- Email: [email protected]
Developmental changes in Ca2+ sensitivity of sea-urchin embryo cilia
Camp. &&em. Physioi.Vol. 85A, No. 1, pp. 8390, 1986 Printed in Great Eiritain
$3.00+ 0.00 Pergamon Journals Ltd
03~-9629j8b
DEVELOPMENTAL CHANGES IN Ca2+ SENSITIVITY OF SEA-URCHIN EMBRYO CILIA MIHO DEGAWA, YOSHIHIROMOGAMIand SHOJIA. BABA Department of Biology, Ochanomizu University, Otsuka, Tokyo 112, Japan (Received 3 Januury 1986)
Abstract-1. The swimming velocity and direction of sea-urchin embryos were directly measured by multiple-exposure photography. 2. An increase in the Ca*+ concentration of the medium decreased the velocity in the stages from blastula through pluteus. 3. The calcium ionophore A23187added in the medium enhanced this Ca*+effect throughout all stages, and further significantly increased the number of embryos that stopped swimming and the ratio of backward vs forward, only when added after a critical stage, where spontaneous backward swimming began to occur. 4. These findings suggest that the regtdatory mechanism of ciliary activity changes in its sensitivity to Ca”+ during deveiopm&.
through pluteus. In this paper we directly measured the swimming velocity and its direction with respect to the orientation of embryos, i.e. forward or backward, by means of multiple-exposure photography. Because the locomotor activity of embryos is mainly dependent on the ciliary movement on their body surface, changes in these parameters of swimming must indicate those in ciliary movement, e.g. backward swimming can be regarded as with ciliary reversal. Thus, we found a qualitative as well as quantitative difference in the sensitivity to Ca*+ and A23 187 of the cilium between the pluteus and earlier stages. This finding may suggest an advantage of using a development-biological approach to seaurchin embryo cilia in the study of the Ca*+-dependent regulatory mechanism of ciliary movement.
Sea-urchin embryos change their slung hehaviour in the course of development. Backward swimming begins to occur at the early pluteus stage and thereafter characterizes the rather complicated swimming hehaviour of plutei. This fact shows that the ciliary motile mechanism may differentiate to acquire the ~pab~lity for ciliary reversal at a speciiic developmental stage (Baba, 1975). It has heen suggested that the ciliary reversal of sea-urchin embryos, similarly to the ciliary responses of many other organisms, follows the bioelectric activity of the ciliated cell (Mackie et al., 1969; Markman, 1972; Baba, 1975). It has heen argued that in ~u~~e~~~ the influx of Cat+ is induced by membrane depolarization and increases intracellular Ca2+, which in turn plays a major role in inducing ciliary reversal (Eckert, 1972). It has been further demonstrated by a substantial number of experiments on demembranated cilia and flagella, first with Put-ume&m (Naitoh and Kaneko, 1972) and then with a variety of organisms (Tsuchiya, 1977; Holwill and McGregor, 1976; Hyams and Borisy, 1978; Walter and Satir, 1979; Brokaw, 1979; Gibbons and Gibbons, 1980), that intracellular Ca*+ thus increased actually modulates ciliary and flagellar bending. It is, therefore, not unlikely that the influx of Ca*+, which is probably associated with the action potential that has been recorded from the pluteus (Mackie et ai., 1969), may trigger ciliary reversal by acting on the internal motile machinery of cilia. With sea-urchin embryos, however, little is known about the control activity of Ca*+ in the ciliary movement, though it has been reported that extracellular Ca*+ affects the swimming activity of the embryos of early developments stages (Soliman, 1984a). In order to gain a deeper insight into the mechanisms of ciliary responses, we investigated in vivo the effects of Ca*+ and the calcium ionophore A23187 on the ciliary movement of sea-urchin embryos at various developmental stages from blastula
MATERIALSAND METHODS Sea-urchin embryos
Sea-urchins (Hemicentrorur padcherrimup) were maintained in circulating natural sea-water at 17°C. Gametes were obtained by injection of 0.5s M KC1 into the body cavity. Eggs were shed into artificial sea-water (lamarin U, Jamarin Lab., Osaka) and inseminated in the same medium. Fertilized eggs (at least 95% fertilization, judged from the elevation of the fertilization membrane) were washed twice to remove excess sperm and incubated at 17°C with paddle stirring. Under these conditions, embryogenesis proceeded almost s~chronously up to 60 hr. The developmental stages were determined from the morphology of embryos according to the table of the early developmental stages of H. pulcherrimus in Okada and Miyauchi (1954). Experimental solutions
Artificial sea-water (ASW), prepared for the observation and recording of the swi~in8 behaviour of embryos, contained 450 mM NaCl, IOmM KCI, IO mM CaCl,, 25 mM MgCl,, 28 mM MgSO,, 10 mM Tris-HQ, PH. 8.0. The concentration of calcium ion was 1OmM in the standard ASW as above, and ranged 0-4OmM in modified ASWs with the total osmolarity kept constant by adjusting the concentration of Na+. 83
MIHO DEGAWAet al.
84
A23187 (Calbiochem-Behring Corp., LaJolla, CA) was dissolved as 1.0 and 10.0 mg/ml stock solutions in dimethylsulfoxide (DMSO), and diluted in ASW. A23187 formed a fine particulate suspension upon addition to ASW. To disperse A23187, ASW was sonicated for 10min in an ice bath, which was then used within 11 hr. The final concentration of DMSO in ASW used was below 2.5 ,ul/ml. In control experiments, DMSO at such a low concentration did not affect the swimming behaviour of the embryos (not shown). All chemicals (reagent grade) were from Wako Pure Chemical Co., Tokyo, unless otherwise specified. Recording of the swimmingbehaviour Every S hr after hatching, embryos were collected by hand centrifuge from 30 ml of the culture, resuspended in 3 ml ASW, and left for about 10min at room temperature (ca 17°C) to adapt to ASW. Several drops of the condensed embryos were then transferred to about 3 ml of the eltperimental solution in a 6-cm Petri dish, in which the solution made a thin layer of ca 1 mm at the bottom. For recovery from the mechanical disturbance upon mixing, the embryos were left for a further 7min in the Petri dish placed in a constant-temperature chamber, which was also kept at 17°C. Then the swimming behaviour was recorded within 30 min after the embryos were transferred. The swimming behaviour of the embryos in the Petri dish was observed by a binocular microscope (X-Tr, Olympus, Tokyo) and recorded on Tri-X film (Eastman Kodak) under a dark-field stroboscopic illumination obtained using a circular xenon flash tube (MFT-48, Miyata Elec. Works Inc., Tokyo) as shown in Fig. 1. Throughout observation and recording, the temperatures of the microscope stage and Petri dish were kept at 17°C with a water jacket (Fig. 1). For measurements of the swimming velocity and direction, the swimming embryos were photographed with five consecutive flashes at fixed time intervals (1 or 2 s), giving multiple-exposure photographs as shown in Fig. 2. The first of these five flashes was brighter than the others, so that the first image along the path of locomotion recorded was clear
and, therefore, the swimming direction of individual embryos could be determined. For both forward and backward swimming embryos, the velocity was calculated from the average distance between sequential images divided by the time interval mentioned above. From enlarged film, the coordinates of the points, fixed in individual embryos, were measured along the swimming path on a digitizer (Gradimate U4-30, Oscon Co., Tokyo) interfaced with a minicomputer (OKITAC SO/lo, Oki Elec. Ind. Co., Tokyo), where the velocity was computed.
