Beating of Cilia of Sea Urchin Embryos: A Critical Comparison of the Normal and Reversed Beating of Cilia of Isolated Cells

Beating of cilia and eukaryotic flagella occurs as a result of sliding displacement between axonemal doublet-microtubules. In order to execute a specific pattern of bend formation, which, depending on the various environments of individual organelles, is well specialized for the effective transport of the surrounding medium, there must be a mechanism controlling the sliding movement of the doublet-microtubules. Comparative approaches analyzing bend formation in different types of beat patterns provide information relevant to the control mechanism. Analyses of bend formation of flagella using mutants of Chlamydomonas with functionally defective motile components (Brokaw et al. 1982; Brokaw and Luck, 1985; Brokaw and Kamiya, 1987) and of voltage-controlled ciliary beating in ciliates (Machemer, 1986; Sugino and Machemer, 1988, 1990; Mogami and Machemer, 1991) have increased our knowledge about the properties of the mechanism underlying the dynamic control of sliding movement.

Cilia of sea urchin embryos are a useful tool for a comparative study of ciliary bend formation. The cilia change their type of response during the course of development, responding to electrical (Baba, 1975; Baba and Mogami, 1987) and Ca²⁺ stimulation (Degawa et al. 1986). Because the bend remained planar during the response, the bending transient could be analyzed with high time resolution (Baba and Mogami, 1987). In addition, an examination of the morphology of just a single cilium per cell made it possible to analyze the bend forms with high space resolution (Baba and Mogami, 1985).

In this paper, we focus on the reversal response of the cilia of pluteus larvae. Isolated cells from the ciliated epithelia rotate under the influence of their own ciliary movement with a beat plane constrained to be parallel to the surface of the microscope slide. Ciliated cells from pluteus larvae spontaneously change the net direction of rotation as a result of reversal of the beating direction. Reversed beating occurs with a different configuration from that of normal beating. This suggests that the regulation of the motile apparatus differs in normal and in reversed beating. We compared reversed beating with normal beating of the same cilium on a rotating cell, and characterized the difference between the two types of beating in terms of the temporal and spatial properties of local oscillatory activity along the length of the cilium.

Eggs and sperm of the sea urchin, Hemicentrotus pulcherrimus, were obtained by injection with 0.55mol l⁻¹ KCl. Eggs were fertilized in Jamarin U artificial sea water (ASW, Jamarin Laboratory, Osaka) and developed at 17°C with paddle stirring (Degawa et al. 1986).

Pluteus larvae (40–60h after insemination) were collected by hand centrifuge and suspended in a dispersion medium (0.9mol l⁻¹ glycine, 90mmol l⁻¹ KCl, 2mmol l⁻¹ EDTA, pH8.0). After 15min incubation at room temperature, the embryos became deformed and a few ciliated cells were dissociated by their own ciliary movement. To encourage dissociation, the suspension of embryos was pipetted in and out of a large-bore pipette (>3mm inner diameter). Aggregated cells, mainly of endodermal origin, and spicules were removed by low-speed centrifuge. Isolated cells in a dispersion medium were spun down and washed twice with Ca²⁺-free ASW (465mmol l⁻¹ NaCl, 10mmol l⁻¹ KCl, 25mmol l⁻¹ MgCl₂, 28mmol l⁻¹ MgSO₄, 10mmol l⁻¹ Tris–HCl, pH8.0) and suspended in the same medium. Cells were kept in suspension before use.

Before recording the ciliary movement, ASW containing an appropriate amount of CaCl₂ was added to produce a final composition in the medium of 450mmol l⁻¹ NaCl, 10mmol l⁻¹ KCl, 25mmol l⁻¹ MgCl₂, 28mmol l⁻¹ MgSO₄, 10mmol l⁻¹ CaCl₂, 10mmol l⁻¹ Tris–HCl, pH8.0 (standard ASW). A drop of the suspension was plated on the slide and covered with a coverslip using a 50 μm plastic film as a spacer. To allow cells to move freely, the glass surface was coated with agarose gel by flowing 0.05% hot agarose solution on its tilted upper surface, followed by air-drying.

