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Calaxin establishes basal body orientation and coordinates movement of monocilia in sea urchin embryos.
Mizuno K
,
Shiba K
,
Yaguchi J
,
Shibata D
,
Yaguchi S
,
Prulière G
,
Chenevert J
,
Inaba K
.
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Through their coordinated alignment and beating, motile cilia generate directional fluid flow and organismal movement. While the mechanisms used by multiciliated epithelial tissues to achieve this coordination have been widely studied, much less is known about regulation of monociliated tissues such as those found in the vertebrate node and swimming planktonic larvae. Here, we show that a calcium sensor protein associated with outer arm dynein, calaxin, is a critical regulator for the coordinated movements of monocilia. Knockdown of calaxin gene in sea urchin embryos results in uncoordinated ciliary beating and defective directional movement of the embryos, but no apparent abnormality in axoneme ultrastructure. Examination of the beating cycle of individual calaxin-deficient cilia revealed a marked effect on the waveform and spatial range of ciliary bending. These findings indicate that calaxin-mediated regulation of ciliary beating is responsible for proper basal body orientation and ciliary alignment in fields of monociliated cells.
Figure 1. Ciliary beating direction and basal structure orientation are initially random and then become aligned. (A) Swimming trajectories of embryos. Ten images acquired at 0.3 second intervals are superimposed. hps, hours post fertilization. Scale, 0.5 mm. (B) Mean swimming velocities of embryos of different ages. N = 45–88 from 3–5 embryos. (C) Quantitative comparison of ciliary beating directions. A, anterior; P, posterior. N = 158 (14 h), 125 (24 h) from 8–9 embryos. *p < 0.001. (D) Schematic of angular analysis plotted in E and F. A vector drawn from the cilia transition zone (green) towards the centriole (magenta) gives the direction of ciliary basal structure (black) with respect to the anterior (A) - posterior (P) embryonic axis. A typical immunofluorescence image is shown. (E,F) Phase contrast (left), immunostaining of ciliary basal structures (middle) and circular histograms (right) in two representative embryos at 14 hpf (E) and 24 hpf (F). Yellow arrows indicate the direction in which ciliary basal structures are extended. Scale, 50 μm (left), 10 μm (middle). Circular histograms show the orientation of ciliary basal structures for 23 (14 hpf) and 22 (24 hpf) cilia. CSD, circular standard deviation. (G) Immunoblot of whole embryo proteins with anti-calaxin antibody.
Figure 2. Knockdown of calaxin results in serious damage of embryonic swimming without changes in ciliary structures. (A) Immunoblots of control or MO-injected embryos (24 hpf) by anti-calaxin and anti-Ac-α-tubulin antibodies. (B) Swimming trajectories of sea urchin embryos at early gastrula stage; images acquired at 0.3 second intervals for 10 seconds are superimposed. Scale, 5 mm. (C) Comparison of mean swimming velocities of embryos injected with different concentrations of calaxin morpholino (MO). N = 61 (control), 38 (0.5 mM MO), 55 (1 mM MO), 28 (2 mM MO). *p < 0.001 vs control (0 mM). (D,E) Immunofluorescence comparison between control (D) and MO (1 mM)-injected (E) embryos. The upper rows, whole embryos; lower rows, enlarged images of squared regions. Scale, 50 μm (upper), 10 μm (lower). (F,G) Control embryo (F) and calaxin morphant (G) immunostained with antibodies against outer arm dynein, showing the presence of outer arm dyneins in morphant cilia. Scale, 50 μm. (H) Thin-section electron microscopy of cilia shows both outer (arrows) and inner arm dyneins in calaxin morphants. Scale, 100 nm.
Figure 3. Lack of calaxin leads to disoriented ciliary movement with abnormal bend curvatures but normal beat frequency. (A) Typical waveforms during ciliary beating in control embryos and embryos injected with calaxin MO (1 mM). Motions of one cycle of beating are represented by the superimposition of images acquired at 5 msec intervals. (B) Definition of ciliary curvature and the angle of effective stroke. Both values were measured using video recordings of individual cilia and statistically analyzed as shown in Table 1. (C,D) Sequential images from high-speed videos (10 msec intervals) in a control embryo (C) and an embryo injected with calaxin MO (2 mM) (D). Arrowheads indicate the tip positions of individual cilia. Control cilia showed directional and coordinated beating but those from MO-injected embryo showed irregular ciliary beatings. Scale bar, 50 μm. (E) Quantitative comparison of ciliary beating directions, categorized as beating in anterior-posterior (A-P) direction, posterior-anterior direction (P-A) or other direction. N = 350 (control), 296 (0.5 mM MO), 424 (1 mM MO) and 212 (2 mM MO) from 16–29 embryos. *p < 0.001 vs control.Table 1. Properties of ciliary beating in control embryos and in those injected with calaxin-MO.
Figure 4. Calaxin morphants are deficient in the coordinated orientation of ciliary basal structures. (A,B) Phase contrast (left), immunostaining of ciliary basal structures (middle) and circular histograms (right) of control (A) and MO (2 mM)-injected (B) embryos at 24 hpf. Magenta, centrioles (γ-tubulin); green, transition zones (aPKC). Yellow arrows, direction of ciliary basal structures. Circular histograms show the orientation of ciliary basal structures. N = 89 (control) and 95 (2 mM MO), using 7–9 different embryos. Scale, 50 μm (left), 10 μm (middle). (C) Swimming trajectories of embryos treated with 100 μM GdCl3. 10 images acquired at 0.2 second intervals are superimposed. Scale, 1 mm. (D) Comparison of mean swimming velocities of control and Gd3+-treated embryos. N = 30–40 from 3 embryos. (E,F) Phase contrast (left), immunostaining of ciliary basal structures (middle) and circular histograms (right) of control (E) and Gd3+-treated (F) embryos at 20 hpf. Circular histogram, N = 502 (control) and 693 (100 μM Gd3+) using 24–25 different embryos. Scale, 50 μm (left), 10 μm (middle).
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