RESULTS Swimming behaviour of sea-urchin embryos
Sea-urchin embryos swam freely in the Petri dish, but the vertical locomotion was limited because of the small depth (ca 1 mm) of ASW. The swimming of the embryos recorded as described above was therefore virtually two-dimensional, as shown in Fig. 2, so that it was easy to analyse the velocity and direction of swimming accurately. Although the embryos were observed to swim forming helical paths and rotating on the animal-vegetal axis, as reported by other authors (Maruyama, 1981a, b; Soliman, 1983), the method of recording used in the present study was not appropriate to analyse the detailed patterns of swimming path and rotation. Therefore, we analysed mainly the velocity and direction of swimming. Forward swimming was observed and recorded throughout the developmental stages, while spontaneous backward swimming only in the pluteus stage, as shown in Figs 2-5 and 7. The velocity of forward swimming increased in the early stages and decreased later, making a maximum of about 0.3 mm/s at the late gastrula stage. In the pluteus stage, the embryos often ceased to swim and the velocity of actively swimming embryos also gradually decreased during further development. The velocity of backward swimming was always lower than that of forward swimming, and among preparations as well as with the time of development, these two velocities varied parallelly to one another (Figs 3-5). Effects of extracellular swimming behaviour
X8L
Fig. 1. Setup for recording of the swimming behaviour of sea-urchin embryos. Light from a circular xenon flash lamp (Xel) is limited by a central cylinder (CL) and a hole of the bottom of a constant-temperature chamber (CH) to effect dark-field illumination. The lamp is operated stroboscopically with pulses produced by a pulse generator (PG). Another pulse, which is synchronized with opening of the camera shutter (SH), triggers, to operate the lamp for recording, programmed sequential five pulses, the first of which is used for the brightest flash by an intensitv modulator (IM). CA, camera; MO, micrdscope objective; W, water jacket; T, semiconductor thermometer; PS, power supply for flashing.
Ca2+ and A23187
on the
Figure 4 shows changes in the velocities of forward and backward swimming in ASWs of different Ca2+ concentrations as a function of the time after insemination. The time course of the changes for forward swimming in ASWs of Ca2+ concentrations higher or lower than 10 mM (standard) is nearly parallel to that in the standard ASW, with the velocity at higher Ca2+ concentrations being smaller (see also Fig. 6). The velocity of backward swimming was observed to decrease in ASWs of higher Ca2+ concentrations, although such decreases were not clearly detected by measurements as in forward swimming. Spontaneous backward swimming itself was never observed in ASW not containing Ca’+ added (not shown). The addition of A23 187 to ASWs induced changes in the swimming behaviour of sea-urchin embryos, particularly in the velocity and direction of swimming. When A23187 was added, the velocities of both forward and backward swimming were reduced at
Ca*+ sensitivity
of cilia
85
Fig. 2. Stroboscopic recordings of swimming embryos of H. pulcherrimus with five successive xenon flashes. The first image in each sequence is the brightest. (a) Embryos at the prism stage (43 hr after insemination), recorded with flash intervals of 1 s; (b) at the pluteus stage (60.5 hr), with flash intervals of 2 s. Beside selected several sequences, the swimming direction is represented by open arrows (forward swimming) and by closed arrows (backward). Inset: enlarged print of a pluteus, which is swimming backward. The shape of embryo is a key factor in determination of its swimming direction as shown here. Scale bars, 0.2 mm.
L
13
a
1
23 TIME
33 AFTER
43
53
INSEMINATION
63 ( hrs }
Fig. 3. Swimming velocity of embryos of H. pulcherrimus in the course of development. Embryos are bathed in the standard ASW. The mean values of the velocity with SD bars from five separate experiments (marked with different symbols) are shown. The upper half of the figure corresponds to the velocity of forward swimming and the lower half to backward swimming. Developmental stages determined from the morphology of embryos are shown at the top of the figure.
MIHO DEGAWAet al.
86
prism
gaatrula
I
1
20.5
30.5 TIME
,
50.5
40.5
AFTER
INSEMINATION
60.6
(hrs)
Fig. 4. Effect of Ca*+ on the swimming velocity of embryos of If. puicherrimus in the course of development. Embryos are bathed in ASWs containing Ca’+ at different ~ncentrations, 0: 2.5 mM, q !: IO mM (standard), 0: 20 mM. Mean values from a typical experiment are shown. The upper half of the figure is for forward swimming and the lower half for backward swimming. Stages at the top of figure. Figure 7 shows the ratio of the number of actively swimming embryos out of all embryos recorded, i.e. the percentage swimming, in ASWs of various concentrations of Ca2+ and A23187. The percentage swimming was usually as high as 70400% in ASWs containing Ca2+ at 10mM (standard) and lower concentrations in the absence of A23187, though in
any developmental stage, as shown in Figs 5 and 6. Figure 6 shows the effect of A23187 on the relation of the velocity of forward swimming and the Ca2+ ~n~ntration in ASW. A23 187 enhanced the effect of Ca’” in reducing the velocity of forward swimming, especially in the range of the Ca2+ concentrations higher than 2.5 mM, in all the developmental stages. An extreme consequence of the enhancement of the effect of Ca2+ by A23 187 was a complete cessation of swimming in the pluteus stage, though this was observed only for high Ca*+ con~ntrations (Fig. 6).
8
/an'am /
/iaatrula
t2katula
1
*
20.6
TIME
““I
30.5
AFTER
40.1
I
plutaua
so.4
INSEMINATION
”
00.~
1
( hrs)
Fig. 5, Effect of A23187 on the swimming velocity of embryos of H. ~Ze~e~~~~ in the course of development. The concentrations of A23187 in ASW are 0 M (O), lo-’ M (c]) and 10e6 M (a). The mean values of the velocity with SD bars from a typical experiment are shown. The upper half of the figure is for forward swimming and the lower half for backward swimming.
Caz+ CONCENTRATION (mM )
Fig. 6. Effect of A23187 on the velocity of forward swimming of embryos of H. ~~~rrirn~ as a function of Ca** concentration. The concentrations of A23187 in ASWs are 0 M (O), lo-‘M (0) and 10m6M (a). The mean values of the velocity from a typical experiment are shown. The number at the upper right of each graph represents the time after insemination.
Ca2+ sensitivity of cilia
a TIME
AFTER
87
0 15.5 25.5 35.5 45.5 55 5
65.5
b
INSEMlNATlON ( hrs )
Fig. 7. Effects of Cal+ and A23 187 on the swimming activity of embryos of H. pulcherrimur in the course of development. (a) Percentage swimming; (b) fraction of backward swimming. The Ca*+ concentrations in ASWs are 2.5 mM (O), IO mM (D) and 20 mM (0). The A23187 concentrations in ASWs are 0 M (upper), 10-‘M (middle) and 10V6M (lower}.