The movement of the ciliated cells was observed through a microscope (BH-2, Olympus Optical Co., Tokyo) with phase-contrast optics (objectives, ×40NH, N/A 0.70) under stroboscopic illumination from a xenon flash tube (Strobex system 236, Chadwick-Helmuth, El Monte, CA). The movement was recorded by focusing the microscopic images through an ocular (×5FK, Olympus) and a 13-mm slit on 35-mm films (Plus-X or Tri-X, Eastman Kodak) with an oscilloscope camera (PC-2B, Nihon Koden, Tokyo) in running film mode at a film speed of 1 ms⁻¹ and a stroboscope flash rate of 80Hz.

We selected from the recordings images of cilia showing spontaneous reversed beating and normal beating recorded just before the response. Images that corresponded to the transition between normal and reversed beating were excluded (Baba and Mogami, 1987). Almost equal numbers of images of normal and reversed beating were used for the analysis of the movement.

Recorded films were enlarged with a microfilm reader to a final magnification of ×4000. Direct photographic prints and tracings on the screen of the microfilm reader were used for the point by point analysis, where the coordinates of the ciliary shaft were measured along the length of the cilium at intervals of 0.2–0.3 μm on a digitizer (Gradimate U4-30, Oscon Co., Tokyo) interfaced with a minicomputer (OKITAC 50/10, Oki Elec. Ind. Co., Tokyo).

For the assessment of rotational movement of ciliated cells, the orientation of the cell, which was defined by a vector directed from the centre of the cell (the centre of rotation) to the base of the cilium, was measured using fixed geometrical markers in the focal field. Parameters of the rotational movement were obtained by fitting analytical equations to the data (see Results) by the method of least squares for non-linear functions (Powell, 1965), using a program written by Dr Y. Oyanagi and filed in the library (C7/TCPOW_I) of the Computer Center of the University of Tokyo.

Shear angles (i.e. tangential angles) of a cilium, as a function of distance along the cilium, were calculated following a method described previously (Baba and Mogami, 1985), using a smoothing parameter of n=6 (cf. Fig. 2 in Baba and Mogami, 1985). The method is briefly summarized as follows.

In order to reduce noise due to errors of digitization, resultant shear curves were smoothed once by a method of moving averages on linear functions (SG13, in the library supplied by Oki Elec Ind. Co.); smoothed values for each data point were calculated by linear regression of the point and its nearest neighbours on both sides. Ciliary bending shapes shown in this paper were drawn by computer, utilizing the smoothed values. For the analysis of local oscillatory activity of the cilium, shear angles at a given distance from the base were calculated by interpolation from the data set of the shear curves, and were plotted as a function of the percentage of the beat period, which was determined by the analysis of the rotational movement of the cell. The mean course of the plot (curves in Fig. 5A) was determined by the smoothing method described above. The amplitude and centre of the oscillation were calculated from the maximum and minimum values found in the mean course.

Isolated cells were observed to rotate on the slide surface by their own ciliary movement (Fig. 1). The rotation was observed to occur around a fixed point, with little translocation of the cell body parallel to the surface. While the cell rotated steadily, ciliary beating occurred parallel to the slide surface. Since embryonic cilia show a planar bend configuration (Baba, 1975; Baba and Mogami, 1987), we can observe the whole length of the cilium in the same focal plane (Fig. 1). The planar bend configuration was further confirmed by recording with an objective of higher numerical aperture (1.25, ×100NH, Olympus).

