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. 2004 May;15(5):2105-15.
doi: 10.1091/mbc.e03-11-0854. Epub 2004 Feb 20.

A Tektin Homologue Is Decreased in Chlamydomonas Mutants Lacking an Axonemal Inner-Arm Dynein

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A Tektin Homologue Is Decreased in Chlamydomonas Mutants Lacking an Axonemal Inner-Arm Dynein

Haru-aki Yanagisawa et al. Mol Biol Cell. .
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Abstract

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found approximately 80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a-g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.

Figures

Figure 1.
Figure 1.
Microtubule overlay analysis of axonemes isolated from wild-type (WT) and mutants lacking inner-arm or outer-arm dyneins. Mutants used are listed in Table 1. (A) SDS-PAGE of axonemes stained with CBB. (B) Same samples detected with biotinylated microtubules and avidin-conjugated horseradish peroxidase. A 58-kDa band is missing in pf3 and ida6 (asterisks).
Figure 2.
Figure 2.
Extraction and purification of the 58-kDa polypeptide. (A) KCl extraction of wild-type axonemes. Insoluble fractions after the extraction were subjected to a microtubule overlay assay. The 58-kDa polypeptide was not extracted with KCl of up to 0.6 M. (B) Urea extraction of wild-type axonemes. Supernatants (S) and precipitates (P) from the centrifuged samples were analyzed by the microtubule overlay assay. The 58-kDa polypeptide remained in the insoluble fraction after extraction with 2 M urea but was almost completely extracted with 8 M urea (asterisks). (C) Purification of the 58-kDa polypeptide. SDS gel patterns stained with CBB. Lane Ax, intact axonemes. Lane K, precipitate after 0.6 M KCl extraction. Lane U, precipitate after 2 M urea extraction of the KCl-extracted sample. Lane E, eluate from a cation-exchange chromatography column.
Figure 3.
Figure 3.
Northern blots of total RNA isolated from wild-type cells after deflagellation. Numbers on the lanes indicate the time after deflagellation. (A) The 3′-UTR fragment of the p58 cDNA used as the probe hybridized with a 2.7-kb band, which was up-regulated after deflagellation. (B) Same blot probed with an α-tubulin sequence, which is known to be up-regulated after deflagellation. (C) Same blot probed with a 16S rRNA sequence for a loading control.
Figure 4.
Figure 4.
Sequence analysis of p58. (A) ClustalW (Thompson et al., 1994) alignment of the deduced amino acid sequence of p58 with the sequence of sea urchin tektins. The characters with black and gray backgrounds represent identical and conservatively substituted amino acids, respectively. Amino acid positions conserved in all four sequences are marked with asterisks. The identities/similarities (identities + conservative substitutions) between p58 and sea urchin tektins are as follows: tektin A1, 23%/39%; tektin B1, 25%/41%; and tektin C1, 23%/40%. The sequence motif RPNVELCRD (referenced to tektin A1 at residues 378–386, underlined) conserved in all sea urchin tektins is only partially preserved in p58. However, several blocks of amino acid residues are conserved between sea urchin tektins and p58 (e.g., ADLRDKTEA in tektin B1 and p58, referenced to tektin B1 at residues 147–155). The GenBank accession nos. for the sequences are as follows: Strongylocentrotus purpuratus (Sp) tektin A1, AAF14818; Sp tektin B1, Q26648; Sp tektin C1, AAB02680; and Chlamydomonas reinhardtii (Cr) p58, BAC77347. (B) Predicted coiled-coil structure of p58 according to the program COILS (Lupas et al., 1991). Probability of coiled-coil formation was calculated for a 28-residue window. Plots are aligned according to the ClustalW result. The horizontal axis shows the amino acid number referenced to tektin A1.
Figure 5.
Figure 5.
(A) Structure of the p58 gene. Boxed areas indicate sequences contained in the p58 cDNA clone. The predicted translation start site (ATG), stop site (TAA), and the putative polyadenylation signal (Poly A) are indicated. (B) Southern blot of genomic DNA digested with the indicated restriction enzymes and hybridized with the 417-base pair HinfI fragment of p58 cDNA.
Figure 6.
Figure 6.