this figure it happened to be low in the early blastula stage. Ca2+ at higher concentrations (20 mM in this figure, closed circle) tended to induce a rapid decrease in the percent swimming. This phenomenon was usually found after a critical developmental stage (cu 40 hr after insemination in Fig. 7a). It should be noted that, after this critical stage, backward swimming began to be induced by A23187 (Fig. 7b). A consequence of addition of A23187 up to lob6 M in ASWs containing Ca2+ at 10mM and higher concentrations was a further decrease in the percent swimming, sometimes to nearly 0% with 10e6M A23187. A23187 had little effect on swimming in the embryos, at least at the early stages, in al1 ASWs used and throu~out development in ASWs cont~ning below 10mM Ca2+ (Fig. 7a). Direct observation by a dark-field microscope at a higher magnification showed that the cilia of the non-swimming embryos at higher Ca2+ concentrations did not move. At lower CaZC concentrations, on the other hand, the cilia were beating weakly even in the embryos which appeared to be scored as non-swimming; such embryos only rotated on the animal-vegetal axis and readily restored their propulsive activity on a mechanical agitation such as a tap of the Petri dish, although they stopped swimming and began to rotate again a few seconds later. A23187 increased the fraction of backwardswimming embryos among the actively swimming ones in a CL?+-dependent manner (Fig. 7b), although it could not induce backward swimming sence of Ca2+ (not shown). A maximum
in the ab-
increase in the fraction was observed at Ca2+ concentrations between 5 and 1OmM. At higher Ca2+ concentrations, backward swimming could hardly be detected because the swimming activity itself was heavily diminished. It should be noted that backward
swimming was induced by A23187 only at the late developmental stages, where spontaneous backward swimming was observed (Fig. 7b). DISCUSSION
The aim of this paper is to test the action of Ca ions on the ciliary movement of sea-urchin embryos. In order to manipulate the intracellular Ca*+ concentration, two methods were employed, raising the extracellular Ca2+ concentration and adding the calcium ionophore A23187 to external solutions. Since plasma membranes are, more or less, capable of leaking Ca2+ or having leakage conductor to Ca2+, raising the extracellular ~on~ntration of the ion may lead to its accumulation within the cell according to the electrochemical gradient. A23187, on the other hand, which is known as an intramembranous Ca2+ carrier, may enhance the entry of Ca2+ into cells. The effects of the increased Ca2+ concentration and A23187 on the swimming behaviour of sea-urchin embryos were a decrease in swimming speed, which was observed throughout all developmental stages examined, and an increase in the fraction of backward swimming, which was observed only after the pluteus stage. Since the locomotor activity of the swimming embryos is derived from the movement of cilia on their body surface, this fact can be interpreted in terms of the action of Ca2’ on ciliary movement, i.e. intracellular or possibly intra~lia~.~~~ulation of Ca2+ resulted in inactivating the ciliary activity or inducing ciliary reversal. Spontaneous occurrence of ciliary reversal and an increase in its frequency induced experimentally by Ca2+ stimulation were seen only after the pluteus stage (ca 40 hr after insemination) but never before this critical developmental stage. The strong stage
88
MIHO DEGAWA et
specificity of the appearance of ciliary reversal in the course of development is in line with the results of previous experiments on electric stimulation (Baba, 1975). The increase induced by Ca2+ stimulation in the fraction of embryos showing ciliary reversal, in combination with the fact that ciliary reversal was not found in Ca-free solutions, indicates that ciliary reversal, a newly acquired faculty developed first at the pluteus stage, is mediated by Ca2+. Another action of Ca*+, i.e. inactivation of ciliary activity, was qualitatively different from that on ciliary reversal, in that it was not stage-specific. Thus, the decrease in ciliary activity measured as that in swimming velocity was found in both forward and backward swimming embryos, while it was not always accompanied by an increase in the fraction of backward swimming. In the pluteus stage, elevation of the external Ca*+ concentration led to decreased forward and backward swimming velocities without increase in the fraction of backward swimming (Figs 47), while A23187 increased this fraction, which was accompanied by decreased swimming activity (Figs 5 and 7). One simple explanation for these findings is that ciliary beating would be inactivated at moderately elevated intracellular Ca2+ concentration and ciliary reversal would be induced by further elevation in this concentration. However, this is not in agreement with the results of experiments on electric stimulation (Baba, 1975; Baba and Mogami, in preparation), in which the ciliary reversal induced by electric stimulation occurred with rather slightly increased beating activity. An alternative possibility for these facts is a distinct separation of two regulatory systems in the ciliary motile machinery, at least with regard to the sensitivity to Ca2+. One is a beating pattern-modulation system, and another is a beating activity-modulation system. The former system may be assumed to be located in the ciliary axoneme, according to recent investigations on reactivation of the ciliary axoneme, especially with Chlurnydomonas in which isolated ciliary axonemes show Ca2+-dependent changes in beating pattern (Bessen et al., 1980). The latter may be assumed to distribute more generally in the cell interior. On the other hand, it has been demonstrated that voltage-sensitive Ca2+ channels, in Paramecium, reside in the ciliary membrane, while resting conductance involving Caleakage channels is located in the somatic membrane (Ogura and Takahashi, 1976; Machemer and Ogura, 1979). If similar localization of these different species of channels is assumed for the ciliated cells of seaurchin embryos, the apparent difference in the sensitivity to Ca 2+ of the ciliary regulatory systems postulated above can be explained as below (see also Fig. 8). In the absence of Ca2+ stimulation, i.e. in the normal ASW, Ca2+ enters through the voltagedependent channels in the ciliary membrane when they are opened spontaneously or experimentally by electric stimulation, accumulates within the cilium, and acts only on the beating pattern-modulation system (Fig. 8b). In this case, reversal beats would not be accompanied by decreased beating activity, because the second system postulated apart from the ciliary motile machinery would be little affected by this entry of Ca 2+ into the cilium. When the external Ca2+ concentration was raised, the influx of Ca2+
al.
A 23187 \
d Fig. 8. Schematic illustration of a model for the local accumulation of Ca*+ into the ciliated cell of sea-urchin embryos. Thin arrows indicate the transition induced by an indicated experimental procedure. Open arrows indicate the transition by spontaneous opening of Ca-channels in the ciliary membrane. Shaded areas represent the site of Ca2+accumulation. through the leakage channels in the somatic membrane would exceed the efflux by Ca-pumps, resulting in an accumulation of Ca2+ within the cell body and in decreased beating activity by acting on the activitymodulation system (Fig. 8~). The entry of Ca2+ mediated by A23187, on the other hand, would occur randomly through the ciliary and somatic membranes increasing Ca2+ concentrations within both the cilium and cell body, so that the two modulation systems could be activated simultaneously (Fig. Sd). A consequence of this case would be reversed beats with low beating activity, as observed in this study. Similar situations of Ca2+ increase within both the cilium and cell body and of reversed beats with low activity could be brought about by the spontaneous opening of the voltage-dependent Ca2+ channels in the elevated Ca2+ medium, which is shown as transition from c to d in Fig. 8. If the frequency of spontaneous opening of the channels in the transition from c to d was the same as from a to b in the normal ASW, there would be no increase in the fraction of ciliary reversal. It can be explained by assuming higher Ca2+ transport rate effected by A23187 than under normal circumstances that the fraction of backward swimming increased when the embryos were treated with A23187. The subsequent processes following the Ca2 + entry into the cilium and cell body have not been clarified. The first action of Ca2+ on the beating pattemmodulation system might be its binding to calmodulin, which has been found in cilia and flagella of many organisms (Maihle et al., 1981; Walter and Schultz, 1981; Ohnishi er al., 1982; Stommel et al., 1982; Gitelman and Witman, 1980). The fact that anti-calmodulin drugs inhibit the Ca2+-dependent alteration of beating pattern (Reed et al., 1982; Otter et al., 1984) may support the actual function of Ca-calmodulin complex in the beating-pattern modulation. For the beating activity-modulation system CAMP must likely be considered as an actual intracellular modulator instead of Ca2+. According to Soliman (1984a, b) the swimming activity of young
Ca*+ sensitivity of cilia
sea-urchin embryos (up to gastrula stage) is enhanced by treatments which may induce an increase in intracellular cyclic nucleotides, especially CAMP. He also suggested that increased intracellular Ca2+ may decrease CAMP within the cells of embryos. These results suggest that the primary function of Ca2+ in the beating-activity modulation is to modulate the CAMP concentration. Calmodulin, therefore, may also be the counterpart of CaZ+ in this system, since the CAMP concentration could be decreased via the activation of phosphodiesterase by Ca-calmoduhn complex, a pronounced function of calmodulin (Cheung, 1980). The fact that non-swimming embryos induced by Ca2+ stimulation markedly increased at the pluteus stage, where ciliary reversal appears, suggests that the system for ciliary reversal may also function as a system for ciliary quiescence, although the complete inhibition of ciliary beating can also be an extreme consequence of execution of the activity-modulation system. Although we have discussed only about the direct action of Caz+ on the ciliary cells of sea-urchin embryos, indirect effects of the ion should not be neglected. The possible indirect effects would be derived from altered nervous control of the ciliary cells. It should be noted that the inactivation of ciliary beating, a response to CaZ+ of the ciliated cells, appears much earlier than the formation of functional nervous systems, which differentiate first at the pluteus stage (Ryberg, 1977; Burke, 1983). The inactivation of ciliary beating by Ca2+ stimulation, therefore, must be assumed to be independent of the nervous system. However, we cannot at present clarify whether the increased frequency of ciliary reversal observed in ASW containing A23187 is caused by the nervous activity enhanced by Caz+ stimulation, since ciliary reversal appears simultaneously with the nervous system in the course of development. Although the indirect effects through neuronal cells cannot be completely excluded, the direct effects on the ciliated cells may be most probable because these cells were directly exposed to external solutions so that Ca2+ and A23187 could act more easily on these cells than the underlying neuronal cells. The real action of Ca2+ on the ciliated cells of sea-urchin embryos will be best revealed by experiments on the isolated ciliary cells, which are now in progress in our laboratory.
