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Cytoskeleton (Hoboken)
2013 Aug 01;708:453-70. doi: 10.1002/cm.21127.
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Glutathione transferase theta in apical ciliary tuft regulates mechanical reception and swimming behavior of Sea Urchin Embryos.
Jin Y
,
Yaguchi S
,
Shiba K
,
Yamada L
,
Yaguchi J
,
Shibata D
,
Sawada H
,
Inaba K
.
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An apical tuft, which is observed in a wide range of embryos/larvae of marine invertebrates, is composed of a group of cilia that are longer and less motile than the abundant lateral cilia covering the rest of the embryonic surface. Although the apical tuft has been thought to function as a sensory organ, its molecular composition and roles are poorly understood. Here, we identified a glutathione transferase theta (GSTT) as an abundant and specific component of the apical tuft in sea urchin embryos. The expression of GSTT mRNA increases and becomes limited to the animal plate of the mesenchyme blastula, gastrula, and prism larva. Electron microscopy and tandem mass spectrometry demonstrated that the apical tuft contains almost every axonemal component for ciliary motility. Low concentrations of an inhibitor of glutathione transferase bromosulphophthalein (BSP) induce bending of apical tuft, suggesting that GSTT regulates motility of apical tuft cilia. Embryos treated with BSP swim with normal velocity and trajectories but show less efficiency of changing direction when they collide with an object. These results suggest that GSTT in the apical tuft plays an important role in the mechanical reception for the motility regulation of lateral motile cilia in sea urchin embryos.
Figure 1. Cilia of normal and Zn-treated sea urchin embryos. (A) Phase contrast microscopic images of normal and Zn-treated embryos at 24- and 36-h post fertilization, respectively. The asterisk indicates the apical tuft. The Zn-treated embryos were animalized and bore long cilia that resemble apical tuft cilia. Bar, 50 μm. (B) Differential interference contrast images of isolated cilia from normal and Zn-treated embryos. Cilia were obtained by deciliation with high-salt seawater. Note that the cilia from Zn-treated embryos are as long as the apical tuft cilia. Bar, 50 μm. (C) Distribution of the length of cilia isolated from normal (open bar) and Zn-treated (closed) embryos. Percentages of cilia in whole isolated cilia are shown. The horizontal bar represents the range of apical tuft cilia directly measured from normal embryos before deciliation. (D) Typical images of cilia from normal (top) and Zn-treated (bottom) embryos by thin-sectioned electron microscopy. Bar, 100 nm. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
Figure 2. Comparison of ciliary proteins between normal and Zn-treated embryos. (A) SDS-PAGE of ciliary proteins (∼20 μg) from normal (N) and Zn-treated (Zn) embryos. The arrowhead shows ∼25-kDa protein specifically present in cilia from Zn-treated embryos. B, successive extraction of cilia isolated from normal (N) and Zn-treated (Zn) embryos. Cilia were successively demembranated with a buffer containing 0.1% Triton X-100 (TX), a high salt buffer (KCl) and then a low salt buffer (TD). PPt represents the axonemal residues. The ∼25-kDa protein (asterisk) shows present in TX fraction. (C) and (D) 2DE patterns of ciliary proteins from normal and Zn-treated embryos. Horizontal numbers represent pH ranges for isoelectric focusing. The two lower panels show magnified images of the ∼25-kDa regions. The red arrowheads show three spots of ∼25-kDa proteins specifically present in cilia from Zn-treated embryos.
Figure 3. Phylogenetic analysis of GSTs. The consensus phylogenetic tree was constructed by the Neighbor-Joining method from sea urchin and mammalian proteins. The numbers at each node are the percentage bootstrap value of 100 replicates. The accession numbers of the protein sequences used are given in Materials and Methods. Blue letters: proteins of sea urchins. Hp-GSTT was identified in this study with the sea urchin H. pulcherrimus. The phylogenetic analysis results indicate that the ∼25-kDa protein identified in the apical tuft is GST theta (GSTT).