Cells rotated with a back-and-forth movement, which depended on the alternately reversing force generation corresponding to the effective and recovery strokes of the beat (Figs 1 and 2A,B). The net direction of the rotation was constant in Ca²⁺-free ASW and changed spontaneously in standard ASW containing Ca²⁺. It was observed that steady rotation was often interrupted by reversals of the rotational direction usually lasting for several hundred milliseconds. Because there is a single cilium per cell, changes in the net direction of cell rotation should correspond to a change in the effective direction of water transport generated by the cilium. In the present study, we compared the ciliary movements recorded from cells that spontaneously changed their net direction of rotation.

Spontaneous reversed beating usually lasted for several hundred milliseconds. In such a short time, we observed only a few cycles of beating. In order to obtain an averaged bending motion, we reconstructed beating within one cycle by sorting the recorded images of different beat cycles by the percentage of the beat period. We used the value of f_r obtained from the least-squares fitting of the rotational movement of cell bodies to calculate the percentage value within the averaged beat period, defining the ‘zero time’ (0% of beat period) at the transition from R-rotation to E-rotation (Fig. 2C). Images were sorted in the same way for normal beating. Cells that continued reversed beating for more than two cycles were used for analyses.

Figs 3 and 4 show the shear curves of a cilium in normal and reversed beating, respectively. In these curves, descending limbs (regions between open circles) correspond to bends with their convex side towards the direction of the effective stroke of normal beating (E-bend) and ascending limbs (regions between filled circles) correspond to bends in the opposite direction (R-bend). The slope of the limb represents the curvature of the bend. In normal beating, both descending and ascending limbs retain an almost constant slope as they shift along the length of the cilium, indicating that both E- and R-bends propagate with constant curvatures (Fig. 3). In reversed beating, in contrast, such constancy of curvature was observed only during the propagation of the E-bend. Fig. 4 shows that the R-bend of reversed beating gradually increased in curvature during tipward propagation, reaching a similar value to that of the R-bend of normal beating. The increment of curvature during the propagation of the R-bend was found in all recordings of the spontaneous reversed beating.

According to the sliding filament hypothesis, shear angle is proportional to the amount of sliding displacement (u) between peripheral doublet-microtubules. The parallel shift of a portion of shear curves within a fraction of time (Δt) indicates that the rate of sliding displacement (Δu/Δt) is constant within the region that is covered by this portion: Δu/Δt>0 during the propagation of an E-bend (E-sliding) and Δu/Δt<0 during the propagation of an R-bend (R-sliding). It therefore follows that, in normal beating, the rate of sliding displacement was constant along individual bending regions in either direction. In reversed beating, however, the increment of curvature during the propagation of the R-bend indicates that the rate of R-sliding in reversed beating was lower in the distal part of the R-bends, while E-sliding occurred at a constant rate along the whole length of the bending region.

Fig. 5 shows plots of shear angles as a function of the percentage of the beat period in normal and reversed beating, measured at various distances from the base. This figure indicates that, when normalized to the fraction of the beat period, the local oscillation in both normal and reversed beating shows similar patterns among individual cilia. It also suggests that the mean course in each plot, obtained by moving average (curves in Fig. 5), is a good representation of the oscillatory movement. Fig. 6 shows the parameters of the angular oscillation calculated from the mean course.

The time courses of the angular oscillation of normal and reversed beating appear to be similar at the proximal portion (about 2.6 μm from the base; Fig. 5 bottom), where both patterns possess a period with little sliding activity, before the onset of E-sliding. In reversed beating, the pause appears, with almost equal duration, between R- and successive E-sliding, irrespective of the position along the length of the cilium, although its temporal position is gradually delayed from base to tip. In normal beating, however, the length of the pause before the onset of E-sliding becomes gradually shorter in the more distal portions and another pause appears after the end of E-sliding. Fig. 6A shows changes in the temporal pattern of the local oscillation in terms of the fraction of the period between the intersections of the descending limb (R-sliding) and the ascending limb (E-sliding) through the centre line of the oscillation (dashed lines in Fig. 5). The duration of the pause after the end of R-sliding of normal beating gradually decreases towards the tip, so that there appears to be almost a mirror-image relationship at the distal portion between the time courses of the shear angles for normal and reversed beating (Fig. 5, top). Fig. 6B,C indicates that the differences in the amplitudes and in the centre of the oscillation are small in the proximal portion (up to a distance of about 4 μm from the base). In the more distal portion, the amplitude of oscillation of reversed beating is smaller, and the centre of oscillation of reversed beating is more negative than that of the normal beat. The parameters of the local oscillation of normal and reversed beating have almost equal values at the proximal portion, which suggests that the change in bending pattern between normal and reversed beating involves little (or no) change in the oscillatory activity at the base of the cilium.