89
Eckert R. (1972) Bioelectric control of ciliary activity. Science 176, 473-481. Gibbons I. R. and Gibbons B. H. (1980) Transient flagellar wavefo~s during remittent swimming in sea urchin sperm: I. Wave parameters. J. MUX. Res. CeN Motil. 1, 31-59. Gitelman S. E. and Witman 0. Ii. (1980) Purification of calmodulin from Chlamydomonas: Calmodulin occurs in cell body and flagella. .I. Cell Biol. 87, 764-770. Holwill M. E. J. and McGregor J. L. (1976) Effects of calcium on the flagellar movement in the trypanosome Crithidia oneopeiti. J. exp. Biof. 65, 229-242.
Hyams J. S. and Borisy G. G. (1978) Isolated flagellar apparatus of Ch~amydomo~: Characteri~tion of forward swimming and alteration of wave form and reversal of motion by calcium ions in vitro. J. Cell Sci. 33, 235-275.
Machemer H. and Ogura A. (1979) Ionic conductances of membranes in ciliated and deciliated Paramecia. J. Physiol. 296, 49-60.
Mackie G. O., Spencer A. N. and Strathmann R. (1969) Electric activity associated with ciliary reversal in echinoderm larva. Nature, Land. 223, 13841385. Maihle N. J., Dedman J. R., Means A. R., Chafouleas I. G. and Satir B. H. (1981) Presence and indirect immunofluorescent localization of calmodulin in Faramecium tetruurelia. J. Ceil Viol. 89, 695-699. Markman B. (1972) Swimming activity of sea urchin embryos subjected to increased concentration of potassium ions. Acta Embryoi. Exp. 1972, Suppl, 407-414. Maruyama Y. K. (1981a) Developmentof swimming behavior in sea urchin embrvos. I. J. exp. Zool. 215, 163-171. Maruyama Y. K. (198 1bjDevelopment of swimming behavior in sea urchin embryos. II. j. exp. Zool. 217,251-259. Naitoh Y. and Kaneko H. (1972) Reactivated Tritonextracted models of Paramecnon: ‘Modification of ciliary motility by calcium ions. Science 176, 523-524. Ogura A. and Takahashi K. (1976) Artificial deciliation causes loss of calcium-dependent response in Paramecium. Nature 264, 170-172. Ohnishi K.. Suzuki Y. and Watanabe Y. (1982) Studies on calmodulin isolated from Tetruhy~nu cilia and its localization within the cilium. Exp. Cell Res. 137, 217-227. Okada K. and Miyauchi H. (1954) Normal table of the early developmental stages on the sea urchin Hemicentrotus pulcherrimus. J. Gakugei, Tokushima Univ. 5, 57-68.
B. H. and Satir P. (1984) Otter T., Satir TriIluoperazine-induced changes in swimming behavior of Paramecium: Evidence for two sites of drug action. Cell Motii. 4, 249-267. Reed W., Lebduska S. and Satir P. (1982) Effects of tritluoperazine upon the calcium-dependent ciliary arrest response of fresh water mussel gill lateral cells. Cell Motil. 2, 405-427.
REFERENCES
Baba S. A. (1975) Developmental changes in the pattern of ciliary responses and the swimming behavior in some invertebrate larvae. In ~w~ming
and Flying in Nature
(Edited by Wu T. Y.-T., Brokaw C. J. and Brennen C.), Vol. 1, pp. 317-323. Plenum, New York. Besscn M., Fay R. B. and Witman G. B. (1980) Calcium control of wave form in isolated flagellar axonemes of ~~i~y~rno~. .I. Celf B&l. Sa, 446-45s. Brokaw C. J. (1979) Calcium induced asymmetrical beating of Triton-demembranated sea urchin sperm flagella. J. Cefl Biol. 82, 401411. Burke R. D. (1983) Development of the larval nervous system of the sand dollar, De&raster excentricus. Cell Tissue Res. 229, 145-154. Cheung W. Y. (1980) -.Calmodulin plays a pivotal role in ____^__ cellular regulatton. Science ZU7, 19-27.
Ryberg E. (1977) The nervous system of the early echinopluteus. Cell Tiss. Res. 179, 157-167. Soliman S. (1983) Pharmacological control of ciliary activity in the young sea urchin larva. Effects of choline& agents. Corni. Biochem. Physiol. 74C, 397-407. Soliman S. 11984a) Pha~acolo~~l control of ciliary activity in the‘young sea urchin l&a. Studies on the-role of Ca’+ and cyclic nucleotides. Comp. Biochem. Physiol. 78c, 183-191. Soliman S. (1984b) Pharmacological control of ciliary activity in the young sea urchin larva: Chemical studies on the role of cyclic nucleotides. Comp. Biochem. Physiol. 79C, 175-181.
Stommel E. W., Stephens R. E., Masure H. R. and Head J. F. (1982) Specific localization of scallop gill epithelium calmodulin in cilia. J. Cell Eiol. 92, 622628. Tsuchiya T. (1977) Effects of calcium ions on Tritonextracted lamellibranch gill cilia: Ciliary arrest response in a model system. Camp. Biochem. Physiof. SA, 353-361.
90
MIHO DEGAWA et al.
Walter M. F. and Satir P. (1979) Calcium does not inhibit active sliding of microtubules from mussel gill cilia. Nature, Lond. 278, 69-70.
