ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly

Abstract

Dynein motors of cilia and flagella function in the context of the axoneme, a very large network of microtubules and associated proteins. To understand how dyneins assemble and attach to this network, we characterized two Chlamydomonas outer arm dynein assembly (oda) mutants at a new locus, ODA16. Both oda16 mutants display a reduced beat frequency and altered swimming behavior, similar to previously characterized oda mutants, but only a partial loss of axonemal dyneins as shown by both electron microscopy and immunoblots. Motility studies suggest that the remaining outer arm dyneins on oda16 axonemes are functional. The ODA16 locus encodes a 49-kDa WD-repeat domain protein. Homologues were found in mammalian and fly databases, but not in yeast or nematode databases, implying that this protein is only needed in organisms with motile cilia or flagella. The Chlamydomonas ODA16 protein shares 62% identity with its human homologue. Western blot analysis localizes more than 90% of ODA16p to the flagellar matrix. Because wild-type axonemes retain little ODA16p but can be reactivated to a normal beat in vitro, we hypothesize that ODA16p is not an essential dynein subunit, but a protein necessary for dynein transport into the flagellar compartment or assembly onto the axoneme.

Figure 1.

(A) Beat frequency analysis of flagella on wild-type (137c), 19F11, and 14E2 cells show that these two new mutants both swim at half the wild-type beat frequency. Wild type (B and C), 19F11 (D and E), and 14E2 (F and G) axonemal cross sections show that both mutants have a reduction but not an absence of outer arm dyneins on the axoneme (black arrows). Outer arm dynein distribution appears to be random around the circumference of the axoneme in both 14E2 and 19F11. (H and I), electron micrographs of longitudinal-sections of mutant axonemes show that outer arm dyneins (arrows), when present, extend in rows along each doublet microtubule. Values in A are mean ± SD (n = 20). Scale bars, (B-I) 100 nm.

Figure 2.

Molecular analysis of the ODA16 locus. (A) Restriction maps of plasmid p19F11-Sal (top), which was rescued from insertional mutant oda16-2 and includes pUC119 vector sequences and genomic sequences flanking the insertion site, and the wild-type genomic ODA16 locus (bottom) showing coding exons (▪), noncoding exons (□), and the insertion break point in oda16-2 (arrowhead). Bar below the p19F11-Sal map indicates the SalI-XmaI fragment used as a hybridization probe for Southern blots and for selection of BAC clones. (B and C) Southern blots of wild type, oda16-1, and oda16-2 genomic DNA digested with SalI and probed with the 19F11-Sal-Xma fragment (B), or digested with XmaI and probed with the coding region from pOda16-cDNA (C). (D) Electron micrographs of axonemes from oda16-1 transformed with pGenD-oda16 to generate strain oda16-1R, in which outer row dynein assembly has been restored. S, SalI; X, XmaI. Scale bar, (D) 100 nm.

Figure 3.

ODA16 encodes a WD repeat domain protein. (A) ODA16p is a 449 amino acid protein that contains a central domain with 8 WD repeats (gray boxes). (B) The Chlamydomonas (Cr) ODA16p shares 58% identity (yellow shading), and 70% similarity (green shading) with an uncharacterized human protein (Hs). Amino acid pairs that define each repeat (GH...WD) are shown in bold face, and each repeat is set off by a space and numbered (1-8). The insertion of pMN24 in oda16-2 disrupts regions 3′ of codon 44 (arrow). Accession numbers are FLJ25955 for Hs and DQ151642 for Cr.

Figure 4.

(A) Western blots compare the reduction in dynein and docking complex proteins in oda16 flagellar fractions. Blots of stoichiometrically equivalent amounts of wild-type and oda16 flagella (F) or detergent extracted flagellar axonemes (A) show a reduction in IC2 outer arm dynein IC in both oda16 mutants. There is also a slight reduction in DC2 (62-kDa docking complex protein). An anti-α-tubulin antibody was included as a loading control. (B) Signal intensities of wild-type (filled bar), oda16-1 (striped bar), and oda16-2 (empty bar) axonemal lanes from blots similar to those in A were quantified on a phosphorimager and normalized to tubulin. Each value is an average of four measurements based on two blots from each of two independent preparations of axonemes.

Figure 5.

Oda16 and oda2 display a similar reduction in beat frequency when compared with wild type (137c). The beat frequency is further reduced in oda16oda2 double mutants below the level seen with either mutation alone (significant at p < 0.05; Duncan's multiple range test). Each measurement is the mean ± SD (n = 20).

Figure 6.

Dikaryon complementation analysis. Beat frequencies were measured in wild-type, oda1, and oda16 gametes (□) and in temporary diploids (dikaryons) between the oda16 mutants and other previously characterized outer arm dynein assembly mutants (▪). All dikaryons between other oda mutants and oda16 show complementation (beat frequencies restored to near wild type). Error bars indicate std dev (n = 15).

Figure 7.

Characterization of anti-ODA16p antibodies and analysis of ODA16p distribution in flagellar fractions. (A) A Coomassie Blue-stained SDS-PAGE gel (CB) and corresponding Western blot of wild-type flagella shows that the anti-Oda16p antibody recognizes a single 50-kDa band (arrow). (B) A Western blot of wild-type, oda16, and oda16-1R (rescued strain) axonemal protein using the anti-ODA16p antibody confirms that the oda16 mutants are knockouts. An anti-α-tubulin antibody was used as a loading control. (C) A Western blot comparing stoichiometrically equivalent amounts of whole wild-type flagella (F) or flagella separated by centrifugation into pellet (P) and supernatant (S) fractions after treatment with the indicated concentration of NP40 detergent (NP) or after freeze-thaw treatment in the absence of detergent (F/T). After NP40 treatment, the pellet fraction contains axonemes and the supernatant fraction contains membranes and the matrix. After freeze/thaw (F/T) treatment, the pellet contains both axonemes and membranes and the supernatant contains the matrix. ODA16p fractionates predominately with the matrix. (D) A Western blot of stoichiometrically equivalent loads of whole flagella (F), axoneme (P), and membrane/matrix (S) fractions of NP40-treated wild-type and oda2 flagella shows that ODA16p localization is unchanged in the absence of outer arm dyneins. All lanes used for C and D are from the same blot but additional lanes in the center were removed for clarity.