In this paper, ciliary movement was investigated in normal and reversed beating recorded from cells isolated from echinoplutei. Isolation of the cells may change the viscous load on the beating cilium, because the cell body rotates free from the connection to the surface of the embryos. The difference between the hydrodynamic situations of ciliated cells in free rotation and those tethered on the surface of embryos could be compared to that of sperm swimming freely or attached by the head to the substratum. In normal beating, the amplitude of shear angle oscillation at the basal region (2–3 μm from the base) is almost equal in rotating and tethered cells; 0.7–1.5rad in normal beating of rotating cells (Fig. 6B) and about 1.2rad in tethered cells (Baba and Mogami, 1987). In rotating cells, the amplitude increases with the distance from the base, having a maximum at the middle of the cilium, and is attenuated in the more distal region to a smaller value at the tip (0.8–1.1rad) (Fig. 6B) than that measured in tethered cells (about 1.6rad). This may correspond to the constriction of shear amplitude that is observed in the flagellar beating of free-swimming sperm (Brokaw, 1991) but not in sperm attached by the head to the substratum (Hiramoto and Baba, 1978). Changes in the hydrodynamic environment might emphasize the feature of the propagation of the R-bend of reversed beating, with an increase in curvature, which is not very evident when the beat occurs on the tethered cells (Baba and Mogami, 1987).

Rotational movement of the isolated cells can be used as a measure of the water transport by the cilium, as in the case of the rotation of uniflagellated mutant Chlamydomonas (Brokaw and Luck, 1983). Since the rotation of the cell occurs under conditions of low Reynolds number (of the order of 10⁻³), it is inferred that the rotation occurs in close relation to the propulsive thrust exerted by the cilium, which varies within a cycle of beating. Results of the least-squares fitting indicate that the rotational movement caused by normal and reversed beating can be characterized by separate equations. Equation 2 involves a rapid change in the direction between successive cycles.

This may represent the sharp transition between E- and R-rotations in reversed beating, which occurs at the same time as the rapid propagation of E-sliding, followed shortly after by R-sliding (Fig. 5). In normal beating, delays in the onset of R-sliding along the distance from the base (Fig. 5) may result in the gradual transition between E- and R-rotation, as represented by equation 1. The lower value of the mean rotation velocity in reversed beating (Table 1) suggests that reversed beating has a lower efficiency in water transport than normal beating has. This may explain the lower speed of backward swimming of the plutei (60–80% of the speed of forward swimming) (Degawa et al. 1986; Mogami et al. 1992). The reduced efficiency seems to be related to symmetrical bend formation in reversed beating compared to highly asymmetrical bend formation in normal beating (Figs 3 and 4).

Analyses of the shear angle of normal- and reversed-beating cilia demonstrate that changes in the doublet-microtubule sliding activity depend on the position along the length of the cilium. This is illustrated in the propagation of the R-bend in reversed beating, which involves differences in sliding rate within a bending region (Fig. 4), and in the temporal pattern of the local angular oscillation (Fig. 5). In normal beating, the oscillation was observed to change its temporal pattern along the length of the cilium, although in reversed beating an almost uniform pattern was observed throughout the length. As a result, the difference in the profiles of beating oscillation between normal and reversed beating was greater towards the tip, whereas the proximal region maintained a fixed profile irrespective of the beating pattern. This suggests that the change in bending pattern between normal and reversed beating does not involve changes in the initiation of oscillation activity at the base, but is entirely caused by modification of the propagation of the activity.