Walter M. F. and Schultz J. (1981) Calcium receptor protein calmodulin isolated from cilia and cells of Paramecium tetraurelia. Eur. J. Cell. Biol. 24, 97-100.
$3.00+ 0.00 Pergamon Journals Ltd
03~-9629j8b
DEVELOPMENTAL CHANGES IN Ca2+ SENSITIVITY OF SEA-URCHIN EMBRYO CILIA MIHO DEGAWA, YOSHIHIROMOGAMIand SHOJIA. BABA Department of Biology, Ochanomizu University, Otsuka, Tokyo 112, Japan (Received 3 Januury 1986)
Abstract-1. The swimming velocity and direction of sea-urchin embryos were directly measured by multiple-exposure photography. 2. An increase in the Ca*+ concentration of the medium decreased the velocity in the stages from blastula through pluteus. 3. The calcium ionophore A23187added in the medium enhanced this Ca*+effect throughout all stages, and further significantly increased the number of embryos that stopped swimming and the ratio of backward vs forward, only when added after a critical stage, where spontaneous backward swimming began to occur. 4. These findings suggest that the regtdatory mechanism of ciliary activity changes in its sensitivity to Ca”+ during deveiopm&.
through pluteus. In this paper we directly measured the swimming velocity and its direction with respect to the orientation of embryos, i.e. forward or backward, by means of multiple-exposure photography. Because the locomotor activity of embryos is mainly dependent on the ciliary movement on their body surface, changes in these parameters of swimming must indicate those in ciliary movement, e.g. backward swimming can be regarded as with ciliary reversal. Thus, we found a qualitative as well as quantitative difference in the sensitivity to Ca*+ and A23 187 of the cilium between the pluteus and earlier stages. This finding may suggest an advantage of using a development-biological approach to seaurchin embryo cilia in the study of the Ca*+-dependent regulatory mechanism of ciliary movement.
Sea-urchin embryos change their slung hehaviour in the course of development. Backward swimming begins to occur at the early pluteus stage and thereafter characterizes the rather complicated swimming hehaviour of plutei. This fact shows that the ciliary motile mechanism may differentiate to acquire the ~pab~lity for ciliary reversal at a speciiic developmental stage (Baba, 1975). It has heen suggested that the ciliary reversal of sea-urchin embryos, similarly to the ciliary responses of many other organisms, follows the bioelectric activity of the ciliated cell (Mackie et al., 1969; Markman, 1972; Baba, 1975). It has heen argued that in ~u~~e~~~ the influx of Cat+ is induced by membrane depolarization and increases intracellular Ca2+, which in turn plays a major role in inducing ciliary reversal (Eckert, 1972). It has been further demonstrated by a substantial number of experiments on demembranated cilia and flagella, first with Put-ume&m (Naitoh and Kaneko, 1972) and then with a variety of organisms (Tsuchiya, 1977; Holwill and McGregor, 1976; Hyams and Borisy, 1978; Walter and Satir, 1979; Brokaw, 1979; Gibbons and Gibbons, 1980), that intracellular Ca*+ thus increased actually modulates ciliary and flagellar bending. It is, therefore, not unlikely that the influx of Ca*+, which is probably associated with the action potential that has been recorded from the pluteus (Mackie et ai., 1969), may trigger ciliary reversal by acting on the internal motile machinery of cilia. With sea-urchin embryos, however, little is known about the control activity of Ca*+ in the ciliary movement, though it has been reported that extracellular Ca*+ affects the swimming activity of the embryos of early developments stages (Soliman, 1984a). In order to gain a deeper insight into the mechanisms of ciliary responses, we investigated in vivo the effects of Ca*+ and the calcium ionophore A23187 on the ciliary movement of sea-urchin embryos at various developmental stages from blastula
MATERIALSAND METHODS Sea-urchin embryos
Sea-urchins (Hemicentrorur padcherrimup) were maintained in circulating natural sea-water at 17°C. Gametes were obtained by injection of 0.5s M KC1 into the body cavity. Eggs were shed into artificial sea-water (lamarin U, Jamarin Lab., Osaka) and inseminated in the same medium. Fertilized eggs (at least 95% fertilization, judged from the elevation of the fertilization membrane) were washed twice to remove excess sperm and incubated at 17°C with paddle stirring. Under these conditions, embryogenesis proceeded almost s~chronously up to 60 hr. The developmental stages were determined from the morphology of embryos according to the table of the early developmental stages of H. pulcherrimus in Okada and Miyauchi (1954). Experimental solutions
Artificial sea-water (ASW), prepared for the observation and recording of the swi~in8 behaviour of embryos, contained 450 mM NaCl, IOmM KCI, IO mM CaCl,, 25 mM MgCl,, 28 mM MgSO,, 10 mM Tris-HQ, PH. 8.0. The concentration of calcium ion was 1OmM in the standard ASW as above, and ranged 0-4OmM in modified ASWs with the total osmolarity kept constant by adjusting the concentration of Na+. 83
MIHO DEGAWAet al.
84
A23187 (Calbiochem-Behring Corp., LaJolla, CA) was dissolved as 1.0 and 10.0 mg/ml stock solutions in dimethylsulfoxide (DMSO), and diluted in ASW. A23187 formed a fine particulate suspension upon addition to ASW. To disperse A23187, ASW was sonicated for 10min in an ice bath, which was then used within 11 hr. The final concentration of DMSO in ASW used was below 2.5 ,ul/ml. In control experiments, DMSO at such a low concentration did not affect the swimming behaviour of the embryos (not shown). All chemicals (reagent grade) were from Wako Pure Chemical Co., Tokyo, unless otherwise specified. Recording of the swimmingbehaviour Every S hr after hatching, embryos were collected by hand centrifuge from 30 ml of the culture, resuspended in 3 ml ASW, and left for about 10min at room temperature (ca 17°C) to adapt to ASW. Several drops of the condensed embryos were then transferred to about 3 ml of the eltperimental solution in a 6-cm Petri dish, in which the solution made a thin layer of ca 1 mm at the bottom. For recovery from the mechanical disturbance upon mixing, the embryos were left for a further 7min in the Petri dish placed in a constant-temperature chamber, which was also kept at 17°C. Then the swimming behaviour was recorded within 30 min after the embryos were transferred. The swimming behaviour of the embryos in the Petri dish was observed by a binocular microscope (X-Tr, Olympus, Tokyo) and recorded on Tri-X film (Eastman Kodak) under a dark-field stroboscopic illumination obtained using a circular xenon flash tube (MFT-48, Miyata Elec. Works Inc., Tokyo) as shown in Fig. 1. Throughout observation and recording, the temperatures of the microscope stage and Petri dish were kept at 17°C with a water jacket (Fig. 1). For measurements of the swimming velocity and direction, the swimming embryos were photographed with five consecutive flashes at fixed time intervals (1 or 2 s), giving multiple-exposure photographs as shown in Fig. 2. The first of these five flashes was brighter than the others, so that the first image along the path of locomotion recorded was clear
and, therefore, the swimming direction of individual embryos could be determined. For both forward and backward swimming embryos, the velocity was calculated from the average distance between sequential images divided by the time interval mentioned above. From enlarged film, the coordinates of the points, fixed in individual embryos, were measured along the swimming path on a digitizer (Gradimate U4-30, Oscon Co., Tokyo) interfaced with a minicomputer (OKITAC SO/lo, Oki Elec. Ind. Co., Tokyo), where the velocity was computed.
