Fig 1.
Morphology of branchial cilia and sperm flagella in Ciona intestinalis.
A. An adult C. intestinalis. B. The branchial wall is perforated by eliptical halls called 'gill slits' or 'stigmata.' Scale bar = 100 μm. C. A magnified stigma. st, stigma. Each stigma is lined by ciliated stigmatal cells. Scale bar = 20 μm. D. Differential interference contrast (DIC) microscopy image of Ciona sperm. Scale bar = 10 μm. E. A TEM image of Ciona sperm. Only one mitochondrion is attached to the head, and the flagellum does not have any accessory structure. Putative glycogen particles can be seen between the nucleus and mitochondrion. n, nucleus; mt, mitochondrion; f, flagella. Scale bar = 500 nm. F and G. Transverse sections of branchial cilia (F) and sperm flagella (G). Both axonemes show the typical motile 9 + 2 architecture with outer and inner arem dyneins, radial spokes, and central pair apparatus. Scale bar = 100 nm.
Fig 2.
Comparison of 2DE protein patterns between branchial cilia and sperm flagella.
Proteins were first separated by isoelectric focusing (pH 3–10), followed by SDS-PAGE with 10% polyacrylamide gel. Proteins were stained with silver. Protein spots predominantly seen in branchial cilia (A) or sperm flagella (B) are indicated by arrows and special spot number, SSP.
Table 1.
Proteins present predominantly in branchial cilia.a
Table 2.
Proteins present predominantly in sperm flagella.a
Fig 3.
Recruitment of different tubulin isotypes in branchial cilia and sperm flagella.
(A) Representative mass spectra of the tubulin spot identified as brancial ciliary α- (KH.C11.291.v1.A.nonSL1-1) and β- (KH.L116.85.v1.A.ND1-1) tubulin and sperm flagellar α- (KH.L119.32.v1.A.ND1-1) and β- (KH.C9.606.v1.A.SL1-1) tubulin. Unique and common mass peaks between the two identifiers are indicated with red and black arrowheads, respectively. The mass value and corresponding peptide sequence are shown on each peak. (B) A phylogenetic tree of tubulin isoforms present in branchial cilia and sperm flagella. Ciona proteins (blue) were aligned with human tubulins by CLUSTALW and the tree was constructed by MEGA5. Human γ-tubulin (TBG1) (P23258) was used as the outgroup. The value shown on each branch represents the number of times that a node was supported in 1000 bootstrap pseudo-replications. Accession numbers of protein sequences are: TBA1A (Q71U36), TBA1B (P68363), TBA1C (Q9BQE3), TBA3C (Q13748), TBA3E (Q6PEY2), TBA4A (P68366), TBA8 (Q9NY65), HUMAN, TBAL3 (A6NHL2), TBB5 (P07437), TBB1 (Q9H4B7), TBB2A (Q13885), TBB2B (Q9BVA1), TBB3 (Q13509), TBB4A (P04350), TBB4B (P68371), TBB6 (Q9BUF5), TBB8 (Q3ZCM7). (C) Gene expression pattern of tubulin isoforms in branchial cilia and sperm flagella. Data are based on EST data collected C. intestinalis tissues (http://hoya.zoology.kyoto-u.ac.jp/). The vertical values in the histogram represent occurrence ratio of the gene against total EST number (percentage). EG, unfertilized egg; LV, larvae; AD, young adult; MA, mature adult; BD, blood cell; ES, endostyle; GD, gonad; HT, heart; MC, neural complex; TS, testis.
Fig 4.
Western blots of cilia and flagella in C. intestinalis using several antibodies against axonemal proteins.
Branchial cilia (BC) and sperm flagella (SF) were separated by SDS-PAGE and immunoblotted with antibodies against several axonemal structures. Both IC2 and IC3 are intermediate chains of the outer arm dynein. LRR-LC and Tctex2-LC are light chains of the outer arm dynein. Ap58 is a protein involved in the anchoring of the outer arm dynein. Both IC98 (ortholog of Chlamydomonas IC110) and IC116 (ortholog of Chlamydomonas IC140) are intermediate chains of two-headed inner arm f/I1 dynein. RSP3, AxHsp40, LRR37 and MORN40 are components of the radial spoke. PF16 is a protein of the central pair apparatus. PKAR2, Calaxin is a Ca2+-binding protein that regulates the outer arm dynein. ARM94 and Tctex2-LC are known to be phosphorylated or dephosphorylated at the activation of sperm motility. ODF3 is a component of the outer dense fiber of mammalian sperm. Both TD27 and TD01 are uncharacterized proteins associated with flagellar axonemes. Tektin 3 is a structural protein in the axoneme (see text).
Fig 5.
Predominant localization of tektin 3 to branchial cilia.
Immunofluorescent microscopy was carried out on trimmed brancial wall with anti-Tctex2 dynein light chain antibody (middle) and anti-tektin 3 antibody (bottom). Non-immune anti-serum was used as a control (top). Samples were stained with anti-acetylated α-tubulin (green), anti-Tctex2 or anti-tektin 3 (red), or 4',6-diamidino-2-phenylindole (DAPI; DNA, blue). Left, double staining with DAPI and anti-acetylated α-tubulin; center, double staining with anti-Tctex2 or anti-tektin 3 antibody; right, merged image from DNA staining, staining with anti-acetylated tubulin antibody and staining with anti-Tctex2 or—tektin 3. Bar, 20 μm.
Fig 6.
Predominant localization of Tctex2 dynein light chain to sperm flagella.
Immunofluorescent microscopy was carried out on sperm attached to the glass slide with anti-Tctex2 dynein light chain antibody (middle) and anti-tektin 3 antibody (bottom). Non-immune anti-serum was used as a control (top). Samples were stained with anti-acetylated α-tubulin (green), anti-Tctex2 or anti-tektin 3 (red), or 4',6-diamidino-2-phenylindole (DAPI; DNA, blue). Left, double staining with DAPI and anti-acetylated α-tubulin; center, double staining with anti-Tctex2 or anti-tektin 3 antibody; right, merged image from DNA staining, staining with anti-acetylated tubulin antibody and staining with anti-Tctex2 or—tektin 3. Bar, 20 μm.