Inverse relationship of Ca2+-dependent flagellar response between animal sperm and prasinophyte algae
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Symmetry/asymmetry conversion of eukaryotic flagellar waveform is caused by the changes in intracellular Ca2+. Animal sperm flagella show symmetric or asymmetric waveform at lower or higher concentration of intracellular Ca2+, respectively. In Chlamydomonas, high Ca2+ induces conversion of flagellar waveform from asymmetric to symmetry, resulting in the backward movement. This mirror image relationship between animal sperm and Chlamydomonas could be explained by the distinct calcium sensors used to regulate the outer arm dyneins (Inaba 2015). Here we analyze the flagellar Ca2+-response of the prasinophyte Pterosperma cristatum, which shows backward movement by undulating four flagella, the appearance similar to animal sperm. The moving path of Pterosperma shows relatively straight in artificial seawater (ASW) or ASW in the presence of a Ca2+ ionophore A23187, whereas it becomes circular in a low Ca2+ solution. Analysis of flagellar waveform reveals symmetric or asymmetric waveform propagation in ASW or a low Ca2+ solution, respectively. These patterns of flagellar responses are completely opposite to those in sperm flagella of the sea urchin Anthocidaris crassispina, supporting the idea previously proposed that the difference in flagellar response to Ca2+ attributes to the evolutional innovation of calcium sensors of outer arm dynein in opisthokont or bikont lineage.
KeywordsCalaxin Cilia Dynein Opisthokont Prasinophyte Sperm flagella
Cilia and flagella are oscillating motile machinery in eukaryotes. The internal microtubule structure, called the axoneme, is composed of 9 doublet and 2 singlet microtubules and is well conserved during eukaryotic evolution (Gibbons 1981; Inaba 2003, 2011; Mitchell 2007). Each doublet microtubule possesses two projections, the outer and inner dynein arms. Ciliary and flagellar motility are based on the sliding of doublet microtubules by outer and inner arm dyneins. The sliding of microtubules is converted into bending, of which propagation propels ciliary and flagellar movement.
Ciliated or flagellated cells change the swimming direction in response to several stimuli, including mechanical, chemical and electrical ones. Upon reception of these extracellular signals, cells transiently increase the intracellular Ca2+ concentration. Ca2+ modulates the activity of dyneins through Ca2+-binding proteins. The radial spokes and central pair are involved in the formation of planar waveform through regulation of inner arm dyneins (Bannai et al. 2000; Porter and Sale 2000; Smith and Yang 2004). Several lines of evidence from Ciona sperm (Mizuno et al. 2009, 2012) and Chlamydomonas (Mitchell and Rosenbaum 1985; Kamiya and Okamoto 1985; Wakabayashi et al. 1997) demonstrate that outer arm dynein is also essential for Ca2+-dependent modulation of ciliary and flagellar motility, in particular for symmetry/asymmetry conversion of flagellar waveform.
A Ca2+-binding protein, light chain 4 (LC4), is associated with γ heavy chain of the outer-arm dynein of Chlamydomonas flagella (King and Patel-King 1995). It shows sequence similarity to CaM or CaM-related proteins such as centrin/caltractin and troponin C. On the other hand, an ortholog of LC4 is not found in sperm flagella. Instead, another type of Ca2+-binding protein, showing sequence similarity to neuronal calcium sensor proteins, termed calaxin, is bound to β heavy chain (orthologous to Chlamydomonas γ heavy chain) of outer arm dynein in sperm flagella of the ascidian Ciona intestinalis (Mizuno et al. 2009). These properties suggest that calaxin and LC4 are the distinct Ca2+-sensor for outer arm dynein in Ciona and Chlamydomonas, respectively, although it is still possible that another Ca2+-binding protein, DC3 (Casey et al. 2003), may play a role in the regulation in Chlamydomonas. Responses of flagellar waveform to Ca2+ are inverse between Ciona and Chlamydomonas: the waveform of sperm flagella becomes asymmetric at high Ca2+, whereas that of Chlamydomonas becomes symmetric. Phylogenetic analysis of the calcium sensors clearly separates the eukaryotes into calaxin-user, opisthokonts, and LC4-user, bikonts, with the exception of excavates (Inaba 2015). However further demonstration is necessary for these mirror-image relationships between opisthokonts and bikonts using a variety of organisms.