RESULTS Swimming behaviour of sea-urchin embryos
Sea-urchin embryos swam freely in the Petri dish, but the vertical locomotion was limited because of the small depth (ca 1 mm) of ASW. The swimming of the embryos recorded as described above was therefore virtually two-dimensional, as shown in Fig. 2, so that it was easy to analyse the velocity and direction of swimming accurately. Although the embryos were observed to swim forming helical paths and rotating on the animal-vegetal axis, as reported by other authors (Maruyama, 1981a, b; Soliman, 1983), the method of recording used in the present study was not appropriate to analyse the detailed patterns of swimming path and rotation. Therefore, we analysed mainly the velocity and direction of swimming. Forward swimming was observed and recorded throughout the developmental stages, while spontaneous backward swimming only in the pluteus stage, as shown in Figs 2-5 and 7. The velocity of forward swimming increased in the early stages and decreased later, making a maximum of about 0.3 mm/s at the late gastrula stage. In the pluteus stage, the embryos often ceased to swim and the velocity of actively swimming embryos also gradually decreased during further development. The velocity of backward swimming was always lower than that of forward swimming, and among preparations as well as with the time of development, these two velocities varied parallelly to one another (Figs 3-5). Effects of extracellular swimming behaviour
X8L
Fig. 1. Setup for recording of the swimming behaviour of sea-urchin embryos. Light from a circular xenon flash lamp (Xel) is limited by a central cylinder (CL) and a hole of the bottom of a constant-temperature chamber (CH) to effect dark-field illumination. The lamp is operated stroboscopically with pulses produced by a pulse generator (PG). Another pulse, which is synchronized with opening of the camera shutter (SH), triggers, to operate the lamp for recording, programmed sequential five pulses, the first of which is used for the brightest flash by an intensitv modulator (IM). CA, camera; MO, micrdscope objective; W, water jacket; T, semiconductor thermometer; PS, power supply for flashing.
Ca2+ and A23187
on the
Figure 4 shows changes in the velocities of forward and backward swimming in ASWs of different Ca2+ concentrations as a function of the time after insemination. The time course of the changes for forward swimming in ASWs of Ca2+ concentrations higher or lower than 10 mM (standard) is nearly parallel to that in the standard ASW, with the velocity at higher Ca2+ concentrations being smaller (see also Fig. 6). The velocity of backward swimming was observed to decrease in ASWs of higher Ca2+ concentrations, although such decreases were not clearly detected by measurements as in forward swimming. Spontaneous backward swimming itself was never observed in ASW not containing Ca’+ added (not shown). The addition of A23 187 to ASWs induced changes in the swimming behaviour of sea-urchin embryos, particularly in the velocity and direction of swimming. When A23187 was added, the velocities of both forward and backward swimming were reduced at
Ca*+ sensitivity
of cilia
85
Fig. 2. Stroboscopic recordings of swimming embryos of H. pulcherrimus with five successive xenon flashes. The first image in each sequence is the brightest. (a) Embryos at the prism stage (43 hr after insemination), recorded with flash intervals of 1 s; (b) at the pluteus stage (60.5 hr), with flash intervals of 2 s. Beside selected several sequences, the swimming direction is represented by open arrows (forward swimming) and by closed arrows (backward). Inset: enlarged print of a pluteus, which is swimming backward. The shape of embryo is a key factor in determination of its swimming direction as shown here. Scale bars, 0.2 mm.
L
13
a
1
23 TIME
33 AFTER
43
53
INSEMINATION
63 ( hrs }
Fig. 3. Swimming velocity of embryos of H. pulcherrimus in the course of development. Embryos are bathed in the standard ASW. The mean values of the velocity with SD bars from five separate experiments (marked with different symbols) are shown. The upper half of the figure corresponds to the velocity of forward swimming and the lower half to backward swimming. Developmental stages determined from the morphology of embryos are shown at the top of the figure.
MIHO DEGAWAet al.
86
prism
gaatrula
I
1
20.5
30.5 TIME
,
50.5
40.5
AFTER
INSEMINATION
60.6
(hrs)
Fig. 4. Effect of Ca*+ on the swimming velocity of embryos of If. puicherrimus in the course of development. Embryos are bathed in ASWs containing Ca’+ at different ~ncentrations, 0: 2.5 mM, q !: IO mM (standard), 0: 20 mM. Mean values from a typical experiment are shown. The upper half of the figure is for forward swimming and the lower half for backward swimming. Stages at the top of figure. Figure 7 shows the ratio of the number of actively swimming embryos out of all embryos recorded, i.e. the percentage swimming, in ASWs of various concentrations of Ca2+ and A23187. The percentage swimming was usually as high as 70400% in ASWs containing Ca2+ at 10mM (standard) and lower concentrations in the absence of A23187, though in
any developmental stage, as shown in Figs 5 and 6. Figure 6 shows the effect of A23187 on the relation of the velocity of forward swimming and the Ca2+ ~n~ntration in ASW. A23 187 enhanced the effect of Ca’” in reducing the velocity of forward swimming, especially in the range of the Ca2+ concentrations higher than 2.5 mM, in all the developmental stages. An extreme consequence of the enhancement of the effect of Ca2+ by A23 187 was a complete cessation of swimming in the pluteus stage, though this was observed only for high Ca*+ con~ntrations (Fig. 6).
8
/an'am /
/iaatrula
t2katula
1
*
20.6
TIME
““I
30.5
AFTER
40.1
I
plutaua
so.4
INSEMINATION
”
00.~
1
( hrs)
Fig. 5, Effect of A23187 on the swimming velocity of embryos of H. ~Ze~e~~~~ in the course of development. The concentrations of A23187 in ASW are 0 M (O), lo-’ M (c]) and 10e6 M (a). The mean values of the velocity with SD bars from a typical experiment are shown. The upper half of the figure is for forward swimming and the lower half for backward swimming.
Caz+ CONCENTRATION (mM )
Fig. 6. Effect of A23187 on the velocity of forward swimming of embryos of H. ~~~rrirn~ as a function of Ca** concentration. The concentrations of A23187 in ASWs are 0 M (O), lo-‘M (0) and 10m6M (a). The mean values of the velocity from a typical experiment are shown. The number at the upper right of each graph represents the time after insemination.
Ca2+ sensitivity of cilia
a TIME
AFTER
87
0 15.5 25.5 35.5 45.5 55 5
65.5
b
INSEMlNATlON ( hrs )
Fig. 7. Effects of Cal+ and A23 187 on the swimming activity of embryos of H. pulcherrimur in the course of development. (a) Percentage swimming; (b) fraction of backward swimming. The Ca*+ concentrations in ASWs are 2.5 mM (O), IO mM (D) and 20 mM (0). The A23187 concentrations in ASWs are 0 M (upper), 10-‘M (middle) and 10V6M (lower}.