Antibody production and immunoblot analysis of mutant axonemes. (A) Purification of recombinant p58 produced in E. coli. The gel was stained with CBB. Lane IB, isolated inclusion bodies. Lane E, eluate from a Hitrap chelating column. (B) Western-blot analysis of the wild-type cell body (CB), NFA, and axoneme (Axo). Left, an SDS-PAGE gel stained with silver. Right, the blot immunostained with anti-p58 serum. A 58-kDa band was detected in both nucleoflagellar apparatus and axoneme. (C) Western blot analysis of wild-type and mutants axonemes. The blot was detected with the p58 antibody. Only an ∼58-kDa region is shown. Each lane contains 2.5 μg of axoneme, whereas lane WT (× 0.2) contains 0.5 μg. Densitometry indicated that p58 is decreased to <20% of the wild-type amount in both pf3 and ida6 (four independent experiments). (D) Quantification of p58 in the axoneme with Western blot by using known amounts of recombinant p58 (with a His tag) as a standard. Lane WT axo, wild-type axoneme (0.75 μg). The numbers on the lanes indicate the ratio (wt/wt) of recombinant p58 to the wild-type axoneme. The ratio was estimated to be 1:75.6 ± 7.9 (four independent experiments). Because the recombinant protein has a six-histidine tag and an additional linker, its mobility on the blot differs from that of p58. The p58 antibody did not react with other protein carrying the same tag and linker sequence (our unpublished data).
Figure 7.
Figure 7.
Immunofluorescence microscopy of whole cells and the NFA. Top, differential interference contrast images. Bottom, indirect immunofluorescence light microscopy. Cells and NFAs stained with preimmune serum or p58 antiserum, followed by FITC-conjugated goat anti-rabbit IgG. WT/preimmune, lack of staining with preimmune serum. WT, wild-type cell stained with the p58 antibody. Flagella and the basal body region are stained. WT NFA, wild-type NFA stained with the p58 antibody. Flagella and basal bodies are stained. Inset, intensive staining on flagella that seem partially disrupted. pf3 NFA, ida6 NFA, NFA of mutants stained with the p58 antibody. Staining of flagella and basal bodies is weak compared with wild-type cells. Bar, 5 μm.
Figure 8.
Figure 8.
Presence and absence of p58 in axonemes extracted with Sarkosyl and urea. Top, silver-stained SDS-PAGE gels. Bottom, blots immunostained with p58 antibody. Lanes S and P indicate supernatants and precipitates from the centrifuged samples, respectively. Numbers on the lanes indicate the concentration of Sarkosyl or urea used. (A) Extraction of wild-type axonemes with Sarkosyl in TED buffer. p58 is insoluble in 0.3% Sarkosyl but completely soluble in 0.7% Sarkosyl, the condition wherein the protofilament ribbons remain in the precipitate (see also Figure 9). (B) Extraction of wild-type axonemes with urea in MESH buffer. The precipitate fraction obtained after 2 M urea extraction did not contain Rib72, a prominent 66-kDa band found in the precipitate after 0.7% Sarkosyl extraction (A and B, top, asterisk). (C) Extraction of wild-type axoneme with Sarkosyl and urea in TED buffer.
Figure 9.
Figure 9.
Electron microscopy of axonemes extracted with Sarkosyl or urea. (A) Negativestain image of wild-type axoneme treated with 0.7% Sarkosyl in TED buffer. A stable three-protofilament structure, previously described as the protofilament ribbons, remained. (B) Negative-stain image of wild-type axoneme treated with 2 M urea in MESH buffer. A filamentous structure remained. This filament is approximately two-protofilament wide. (C and D) Immunoelectron microscopy of the axonemes treated with 2 M urea. The specimens were stained with preimmune serum (C) or p58 antiserum (D), followed by 5-nm colloidal gold-conjugated secondary antibody. (E–G) Cross section images of detergent-treated axonemes. (E) Sarkosyl (0.3%) in TED. (F) Urea (1 M) in TED. (G) Sarkosyl (0.3%) and 1 M urea in TED. A-tubules remain intact in all three conditions. Bar, 100 nm.
Figure 10.
Figure 10.
Chemical cross-linking analysis of axonemes. Wild-type axonemes were treated with either EDC or DSS, followed by Western blot. The concentration of cross-linkers used is noted above lanes. Blots were immunostained with p58 antibody (p58) and α-tubulin antibody (α-tub). In the control sample without chemical cross-linking, p58 antibody produced weak bands at 168 kDa (asterisks) in addition to p58 itself (arrow). These bands were probably due to nonspecific staining of the antibody. In the EDC-treated sample, 107- and 150-kDa bands were detected with p58 antibody (arrowheads). In the DSS-treated sample, 112-, 117-, 143-, and 151-kDa bands were detected with p58 antibody (arrowheads).
Figure 11.
Figure 11.
Model of the p58 structure. (A) Model of a p58 homodimer. Boxes indicate helical domains. Following the previous structural model of sea urchin tektins (Norrander et al., 1996), two p58 polypeptides are assumed to form a homodimer. Because p58 has five ∼8-nm-long helical domains, the total length of the homodimer is ∼40 nm. The homodimer could be concatenated to form a filament. (B) Model of p58 filaments on A-tubule. The ratio of p58 to the total axoneme is ∼1:76 (wt/wt) (Figure 6C). From this ratio, the number of p58 molecules within a 40-nm period of one outer doublet is calculated to be ∼5.5. This means that approximately three p58 filaments exist per one outer doublet.

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