The flagellar movement with asymmetric waveform is called “ciliary” type and that with symmetric movement is called “flagellar” type. Cells with the former type move forward but those with the latter move backward (Inaba et al. 2015). In contrast, Ciona sperm always move forward but change their direction by modulating flagellar asymmetry. Inouye and Hori (1991) examined flagellar beat and swimming patterns of 22 green plants and categorized into six different groups depending on the flagellar patterns. Among them, a group with the genera Pedinomonas, Cymbomonas and Pterosperma usually show backward movements with the cell body always at the forefront, of which appearance is quite similar to those of animal sperm. Here we comparatively analyzed the flagellar movements and their responses to Ca2+ between sea urchin sperm and Pterosperma to clearly confirm the above “mirror-image” phylogenetic relationship in the Ca2+-mediated flagellar responses between opisthokonts and bikonts.
Materials and methods
The sea urchin Anthocidaris crassispina was collected near Shimoda Marine Research Center and kept in an aquarium with running seawater until use. Sperm were collected by the intrablastocoelar injection of 0.5 M KCl. The prasinophyte Pterosperma cristatum (NIES-626) was provided by NIES (National Institute for Environmental Studies, Tsukuba, Japan) and cultured in ASW-IMK (Daigo’s artificial seawater containing IMK medium; Wako, Osaka, Japan) at 20 °C on a 14/10 h light/dark cycle. Flagellar movements were recorded and analyzed as previously described (Mizuno et al. 2012). Briefly, the suspension of sperm or Pterosperma was placed on a glass slide coated with 1% BSA to avoid adhesion of sperm to the glass surface. Images were recorded through a phase contrast microscope (BX51, Olympus, Tokyo) with a 10× or 20× objective (UPlan FLN, Olympus) connected to a high-speed CCD camera (HAS D3, Ditect, Tokyo) at 100 or 500 frames s−1. Trajectories of the cells and waveforms were analyzed using Bohboh software (Bohboh Soft, Tokyo, Japan). The shear angle was calculated and drawn by Bohboh as the angle between the axis of basal body (head axis) and the tangent of a position along the flagellum (Brokaw 1991). Artificial seawater (ASW) consisted of 462.01 mM NaCl, 9.39 mM KCl, 10.81 mM CaCl2, 48.27 mM MgCl2, and 10 mM Hepes-NaOH, (pH 8.0). To prepare low Ca2+ sea water (LCSW), we added Ca2+-free sea water [475.7 mM NaCl, 9.39 mM KCl, 10.81 mM CaCl2, 48.27 mM MgCl2, 10 mM EGTA and 10 mM Hepes-NaOH, (pH 8.0)] into the culture medium or ASW equivalently. The Ca2+ ionophore A23187 and ionomycin was purchased from Sigma–Aldrich (St Louis, MO, USA).
We compared the cell morphology and flagellar movements in artificial seawater between the sea urchin Anthocidaris crassispina and the prasinophyte Pterosperma cristatum (Fig. 1a; Table 1; Movies S1, S2 and S3). The long axis of P. cristatum cell body was ~10 μm, which is almost twice as long as that of A. crassispina sperm head. The flagellar length of P. cristatum was also twice as long as A. crassispina. P. cristatum has four flagella but usually swims in aligned or bundled with the cell body positioned at anterior end, which appears similar to sperm swimming. This is defined as backward swimming when compared with normal swimming direction of other green algae (Inouye and Hori 1991). The four flagella were occasionally observed to drift without beating flagella. The flagellar beat frequency was a little higher in P. cristatum flagella than that of A. crassispina sperm flagella, whereas the wavelength of the former was more than twice as large as the latter. In total the number of waves was calculated as a little more than 1 in both cells. As expected from the large size of flagella, the swimming speed of P. cristatum was more than twice as fast than that of A. crassispina sperm (Table 1). Although both cells showed great difference in the size of flagella and swimming speed, they commonly propagated almost one wave with a principal and a reverse bend (Fig. 1b).