this figure it happened to be low in the early blastula stage. Ca2+ at higher concentrations (20 mM in this figure, closed circle) tended to induce a rapid decrease in the percent swimming. This phenomenon was usually found after a critical developmental stage (cu 40 hr after insemination in Fig. 7a). It should be noted that, after this critical stage, backward swimming began to be induced by A23187 (Fig. 7b). A consequence of addition of A23187 up to lob6 M in ASWs containing Ca2+ at 10mM and higher concentrations was a further decrease in the percent swimming, sometimes to nearly 0% with 10e6M A23187. A23187 had little effect on swimming in the embryos, at least at the early stages, in al1 ASWs used and throu~out development in ASWs cont~ning below 10mM Ca2+ (Fig. 7a). Direct observation by a dark-field microscope at a higher magnification showed that the cilia of the non-swimming embryos at higher Ca2+ concentrations did not move. At lower CaZC concentrations, on the other hand, the cilia were beating weakly even in the embryos which appeared to be scored as non-swimming; such embryos only rotated on the animal-vegetal axis and readily restored their propulsive activity on a mechanical agitation such as a tap of the Petri dish, although they stopped swimming and began to rotate again a few seconds later. A23187 increased the fraction of backwardswimming embryos among the actively swimming ones in a CL?+-dependent manner (Fig. 7b), although it could not induce backward swimming sence of Ca2+ (not shown). A maximum
in the ab-
increase in the fraction was observed at Ca2+ concentrations between 5 and 1OmM. At higher Ca2+ concentrations, backward swimming could hardly be detected because the swimming activity itself was heavily diminished. It should be noted that backward
swimming was induced by A23187 only at the late developmental stages, where spontaneous backward swimming was observed (Fig. 7b). DISCUSSION
The aim of this paper is to test the action of Ca ions on the ciliary movement of sea-urchin embryos. In order to manipulate the intracellular Ca*+ concentration, two methods were employed, raising the extracellular Ca2+ concentration and adding the calcium ionophore A23187 to external solutions. Since plasma membranes are, more or less, capable of leaking Ca2+ or having leakage conductor to Ca2+, raising the extracellular ~on~ntration of the ion may lead to its accumulation within the cell according to the electrochemical gradient. A23187, on the other hand, which is known as an intramembranous Ca2+ carrier, may enhance the entry of Ca2+ into cells. The effects of the increased Ca2+ concentration and A23187 on the swimming behaviour of sea-urchin embryos were a decrease in swimming speed, which was observed throughout all developmental stages examined, and an increase in the fraction of backward swimming, which was observed only after the pluteus stage. Since the locomotor activity of the swimming embryos is derived from the movement of cilia on their body surface, this fact can be interpreted in terms of the action of Ca2’ on ciliary movement, i.e. intracellular or possibly intra~lia~.~~~ulation of Ca2+ resulted in inactivating the ciliary activity or inducing ciliary reversal. Spontaneous occurrence of ciliary reversal and an increase in its frequency induced experimentally by Ca2+ stimulation were seen only after the pluteus stage (ca 40 hr after insemination) but never before this critical developmental stage. The strong stage
88
MIHO DEGAWA et
specificity of the appearance of ciliary reversal in the course of development is in line with the results of previous experiments on electric stimulation (Baba, 1975). The increase induced by Ca2+ stimulation in the fraction of embryos showing ciliary reversal, in combination with the fact that ciliary reversal was not found in Ca-free solutions, indicates that ciliary reversal, a newly acquired faculty developed first at the pluteus stage, is mediated by Ca2+. Another action of Ca*+, i.e. inactivation of ciliary activity, was qualitatively different from that on ciliary reversal, in that it was not stage-specific. Thus, the decrease in ciliary activity measured as that in swimming velocity was found in both forward and backward swimming embryos, while it was not always accompanied by an increase in the fraction of backward swimming. In the pluteus stage, elevation of the external Ca*+ concentration led to decreased forward and backward swimming velocities without increase in the fraction of backward swimming (Figs 47), while A23187 increased this fraction, which was accompanied by decreased swimming activity (Figs 5 and 7). One simple explanation for these findings is that ciliary beating would be inactivated at moderately elevated intracellular Ca2+ concentration and ciliary reversal would be induced by further elevation in this concentration. However, this is not in agreement with the results of experiments on electric stimulation (Baba, 1975; Baba and Mogami, in preparation), in which the ciliary reversal induced by electric stimulation occurred with rather slightly increased beating activity. An alternative possibility for these facts is a distinct separation of two regulatory systems in the ciliary motile machinery, at least with regard to the sensitivity to Ca2+. One is a beating pattern-modulation system, and another is a beating activity-modulation system. The former system may be assumed to be located in the ciliary axoneme, according to recent investigations on reactivation of the ciliary axoneme, especially with Chlurnydomonas in which isolated ciliary axonemes show Ca2+-dependent changes in beating pattern (Bessen et al., 1980). The latter may be assumed to distribute more generally in the cell interior. On the other hand, it has been demonstrated that voltage-sensitive Ca2+ channels, in Paramecium, reside in the ciliary membrane, while resting conductance involving Caleakage channels is located in the somatic membrane (Ogura and Takahashi, 1976; Machemer and Ogura, 1979). If similar localization of these different species of channels is assumed for the ciliated cells of seaurchin embryos, the apparent difference in the sensitivity to Ca 2+ of the ciliary regulatory systems postulated above can be explained as below (see also Fig. 8). In the absence of Ca2+ stimulation, i.e. in the normal ASW, Ca2+ enters through the voltagedependent channels in the ciliary membrane when they are opened spontaneously or experimentally by electric stimulation, accumulates within the cilium, and acts only on the beating pattern-modulation system (Fig. 8b). In this case, reversal beats would not be accompanied by decreased beating activity, because the second system postulated apart from the ciliary motile machinery would be little affected by this entry of Ca 2+ into the cilium. When the external Ca2+ concentration was raised, the influx of Ca2+
al.
A 23187 \
d Fig. 8. Schematic illustration of a model for the local accumulation of Ca*+ into the ciliated cell of sea-urchin embryos. Thin arrows indicate the transition induced by an indicated experimental procedure. Open arrows indicate the transition by spontaneous opening of Ca-channels in the ciliary membrane. Shaded areas represent the site of Ca2+accumulation. through the leakage channels in the somatic membrane would exceed the efflux by Ca-pumps, resulting in an accumulation of Ca2+ within the cell body and in decreased beating activity by acting on the activitymodulation system (Fig. 8~). The entry of Ca2+ mediated by A23187, on the other hand, would occur randomly through the ciliary and somatic membranes increasing Ca2+ concentrations within both the cilium and cell body, so that the two modulation systems could be activated simultaneously (Fig. Sd). A consequence of this case would be reversed beats with low beating activity, as observed in this study. Similar situations of Ca2+ increase within both the cilium and cell body and of reversed beats with low activity could be brought about by the spontaneous opening of the voltage-dependent Ca2+ channels in the elevated Ca2+ medium, which is shown as transition from c to d in Fig. 8. If the frequency of spontaneous opening of the channels in the transition from c to d was the same as from a to b in the normal ASW, there would be no increase in the fraction of ciliary reversal. It can be explained by assuming higher Ca2+ transport rate effected by A23187 than under normal circumstances that the fraction of backward swimming increased when the embryos were treated with A23187. The subsequent processes following the Ca2 + entry into the cilium and cell body have not been clarified. The first action of Ca2+ on the beating pattemmodulation system might be its binding to calmodulin, which has been found in cilia and flagella of many organisms (Maihle et al., 1981; Walter and Schultz, 1981; Ohnishi er al., 1982; Stommel et al., 1982; Gitelman and Witman, 1980). The fact that anti-calmodulin drugs inhibit the Ca2+-dependent alteration of beating pattern (Reed et al., 1982; Otter et al., 1984) may support the actual function of Ca-calmodulin complex in the beating-pattern modulation. For the beating activity-modulation system CAMP must likely be considered as an actual intracellular modulator instead of Ca2+. According to Soliman (1984a, b) the swimming activity of young
Ca*+ sensitivity of cilia
sea-urchin embryos (up to gastrula stage) is enhanced by treatments which may induce an increase in intracellular cyclic nucleotides, especially CAMP. He also suggested that increased intracellular Ca2+ may decrease CAMP within the cells of embryos. These results suggest that the primary function of Ca2+ in the beating-activity modulation is to modulate the CAMP concentration. Calmodulin, therefore, may also be the counterpart of CaZ+ in this system, since the CAMP concentration could be decreased via the activation of phosphodiesterase by Ca-calmoduhn complex, a pronounced function of calmodulin (Cheung, 1980). The fact that non-swimming embryos induced by Ca2+ stimulation markedly increased at the pluteus stage, where ciliary reversal appears, suggests that the system for ciliary reversal may also function as a system for ciliary quiescence, although the complete inhibition of ciliary beating can also be an extreme consequence of execution of the activity-modulation system. Although we have discussed only about the direct action of Caz+ on the ciliary cells of sea-urchin embryos, indirect effects of the ion should not be neglected. The possible indirect effects would be derived from altered nervous control of the ciliary cells. It should be noted that the inactivation of ciliary beating, a response to CaZ+ of the ciliated cells, appears much earlier than the formation of functional nervous systems, which differentiate first at the pluteus stage (Ryberg, 1977; Burke, 1983). The inactivation of ciliary beating by Ca2+ stimulation, therefore, must be assumed to be independent of the nervous system. However, we cannot at present clarify whether the increased frequency of ciliary reversal observed in ASW containing A23187 is caused by the nervous activity enhanced by Caz+ stimulation, since ciliary reversal appears simultaneously with the nervous system in the course of development. Although the indirect effects through neuronal cells cannot be completely excluded, the direct effects on the ciliated cells may be most probable because these cells were directly exposed to external solutions so that Ca2+ and A23187 could act more easily on these cells than the underlying neuronal cells. The real action of Ca2+ on the ciliated cells of sea-urchin embryos will be best revealed by experiments on the isolated ciliary cells, which are now in progress in our laboratory.