Figure 4. Expression of GSTT gene during the development of sea urchin embryos. (A) Expression patterns by in situ hybridization of several stages of H. pulcherrimus embryos. GSTT mRNA begins to be highly expressed in the animal plate of mesenchyme blastula and then in the ciliary band of pluteus larva. Bar, 50 μm. B, Expression patterns from in situ hybridization are shown for normal (left), Zn-treated (middle) and Δ cadherin (right) embryos. GSTT mRNA was expressed strongly and ubiquitously throughout Zn-treated or cadherin-depleted embryos. Bar, 50 μm.
Figure 5. Immunoblots of ciliary proteins with an anti-GSTT antibody. (A) A strong 25-kDa signal corresponding to GSTT (arrowhead) was observed in the Zn-treated embryos, whereas the signal was quite faint in the ciliary proteins from normal embryos. (B) Selective deciliation of lateral motile cilia by DID1. After the treatment of 24-h embryos with DID1, lateral motile cilia and a part of apical tuft were detached. Dark field images of embryos without (−) or with (+) DID1 treatment are shown. Arrowheads show apical tufts. Asterisks represent embryos of which apical tuft is detached. Bar, 50 μm. (C) Lateral motile cilia and a part of apical tuft were isolated by DID1. The rest of apical tuft was isolated by 2X ASW (2XSW) without contamination of lateral motile cilia. GSTT was significantly detected in 2XSW. An antibody against a light chain of outer arm dynein (LC3) was used as an internal control [Hozumi et al., 2006].
Figure 6. Bending of apical tuft by a GST inhibitor, bromosulphophthalein (BSP). (A) Phase contrast images of embryonic cilia. Top two panels, normal embryo; bottom two panels, embryo treated with 10 μM BSP. Right panels show magnified image of apical tuft regions. Note the bending of apical tuft cilia in the BSP-treated embryos. Bar, 50 μm. (B) Angles of the cilia of apical tuft relative to the animal-vegetal axis in normal embryos (open bar) and in those treated with 10 μM BSP (closed bar). The angle of each cilium was measured from recorded images. (C) Shear angles at various distances from the base of the cilia. Open circles, control (0 μM BSP); closed triangles, 1 μM BSP; open rhombuses, 5 μM BSP; open squares, 10 μM BSP. Bars represents standard error (SE) (n = 15). (D) Beat frequency of lateral cilia in embryos treated with several concentrations of BSP. Bars: SE. (E) Swimming speed and trajectories of embryos. Multiple images of normal embryos (top) or those treated with 10 μM BSP (bottom) at 0.05 sec intervals are overdrawn by Bohboh software. Bar: 500 μm. (F) Effect of various concentrations of BSP on the swimming velocity of free-swimming embryos. Bars: SE (n = 3).
Figure 7. Effect of BSP on the negative geotactic behavior of sea urchin embryos. (A) Sequential images of embryonic movements in a vertically placed chamber. Images from dark-field stereomicroscopy were processed to draw embryos stuck to the wall and background nonembryonic debris. Top, in the absence of BSP; bottom, in the presence of 10 μM BSP. (B) Percentage of embryos that moved into the half top of the chamber against time. Open or closed bar represents embryos in the absence or presence of 10 μM BSP, respectively. Bars: SE (n = 6).
Figure 8. Escaping response of embryos against mechanical barrier. (A) Sequential images of embryos near the wall of the chamber. The bottom dotted lines show the wall of the silicon chamber. The normal embryos changed their swimming direction after colliding with the chamber wall, whereas the BSP-treated embryos were unable to escape and became trapped at the wall. Right panels represent overdrawn images showing trajectories. (B) Distribution of time required for escaping from the wall after collisions, in normal embryos and those treated with 10 μM BSP. Video images from 336 (-BSP) or 356 (+10 μM BSP) embryos were analyzed. The vertical axis represents the percentages of embryos with escaping times in the range of 0–1, 1–4, 4–7, and over 7 sec. BSP-treated embryos with escaping times over 7 sec include those trapped on the wall.
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