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. 2012 Aug 15;125(Pt 16):3904-13.
doi: 10.1242/jcs.107151. Epub 2012 May 8.

A FAP46 Mutant Provides New Insights Into the Function and Assembly of the C1d Complex of the Ciliary Central Apparatus

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Free PMC article

A FAP46 Mutant Provides New Insights Into the Function and Assembly of the C1d Complex of the Ciliary Central Apparatus

Jason M Brown et al. J Cell Sci. .
Free PMC article

Abstract

Virtually all motile eukaryotic cilia and flagella have a '9+2' axoneme in which nine doublet microtubules surround two singlet microtubules. Associated with the central pair of microtubules are protein complexes that form at least seven biochemically and structurally distinct central pair projections. Analysis of mutants lacking specific projections has indicated that each may play a unique role in the control of flagellar motility. One of these is the C1d projection previously shown to contain the proteins FAP54, FAP46, FAP74 and FAP221/Pcdp1, which exhibits Ca(2+)-sensitive calmodulin binding. Here we report the isolation and characterization of a Chlamydomonas reinhardtii null mutant for FAP46. This mutant, fap46-1, lacks the C1d projection and has impaired motility, confirming the importance of this projection for normal flagellar movement. Those cells that are motile have severe defects in phototaxis and the photoshock response, underscoring a role for the C1d projection in Ca(2+)-mediated flagellar behavior. The data also reveal for the first time that the C1d projection is involved in the control of interdoublet sliding velocity. Our studies further identify a novel C1d subunit that we term C1d-87, give new insight into relationships between the C1d subunits, and provide evidence for multiple sites of calmodulin interaction within the C1d projection. These results represent significant advances in our understanding of an important but little studied axonemal structure.