89
Eckert R. (1972) Bioelectric control of ciliary activity. Science 176, 473-481. Gibbons I. R. and Gibbons B. H. (1980) Transient flagellar wavefo~s during remittent swimming in sea urchin sperm: I. Wave parameters. J. MUX. Res. CeN Motil. 1, 31-59. Gitelman S. E. and Witman 0. Ii. (1980) Purification of calmodulin from Chlamydomonas: Calmodulin occurs in cell body and flagella. .I. Cell Biol. 87, 764-770. Holwill M. E. J. and McGregor J. L. (1976) Effects of calcium on the flagellar movement in the trypanosome Crithidia oneopeiti. J. exp. Biof. 65, 229-242.
Hyams J. S. and Borisy G. G. (1978) Isolated flagellar apparatus of Ch~amydomo~: Characteri~tion of forward swimming and alteration of wave form and reversal of motion by calcium ions in vitro. J. Cell Sci. 33, 235-275.
Machemer H. and Ogura A. (1979) Ionic conductances of membranes in ciliated and deciliated Paramecia. J. Physiol. 296, 49-60.
Mackie G. O., Spencer A. N. and Strathmann R. (1969) Electric activity associated with ciliary reversal in echinoderm larva. Nature, Land. 223, 13841385. Maihle N. J., Dedman J. R., Means A. R., Chafouleas I. G. and Satir B. H. (1981) Presence and indirect immunofluorescent localization of calmodulin in Faramecium tetruurelia. J. Ceil Viol. 89, 695-699. Markman B. (1972) Swimming activity of sea urchin embryos subjected to increased concentration of potassium ions. Acta Embryoi. Exp. 1972, Suppl, 407-414. Maruyama Y. K. (1981a) Developmentof swimming behavior in sea urchin embrvos. I. J. exp. Zool. 215, 163-171. Maruyama Y. K. (198 1bjDevelopment of swimming behavior in sea urchin embryos. II. j. exp. Zool. 217,251-259. Naitoh Y. and Kaneko H. (1972) Reactivated Tritonextracted models of Paramecnon: ‘Modification of ciliary motility by calcium ions. Science 176, 523-524. Ogura A. and Takahashi K. (1976) Artificial deciliation causes loss of calcium-dependent response in Paramecium. Nature 264, 170-172. Ohnishi K.. Suzuki Y. and Watanabe Y. (1982) Studies on calmodulin isolated from Tetruhy~nu cilia and its localization within the cilium. Exp. Cell Res. 137, 217-227. Okada K. and Miyauchi H. (1954) Normal table of the early developmental stages on the sea urchin Hemicentrotus pulcherrimus. J. Gakugei, Tokushima Univ. 5, 57-68.
B. H. and Satir P. (1984) Otter T., Satir TriIluoperazine-induced changes in swimming behavior of Paramecium: Evidence for two sites of drug action. Cell Motii. 4, 249-267. Reed W., Lebduska S. and Satir P. (1982) Effects of tritluoperazine upon the calcium-dependent ciliary arrest response of fresh water mussel gill lateral cells. Cell Motil. 2, 405-427.
REFERENCES
Baba S. A. (1975) Developmental changes in the pattern of ciliary responses and the swimming behavior in some invertebrate larvae. In ~w~ming
and Flying in Nature
(Edited by Wu T. Y.-T., Brokaw C. J. and Brennen C.), Vol. 1, pp. 317-323. Plenum, New York. Besscn M., Fay R. B. and Witman G. B. (1980) Calcium control of wave form in isolated flagellar axonemes of ~~i~y~rno~. .I. Celf B&l. Sa, 446-45s. Brokaw C. J. (1979) Calcium induced asymmetrical beating of Triton-demembranated sea urchin sperm flagella. J. Cefl Biol. 82, 401411. Burke R. D. (1983) Development of the larval nervous system of the sand dollar, De&raster excentricus. Cell Tissue Res. 229, 145-154. Cheung W. Y. (1980) -.Calmodulin plays a pivotal role in ____^__ cellular regulatton. Science ZU7, 19-27.
Ryberg E. (1977) The nervous system of the early echinopluteus. Cell Tiss. Res. 179, 157-167. Soliman S. (1983) Pharmacological control of ciliary activity in the young sea urchin larva. Effects of choline& agents. Corni. Biochem. Physiol. 74C, 397-407. Soliman S. 11984a) Pha~acolo~~l control of ciliary activity in the‘young sea urchin l&a. Studies on the-role of Ca’+ and cyclic nucleotides. Comp. Biochem. Physiol. 78c, 183-191. Soliman S. (1984b) Pharmacological control of ciliary activity in the young sea urchin larva: Chemical studies on the role of cyclic nucleotides. Comp. Biochem. Physiol. 79C, 175-181.
Stommel E. W., Stephens R. E., Masure H. R. and Head J. F. (1982) Specific localization of scallop gill epithelium calmodulin in cilia. J. Cell Eiol. 92, 622628. Tsuchiya T. (1977) Effects of calcium ions on Tritonextracted lamellibranch gill cilia: Ciliary arrest response in a model system. Camp. Biochem. Physiof. SA, 353-361.
90
MIHO DEGAWA et al.
Walter M. F. and Satir P. (1979) Calcium does not inhibit active sliding of microtubules from mussel gill cilia. Nature, Lond. 278, 69-70.
Walter M. F. and Schultz J. (1981) Calcium receptor protein calmodulin isolated from cilia and cells of Paramecium tetraurelia. Eur. J. Cell. Biol. 24, 97-100.