Motility characteristics of the sea urchin Anthocidaris crassispina and the prasinopyte Pterosperma cristatum
Head size (μm)
Flagellar length (μm)
Swimming velocity (μm/sec)
Beat frequency (Hz)
Wave length (μm)
Number of waves
Sea urchin Anthocidaris crassispina
5.64 ± 0.29 (N = 56)
37.18 ± 1.69 (N = 56)
153.53 ± 36.16 (N = 69)
40.73 ± 2.42 (N = 12)
26.37 ± 1.57 (N = 12)
1.40 ± 0.09 (N = 12)
9.97 ± 1.64 (N = 44)
69.95 ± 5.22 (N = 44)
366.55 ± 82.92 (N = 79)
47.58 ± 6.02 (N = 9)
63.52 ± 5.96 (N = 9)
1.12 ± 0.13 (N = 9)
Pterosperma cristatum showed a quick change of the swimming direction in seawater. This response became frequent in the ASW-IMK culture medium (Fig. 2a; Movie S3). This was previously thought as “avoiding reaction” (Inouye and Hori 1991). The change accompanied a “turn” movement with the conversion of flagellar waveform as an highly asymmetric one (Fig. 2b). We tested if this reaction depends on the intracellular Ca2+ using an ionophore. The addition of ionomycin to ASW-IMK culture medium caused the cell to move mostly in a straight path and significantly decreased of turn movement (Fig. 2a). The ASW-IMK culture medium has a composition similar to SW, containing 10 mM Ca2+. Therefore, the result suggests that the increase of Ca2+ by the influx by ionomycin induced the loss of avoiding reaction (Fig. 2c).
The inhibition of avoiding reaction by Ca2+ implies that the conversion of flagellar waveform in P. cristatum from symmetric to asymmetric needs decreased intracellular Ca2+. To elucidate this, we comparatively analyzed the flagellar waveform in ASW, ASW plus a Ca2+ ionophore A23187, and a low Ca2+ SW (LCSW). The sea urchin sperm moved in a circular way in ASW and the radius of the trajectory became smaller in the presence of A23187. The radius of swimming trajectory in LCSW became larger than that in SW (Fig. 3a). In contrast, the prasinophyte P. cristatum moved in rather a straight path in ASW and the addition of A23187 resulted in no clear change in the swimming trajectory (Fig. 3b). However, movement of P. cristatum became circular in LCSW (Fig. 3b). The analysis of average path curvature clearly showed that the response to Ca2+ in the conversion of straight/circular movement is contrary between sperm and prasinophyte (Fig. 3c).
Sperm with relatively planar flagellar beating swim straight or circular when the waveform is symmetric or asymmetric, respectively (Inaba et al. 2015). Therefore, Ca2+-dependent above changes in swimming trajectories indicate inverse conversion of symmetric/asymmetric flagellar waveforms between two organisms. To demonstrate this, we analyzed the shear angle along a flagellum, which is often used to show the microtubule sliding along a flagellum and thereby the wave asymmetry (Fig. 4; Brokaw 1991). The shear curve of sperm flagella from A. crassispina showed down-ward sloping in ASW, where the shear angle became negative at flagellar distance over ~15 μm (Fig. 5a), indicating the propagation of asymmetric waveform. In contrast, a similar profile was obtained in LCSW in the prasinophyte P. cristatum, where the shear angle became negative at flagellar distance over ~30 μm (Fig. 5a). Quantitative analysis of the maximum shear angle between proximal and distal regions of a flagellum clearly showed an inverse response to Ca2+ between A. crassispina sperm and P. cristatum (Fig. 5b).
The regulation of ciliary and flagellar motility are a key mechanism for organisms to perform directed movements and fluid flow. In general, such regulations are thought to be caused by the modulation of axonemal dyneins, resulting in the change of microtubule-sliding and ciliary/flagellar bend propagation. Ca2+ has been for long known to be a critical messenger regulating axonemal dyneins. Calmodulin or centrin is present in the radial spoke/central pair and inner arm dyneins. These structures are essential for the formation of asymmetric waveforms (Bannai et al. 2000; Porter and Sale 2000; Smith and Yang 2004). In Chamydmonas, the conversion of flagellar asymmetry needs the outer arm dynein (Kamiya and Okamoto 1985). A similar case is known in sperm flagella that the propagation of asymmetric flagellar waveform requires Ca2+-dependent regulation of the outer arm dynein (Mizuno et al. 2012). The molecular compositions of the outer arms dynein have been widely studied in Chlamydomonas and sperm flagella and many of the components are present in common (King 2000; Inaba 2007). However, the calcium sensor of the outer arm dyneins are phylogenetically different (King and Patel-King 1995; Mizuno et al. 2009; Inaba 2015).
The distinct calcium sensor, as well as possibly the number of motors, in the outer arm dynein between sperm and Clamydomonas flagella is proposed to be related to the mirror-image response of flagellar asymmetry to intracellular Ca2+. Distinctly separate use of calcium sensors appears to reflect phylogenetic lineages of eukaryotes (Inaba 2015). Chlamydomonas has two flagella and normally show forward swimming in breast-stroke way with asymmetric flagellar waveform. In a photophobic movement, the flagella waveform changes to symmetric, resulting in a backward straight swimming. These swimming patterns of Chlamydomonas using two flagella are not so compatible to that of sperm, although the mirror relationship of Ca2+ concentration and flagellar asymmetry is obvious. Pterosperma cristatum is a prasinophyte that swims like sperm by tightly aligning and coordinating four flagella (Inouye and Hori 1991). It shows clear turn movement with asymmetric flagella beating (Fig. 2), similar to that in sperm chemotactic behavior (Mizuno et al. 2012).