Baba and Mogami (1987) previously reported a tipward propagation of the local response at the transient, from normal to reversed beating, that was induced by electrical stimulation. The apparent change at the proximal region during the transition is not necessarily inconsistent with the present data, which show a nearly identical temporal profile of the proximal oscillation for both normal and reversed beating, bearing in mind that the transient response includes a phase shift of oscillatory activity. The time course of the response (Fig. 4 in Baba and Mogami, 1987) can be interpreted as a consequence of the resetting of local oscillatory movement at each point of the cilium to the beginning of E-sliding of the subsequent reversed beating. Provided that the resetting of the oscillation is delayed increasingly towards the tip, a phase shift resulting from the resetting may be observed as the tipward propagation of the local response upon electrical stimulation.

For the regulation of asymmetry of the ‘flagellar’ beating of sperm and Chlamydomonas, Eshel and Brokaw (1987) proposed the biased baseline model. The model explains the change in the beating asymmetry solely by the change in the centre of the oscillation (static bias) on which a symmetrical metachronous oscillation is superimposed. The present data, however, show that, in the transformation of the bending pattern from normal to reversed beating, the change in the static bias occurs concomitantly with the changes in the temporal pattern of the local oscillation (Fig. 6). Therefore, the change in the bending pattern of sea urchin embryo cilia probably cannot be explained only by an alteration of the centre of oscillation.

From axial view analyses on the basal activity of three-dimensional ciliary beating, Machemer and Sugino (1989) indicated that voltage-activated ciliary responses in Stylonychia consisted of static noncyclic and cyclic responses. This fact also suggests that a simple biased baseline model does not apply to the mechanism regulating the beating of the ‘ciliary’ organelles.

Fig. 6 shows that the difference in the temporal oscillatory activity of the cilium becomes greater in parallel with the increment of the change in the static bias of the oscillation. Do the changes in the two parameters occur independently? Okuno and Brokaw (1981) demonstrated that Ca²⁺ could reversibly change the configuration of a sperm flagellum in the presence of vanadate and ATP. The noncyclic change in the curvature may provide the bias of the propagating metachronous oscillation (Eshel and Brokaw, 1987). In the marginal cirri of Stylonychia, voltage-clamp experiments applying extremely slow voltage ramps showed a noncyclic orientation response below the threshold potential of cyclic activity (de Peyer and Machemer, 1973). These results suggest that the mechanism for shifting the spatial bias of oscillations may function independently from the temporal control of the local oscillation. In fact, a ‘flip-flop’ movement without cyclic activity between two inactive configurations was observed in epaulette cilia of echinoplutei (Mogami et al. 1991).

Previous reports suggest that ciliary responses observed in sea urchin embryos are Ca²⁺-dependent (Baba, 1975; Degawa et al. 1986; Baba and Mogami, 1987). The absence of reversed beating in isolated cells immersed in Ca²⁺-free ASW suggests that an influx of Ca²⁺ into the ciliated cells from the external medium is required for the initiation of reversed beating. Although it is not known whether Ca²⁺ is the primary intracellular regulator of the ciliary beating of sea urchin embryos, the present data indicate that Ca²⁺ would act to regulate the oscillatory activity along the whole length of the cilium.

Reversed beating is performed only after the morphogenesis to pluteus larva. The newly acquired ability to beat in the reversed direction may be needed to alter the ciliary motile apparatus. Detailed analysis of the ciliary beating of the cells isolated from various developmental stages did not show significant differences in the parameters of normal beating, such as the beat frequency, maximum curvature and the rate of E-sliding and the propagation of the R-bend (Sekiguchi et al. 1986). This result suggests that incorporation of the capability for reversed beating occurs without affecting the motile mechanism responsible for generating normal beating.