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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
.
Abstract
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).
Afzelius,
Cilia-related diseases.
2004,
Pubmed
Anstrom,
Organization of the ciliary basal apparatus in embryonic cells of the sea urchin, Lytechinus pictus.
1992,
Pubmed
,
Echinobase
Auclair,
Cilia regeneration in the sea urchin embryo: evidence for a pool of ciliary proteins.
1966,
Pubmed
,
Echinobase
Brokaw,
Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function.
1987,
Pubmed
Böhmer,
Ca2+ spikes in the flagellum control chemotactic behavior of sperm.
2005,
Pubmed
,
Echinobase
Choksi,
Switching on cilia: transcriptional networks regulating ciliogenesis.
2014,
Pubmed
Gibbons,
The effect of partial extraction of dynein arms on the movement of reactivated sea-urchin sperm.
1973,
Pubmed
,
Echinobase
Gueron,
Energetic considerations of ciliary beating and the advantage of metachronal coordination.
1999,
Pubmed
Guirao,
Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia.
2010,
Pubmed
Inaba,
Two states of the conformation of 21S outer arm dynein coupled with ATP hydrolysis.
1989,
Pubmed
,
Echinobase
Inaba,
Calcium sensors of ciliary outer arm dynein: functions and phylogenetic considerations for eukaryotic evolution.
2015,
Pubmed
Kunimoto,
Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet.
2012,
Pubmed
Marshall,
Cilia orientation and the fluid mechanics of development.
2008,
Pubmed
Mitchell,
A positive feedback mechanism governs the polarity and motion of motile cilia.
2007,
Pubmed
Mizuno,
A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella.
2009,
Pubmed
Mizuno,
Calaxin drives sperm chemotaxis by Ca²⁺-mediated direct modulation of a dynein motor.
2012,
Pubmed
Nonaka,
Determination of left-right patterning of the mouse embryo by artificial nodal flow.
2002,
Pubmed
Okada,
Mechanism of nodal flow: a conserved symmetry breaking event in left-right axis determination.
2005,
Pubmed
Prulière,
Atypical protein kinase C controls sea urchin ciliogenesis.
2011,
Pubmed
,
Echinobase
Rompolas,
An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria.
2010,
Pubmed
Shiba,
Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response.
2008,
Pubmed
Sorokin,
Reconstructions of centriole formation and ciliogenesis in mammalian lungs.
1968,
Pubmed
Takamatsu,
Asymmetric rotational stroke in mouse node cilia during left-right determination.
2013,
Pubmed
Wallingford,
Planar cell polarity signaling, cilia and polarized ciliary beating.
2010,
Pubmed
Wirschell,
The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans.
2013,
Pubmed
Wood,
Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm.
2005,
Pubmed
,
Echinobase
Yaguchi,
ankAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo.
2010,
Pubmed
,
Echinobase
Yoshiba,
Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.
2012,
Pubmed