Some compound cilia show distinct structures that connect between multiple cilia (Tamm 2014). However, there seems to be no structural interaction among the four flagella in P. cristatum (Inouye et al. 1990; Inouye and Hori 1991). Recent study indicates that closely positioned cilia/flagella show wave synchronization due to hydrodynamic interaction (Brumley et al. 2014). Experiments using sea urchin sperm indicate that the beating plane and frequency become synchronized to the imposed vibration of the head (Shingyoji et al. 1991). Thus, it is plausible that each flagellum responses to Ca2+ and changes the waveform and that bundled flagella accordingly synchronize the movement by hydrodynamic interaction. Regarding the beating plane and its relationship with the axonemal structure, all four flagella under being bundled and synchronized are considered to have the same structural orientations. Since the axonemes of a prasinophyte show 9 + 2 structure lacking the outer arm dynein at doublet 1 (Hori and Moestrup 1987), like that of Chlamydomonas, further observation by electron microscopy could possibly confirm the axonemal orientation in the bundled flagella. Nevertheless, Pterosperma move with four bundled flagella, which are not the complete counterpart of sea urchin sperm flagella, and thus the use of monoflagellated prasinophyte, such as Cymbomonas (Throndsen 1988; Inouye and Hori 1991), could be desirable.
In this study, we show the inverse relationship between sperm and prasinophyte flagella: conversion to asymmetric flagellar waveform requires high Ca2+ in sperm flagella, whereas high Ca2+ reversely induces symmetric flagellar waveform in prasinophyte flagella (Fig. 6). The study clearly indicates that the relationship is not attributed to either direction of swimming or the relative positions of motile flagella to cell body or head, i.e. posterior in sperm and anterior in algae. Extracellular hair-like structures, called mastigonemes, are prominent in heterokont and cryptophyte algae. They are thought to change the fluid hydrodynamics around the flagellum and cause the reversal of wave propagation (Sleigh 1991). Pterosperma flagella are covered with scales and are also decorated by flexible mastigonemes as seen in Chlamydomonas (Witman et al. 1972; Inouye and Hori 1991; Martin and Melkonian 1994). These could affect the waveform of Pterosperma flagella but a computational simulation demonstrates that flexible mastigonemes, as seen in Pterosperma and Chlamydomonas, are ineffective at least in causing direction reversal (Namdeo et al. 2011).
As previously proposed, mirror-image relationship would be possibly result from the difference in the Ca2+ sensor proteins used for regulation of outer arm dynein (Inaba 2015). Animals and fungi (opisthokonts) use calaxin, whereas bikont species, except excavates and those lacking cilia/flagella or the outer arm dynein, such as embryophytes, moss and ferns use LC4 (Inaba 2015). However, the Ca2+ protein in Pterosperma has not been elucidated in the present study. BLAST search revealed that prasinophyte does not have a ortholog of calaxin. Instead, BLAST search of Chlamydomonas LC4 resulted in a significant hit (E value = 8e-52) with the prasinophyte Micromonas commoda protein with sequence similarity to LC4 (Accession number, XP_003056424). Thus, it is strongly suggested that calaxin and LC4 regulate the activity of outer arm dynein reversely in response to Ca2+. Further studies are needed to obtain direct evidences, such as molecular identification LC4 in the outer arm dynein from isolated flagella and an image analysis of Ca2+ dynamics during the change of flagellar asymmetry in Pterosperma.
We thank National Institute for Environmental Studies (NIES) for providing a strain of Pterosperma cristatum (NIES-626). This work was supported in part by Grant-in-Aid 15H01201 for Scientific Research on Innovative Areas and 22370023 for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).
Movie S1. Flagellar movement of the sea urchin A. crassispina sperm. Five hundred frames were recorded per second. The movie plays at 0.06× speed. (MOV 644 KB)
Movie S2. Flagellar movement of the prasinophyte P. cristatum. Five hundred frames were recorded per second. The movie plays at 0.06× speed. (MOV 222 KB)
Movie S3. Turn movement of the prasinophyte P. cristatum. Five hundred frames were recorded per second. The movie plays at 0.06× speed. (MOV 171 KB)
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