Figures

Fig. 1.
Fig. 1.
FAP46 gene, fap46-1 null mutant strains, and FAP46 rescued strains. (A) Diagram of FAP46 gene and flanking regions; FAP46 and RPL30 exons are shown as grey boxes. Locations of aph7″ insert and synthetic peptide sequences used to make anti-FAP46 antibodies are indicated by arrowheads. The line above the genes represents the fragment used to rescue the fap46-1 strain. (B) Pedigree charts showing generation of fap46-1, fap46-1xFAP74ami, fap46-1::FAP46 and fap46-1::FAP46-HA strains used in this study. DNS11/fap46' is the fap46-1 founder strain; fap46″ is a progeny of a cross between DNS11 and the WT strain 137c. The fap46-1 (mt+) strain was transformed with a plasmid (pFAP46) containing the cloned WT FAP46 gene or a plasmid (pFAP46-HA) containing a gene encoding HA-tagged FAP46, to generate the rescued strains fap46-1::FAP46 or fap46-1::FAP46-HA, respectively. (C) Upper panel: western blot of flagella isolated from WT and the indicated fap46-1 strains, probed with the anti-FAP46 antiserum. The antiserum recognizes a band of the expected size in WT that is missing in the fap46-1 (mt−) and fap46-1 (mt+) strains but restored in the fap46-1::FAP46 rescued strain. FAP46–HA from the fap46-1::FAP46-HA strain migrates slightly slower than WT FAP46 due to the HA tag. Middle and lower panels: blots with loading equivalent to that of the blot in the upper panel were probed with anti-HA (middle panel) or an antibody to the outer dynein arm intermediate chain IC2 as a loading control (lower panel).
Fig. 2.
Fig. 2.
Axonemes of fap46-1 and FAP74ami retain different subunits, but both fail to assemble C1d. (A) Western blots of isolated flagella and axonemes from WT, mutant and rescued cells; fap46-1' is the fap46-1 founder strain DNS11. FAP54, FAP46, FAP74 and FAP221 are C1d subunits. IC2 is an intermediate chain of the outer dynein arms and serves as a loading control. Affinity-purified antibody was used for the FAP221 blot. (B) Diagram showing the identities of the central pair projections; electron micrographs show axonemes from WT, the fap46-1 founder strain DNS11 (fap46-1'), and the FAP74 knockdown strain (FAP74ami). All micrographs are oriented with the axoneme viewed proximal to distal; the C1 central microtubule in all images is to the left. The C1d projection in the diagram and in the electron micrograph of the WT axoneme is indicated by red arrows. The fap46-1' and FAP74ami axonemes lack the C1d density and the sheath connecting C1d to C1b (red arrowheads). Identical defects were observed for fap46-1. n>50 transverse sections resulting from two axoneme preparations for each strain.
Fig. 3.
Fig. 3.
FAP46, FAP54 and FAP74 form a sub-complex in extracts from C1d-defective axonemes. (A) Silver-stained gel of anti-FAP74 immunoprecipitations from high-Ca2+ NaCl extracts from WT, fap46-1, and fap46-1::FAP46 axonemes. The immunoprecipitates from WT and the rescued strain fap46-1::FAP46 are indistinguishable and contain the four previously described C1d subunits; that from fap46-1 contains reduced amounts of FAP54 and FAP74 but no FAP46 or FAP221. (B) Silver-stained gel and corresponding western blots of anti-CaM immunoprecipitations from high-Ca2+ NaCl extracts from WT and mutant axonemes. FAP54 and FAP46 are co-precipitated from the extract of FAP74ami axonemes, FAP54 and FAP74 are co-precipitated from the fap46-1 axonemal extract, and only FAP54 is precipitated from the fap46-1xFAP74ami axonemal extract. FAP221 is not detectable in the immunoprecipitates from any of the mutant extracts. Western blots for each antibody are from the same membrane and exposure; vertical white lines indicate that lanes were removed from between each sample. In both A and B, the red asterisk indicates a 100-kDa protein that is co-precipitated with C1d subunits from WT axonemal extracts but is not present in precipitates from C1d-defective mutants.
Fig. 4.
Fig. 4.
A novel C1d subunit with a WD-repeat domain and an N-terminal domain conserved in organisms with motile cilia. (A) Upper panel: silver-stained gel of anti-HA immunoprecipitates from high-Ca2+ NaCl extracts from WT and FAP46-HA-rescued axonemes. Immunoprecipitation input lanes are on right. FAP46-HA, FAP54, FAP74 and FAP221 are specifically co-immunoprecipitated from the FAP46-HA rescued axonemal extract along with two unknown bands, a and b. Lower panel: Immunoblots of the same immunoprecipitates confirm that FAP46-HA, FAP54, FAP74 and FAP221 are specifically co-immunoprecipitated from the FAP46-HA-rescued axonemal extract. All blots were probed with antiserum. (B) Anti-HA-immunoprecipitate from a similar experiment probed with affinity-purified anti-FAP221 antibody, confirming that FAP221 is present in the immunoprecipitate from fap46-1::FAP46-HA axonemal extracts. (C) Domain maps of C1d-87, identified by mass spectrometry of band b from panel A, and its human homologue WDR93. The N-terminal domain is conserved only in organisms with motile cilia. (D) Sequence alignment illustrating the high degree of conservation of the N-terminal domain of C1d-87. The C. reinhardtii N-terminal region from aa 7–49 is aligned with highly similar regions of proteins from 14 other ciliated organisms ranging from protists to mammals. Protein sequences from which these amino acid sequences were taken are as follows [organism (accession information – all refer to NCBI reference sequences unless otherwise noted)]: C. reinhardtii (Joint Genome Institute Chlamydomonas v. 4.0 protein ID 206529); Volvox carteri (XP_002955486.1); Ciona intestinalis (XP_002126065.1); Branchiostoma floridae (XP_002601619.1); Strongylocentrotus purpuratus (XP_788085.2); Homo sapiens (NP_064597.1); Mus musculus (NP_001033016.1); Bos taurus (GenBank: AAI13295.1); Ornithorhynchus anatinus (XP_001509766.1); Taeniopygia guttata (XP_002197480); Schistosoma mansoni (XP_002577037.1); Nematostella vectensis (XP_001637625.1); Trichoplax adhaerens (XP_002110435.1); Tetrahymena thermophila (XP_001031667.1); Trichomonas vaginalis (XP_001312755.1).
Fig. 5.
Fig. 5.
Cells of fap46-1 and FAP74ami strains have distinct motility phenotypes. (A) Percentage of cells swimming (n>130 for each); (B) average swimming velocity (n>40 for each); (C) percentage photoshock (values are percentages of the motile population; n>80 for each); (D) average asymmetric beat frequency (n>60 for each); (E) average symmetric beat frequency (n>8 for each) of WT, mutant and rescued strains. For each motility parameter, values for all strains were collected in the same experiment; the values for WT and FAP74ami strains were reported previously (DiPetrillo and Smith, 2011) and are included here for comparison. Where tested, asterisks (*) denote a significant difference (P<0.01) compared to WT and fap46-1::FAP46. Hash signs (#) denote a significant difference (P<0.01) from fap46-1. Error bars indicate ± s.e.m. (F) Average in vitro microtubule sliding velocity of WT and mutant axonemes treated with protease and ATP in either low (black bars) or high (grey bars) Ca2+ buffers. *Significant difference (P<0.01) from WT and fap46-1::FAP46; significant difference (P<0.05) from FAP74ami (n>50 for each). Error bars indicate ± s.e.m.
Fig. 6.
Fig. 6.
Model for C1d subunit interactions. Predicted interactions between C1d subunits in WT and fap46-1, FAP74ami and fap46-1xFAP74ami mutant strains in the presence of high Ca2+. 54, FAP54; 46, FAP46; 74, FAP74; 221, FAP221; 87, C1d-87; C, CaM. The thick vertical lines represent C1 microtubules. Faded shading with dashed outlines indicate decreased axonemal content relative to WT. Arrows represent 0.6 M NaCl extraction and dialysis with NaLow. (A) On WT axonemes, FAP46, FAP54, FAP74, FAP221, C1d-87 and CaM form a complex that can be extracted with 0.6 M NaCl and immunoprecipitated from the extract by an anti-CaM antibody. The direct interaction between FAP221 and C1d-87 is speculative, as indicated with a question mark. FAP54 may or may not bind CaM in WT cells, as indicated by a second question mark. (B) In fap46-1 axonemes, FAP54, FAP74 and FAP221 are present in reduced amounts and anti-CaM co-immunoprecipitation of FAP54 and FAP74 without FAP221 suggests formation of a FAP54/FAP74/CaM sub-complex and partial requirement of FAP46 for FAP221 binding to C1d. Although C1d-87 is shown, it is not yet known if this subunit is part of the residual C1d complex in fap46-1. (C) In FAP74ami axonemes, FAP54 and FAP46 are present in reduced amounts; absence of FAP221 indicates that FAP74 is required for FAP221 binding to C1d. Anti-CaM co-immunoprecipitation of FAP54 and FAP46 suggests that FAP46 is included with FAP54, FAP74 and CaM in a C1d sub-complex. (D) In fap46-1xFAP74ami, FAP54 is present in reduced amounts. Axonemal binding of FAP54 in the absence of other C1d proteins suggests direct binding of FAP54 to the C1 microtubule, and immunoprecipitation of FAP54 by anti-CaM suggests direct binding of CaM to FAP54.

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