibition was transient and the cells began to elongate gradually 5 min after the light stimulation was applied.

Effect of PDE-Antagonists Coexistent with Valinomycin The effect of cyclic mononucleotide PDE-antagonists on the cell elongation is shown in Table 2. In this experiment,

Table 2. Effect of POE-antagonists on the cell elongation. Antagonists were dissolved in the test solution. Cells were adapted to the solution containing PDEantagonist for 10 min, and were stimulated by 15 min exposure to 1000 lux light. The column "Test soln." shows that only POE-antagonists were dissolved in the test solution. The column "Test soln. + Val." demonstrates that POE-antagonists and valinomycin (5 x 10-8 M) were dissolved in the test solution. Mean values and SE from 45 specimens are presented. Statistically significant differences (two tailed t-test, P < 0.05) was detected except for papaverine and dibutyril-cAMP. All of these inhibitory effects disappeared when valinomycin was coexistent.

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186 . M. Ishida, Y. Shigenaka and K. Taneda Discussion The present investigation demonstrated that cell elongation of Blepharisma was evoked by increasing K+ transport by valinomycin and inhibited by K+-channel blockers. Furthermore, 10-7 and 10-6 M valinomycin could induce cell elongation even in darkness only when extracellular K+ concentration was less than 1 mM. These results suggest that a change of the membrane conductance for K+ may be required for the process of cell elongation. In general, valinomycin can induce electrical changes of the cell; both hyper- and depolarization of the cell membrane. The cell may be transiently hyperpolarized by K+ diffusion potential, and the following depolarization may be caused and retained by the transmembrane K+-equilibrium. Since the coexistence of a higher concentration of extracellular K+ ions inhibited valinomycin-induced cell elongation, the effect of valinomycin may be attributed to the membrane

hyperpolarization. Antagonists to cyclic mononucleotide PDE showed inhibitory effects on the cell elongation (Table 2). Since these effects of these antagonists are probably due to accumulation of intracellular cyclic mononucleotides, the cell elongation is considered to be mediated by a decrease in intracellular cyclic mononucleotide concentration. These inhibitory effects disappeared when they were coexistent with valinomycin, which suggests that the cyclic mononucleotide, probably cGMP, behaved as a mediator which converts light stimulation to the electrical signal (membrane hyperpolarization). Some investigations have reported that cyclic mononucleotides are involved in ion channel activities. According to Kandel [7], for example, K+ channels in Aplysia punctata may be controlled by cAMP. In the retinal rod photoreceptor cell in vertebrates, Na + channels are probably controlled directly by cGMP [3]. As shown in the present study, Blepharisma seems to be another example in which membrane conductance is controlled by intracellular cyclic mononucleotides. In the process of cell elongation, there may be some mediating steps from light reception to the cell elongation as a mechanical response. The present work has thus demonstrated that the whole process, which requires about 10 min, involves at least four or more successive steps: light reception - decrease in cytoplasmic concentration of cyclic mononucleotide (probably cGMP) - conductance change of the plasma membrane for K+ - membrane hyperpolarization - cell elongation.

References 1 AmosW. B. (1975): Contraction and calcium binding in the vorticellid ciliates. In: Inoue S. and Stephens R. E. (eds.): Molecules and cell movement, pp. 411-436. Raven Press, New York. 2 BannisterL. N. and Tatchel E. C. (1968): Contractility and the fiber systems of Stentor coeruleus. J. Cell Sci., 3, 295-308. 3 Fesenko E. E., Kolesnikov S. S. and LyubaruskyA. L. (1985): Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature, 313, 310-313. 4 HuangB. and Mazia D. (1975):Ciliatecontractility.In: Inoue S.and Stephens R. E. (eds.): Moleculeand cellmovement,pp. 389-409. Raven Press, New York. 5 Huang B.and PitelkaD. R. (1973): The contractileprocessin the ciliate Stentor coeruleus. I. The role of microtubules and filaments. J. Cell Biol., 57, 704-728. 6 IshidaH. and Shigenaka Y. (1988): Cellmodel contraction in the ciliate Spirostomum. Cell Motil. Cytoskel., 9, 278-282. 7 Kandel E. R. (1981): Calcium and the control of synaptic strength by learning. Nature, 293, 697-700. 8 Matsuoka T. (1983a): Distribution of photoreceptors inducingciliaryreversaland swimmingaccelerationin Blepharisma [aponicum. J. Exp. Zool., 225, 337-340. 9 Matsuoka T. and Shigenaka Y. (1985): Mechanism of cell elongation in Blepharisma japonicum, with special reference to the role of cytoplasmic microtubules, Cytobios, 42, 215-226. 10 Randall J. T. and Jackson S. F. (1958): Fine structure in Stentor polymorphus. J. Biophys. Biochem, Cytol., 4, 807-830. 11 Yagiu R. and Shigenaka Y. (1960): Electron-microscopical studieson the fibrillarsystemin the protozoan ciliates. Jpn. J. Exp. Morphol., 14, 1-52. 12 Yagiu R. and Shigenaka Y. (1963): Electron microscopy on the longitudinal fibrillar bundle and the contractile fibrillar system in Spirostomum ambiguum. J. Protozool., 10, 364-369. 13 Yogosawa-Ohara R. and Shigenaka Y. (1985): Twisting contraction mechanism of a heterotrichous ciliate, Spirostomum ambiguum. 1. Role of the myoneme. Cytobios, 44, 7-17. 14 Yogosawa-Ohara R., Suzaki T. and Shigenaka Y. (1985): Twisting contraction mechanism of a heterotrichous ciliate, Spirostomum ambiguum. 2. Role of longitudinal microtubular sheet. Cytobios, 44, 215-230.

Key words: Blepharisma - Cell elongation - K+ channel - Light stimulus - Cyclic mononucleotide YoshinobuShigenaka, Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan