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. 2015 Feb 15;26(4):696-710.
doi: 10.1091/mbc.E14-11-1506. Epub 2014 Dec 24.

FAP206 Is a Microtubule-Docking Adapter for Ciliary Radial Spoke 2 and Dynein C

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Free PMC article

FAP206 Is a Microtubule-Docking Adapter for Ciliary Radial Spoke 2 and Dynein C

Krishna Kumar Vasudevan et al. Mol Biol Cell. .
Free PMC article

Abstract

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.

Figures

FIGURE 1:
FIGURE 1:
FAP206 localizes to the ciliary axoneme, and knockout of FAP206 results in cilia-related defects. (A) TIRF image of a live cell expressing FAP206-GFP under the native promoter. (B) A wild-type (negative control) cell (left) and a cell expressing FAP206-GFP under the native promoter (right) extracted with Triton X-100, fixed with paraformaldehyde, and imaged for GFP using a confocal microscope. (C) Culture growth rates for a wild-type CU428 and an FAP206-KO strain. Each data point represents an average for three independent experiments. (D) Paths of swimming wild-type and FAP206-KO cells recorded for 1 s. The average swim velocities were 170 μm/s for the wild type and 50 μm/s for FAP206-KO. (E) Immunofluorescence images of tubulin for wild-type and FAP206-KO cells. For each genotype, an interphase (left) and a dividing cell (right) are shown. (F) Classical TEM images of cilia cross-sections that are either circular (left) or compressed (right). The percentages represent fractions of either circular or compressed axonemes (n = 52 for wild type, n = 48 for FAP206-KO). Scale bars, 20 μm (A, B, and E), 1 mm (D), 0.2 μm (F).
FIGURE 2:
FIGURE 2:
Deletion of FAP206 leads to loss of RS2 and associated dynein c in the 96-nm repeat. Isosurface renderings (A–F, K, L) and tomographic slices (G–J) show the averaged 96-nm axonemal repeats of wild type (A, C, D, G, H, K) and FAP206-KO (B, E, F, I, J, L) in longitudinal (A, B, D, F, H, J–L), cross-sectional (C, E, G, I), and bottom views looking from the central pair toward the doublet microtubule (D, F, H, J–L); the dotted line in G indicates the orientation of the tomographic slice shown in H and J. There are three radial spokes: RS1 (green), RS2 (blue), and RS3 (orange) in wild type (A), whereas RS2 is missing in FAP206-KO (B, E, I). The RS2 base is composed of three regions: front (red), back (yellow), and side prongs (light blue and/or black arrowheads). These three regions are connected with each other and form the attachment of RS2 to the doublet A-tubule (At). The back prong of RS2 and the RS3 base (yellow) were previously identified as parts of the CSC (Heuser et al., 2012b). IDA c (pink), which anchors with its tail (pink arrowhead in H) to the A-tubule through the front prong of the RS2 base in WT, is also missing in FAP206-KO (white labels in J, where the IDA c with tail should be). (K, L) Densities representing radial spoke heads and stems were removed to visualize the microtubule-attachment sites of the spoke bases. Note that in the FAP206-KO mutant, the main difference from wild type is the complete absence of the RS2 front prong (red), RS2 back prong (yellow), and dynein c; in contrast, RS1, RS3, and the RS2 side prong (light blue, black arrowhead) appear unaffected in FAP206-KO. All structures shown are subtomogram averages without prior classification analysis. Scale bar, 10 nm.
FIGURE 3:
FIGURE 3:
Classification analysis of RS2 reveals that in the absence of FAP206, RS2 can occasionally assemble but lacks the front prong. Cross-sectional tomographic slices (A–D) and isosurface renderings (E, F) of averaged 96-nm axonemal repeats show the presence and absence of RS2 in wild type (WT; A) and FAP206-KO (B–F), respectively. Subtomogram averages of all axonemal repeats (100%) indicate that the density of RS2 is dramatically reduced in FAP206-KO (B) as compared with WT (A). Classification of RS2 resulted in two distinct class averages for FAP206-KO: a large set (83%) of axonemal repeats from FAP206-KO lack RS2 (–RS2), and only the side prong (light blue) remains (D and F). However, in a small subset (17%) of FAP206-KO repeats, RS2 is present (+RS2) yet lacks the front prong density of the RS2 base (C and E); the back prong density also appears slightly reduced. All axonemal repeats from WT showed normal RS2 (A). Scale bar, 10 nm.
FIGURE 4:
FIGURE 4:
Classification analysis of RS3 reveals that in the absence of FAP206, RS3 is destabilized. Longitudinal tomographic slices (A–D) of averaged 96-nm axonemal repeats show the presence (A–C) and absence (D) of RS3 in WT (A) and FAP206-KO (B–D). The density of RS3 is weaker in the average of all axonemal repeats from FAP206-KO (B) as compared with WT (A). Classification of RS3 resulted in two distinct class averages for FAP206-KO: in the majority (89%) of axonemal repeats from FAP206-KO, RS3 was assembled, whereas a small set (11%) of repeats lacked RS3 (D). All axonemal repeats from WT showed a normal RS3 (A). Scale bar, 10 nm.
FIGURE 5:
FIGURE 5:
Dynein c requires FAP206 for its assembly at the base of RS2. Longitudinal tomographic slices of averaged 96-nm axonemal repeats show the presence (A–B) and absence (C–E) of IDA c in WT (A–C) and FAP206KO (D, E). Classification analysis confirms the complete absence of IDA c (both the head and tail [arrowheads] are missing) from all axonemal repeats from FAP206-KO, whereas the remaining IDAs appear intact (E). The majority of the WT subtomograms have a fully assembled dynein c (B), whereas 11% lack dynein c (C). The subtomograms that lack dynein c are randomly located within all nine outer doublets and were likely extracted during the axoneme isolation procedure. Scale bar, 10 nm.
FIGURE 6:
FIGURE 6:
GFP-FAP206 has microtubule-binding activity in vivo. Overexpressed GFP-FAP206 associates with nonciliary microtubules. Cells that overexpress GFP-FAP206 transgene under the MTT1 cadmium-inducible promoter were stained by immunofluorescence using a mix of 12G10 monoclonal anti–α-tubulin and SG polyclonal anti-tubulin antibodies (red) and imaged for GFP (green) using a confocal microscope. Top, a cell before induction; middle and bottom, an interphase cell and a dividing cell, respectively, fixed after 3 h of transgene induction with 2.5 μg/ml CdCl2. The white insets magnify the intracytoplasmic microtubules in the cell body. The yellow insets magnify mature full-length cilia with strong GFP-FAP206 accumulation at the tips. The white arrowheads mark short (likely assembling) cilia with uniform distribution of GFP-FAP206. Scale bar, 20 μm.
FIGURE 7:
FIGURE 7:
FAP206 interacts with CSC. (A) A Western blot of purified cilia isolated from Tetrahymena strains that are wild type, lack genes encoding the Tetrahymena homologues of CSC proteins (FAP61/CaM-IP3 or FAP251/CaM-IP4), or lack FAP206, probed with antibodies specific to FAP91/CaM-IP2 of C. reinhardtii (top; Dymek et al., 2011), 12G10 monoclonal α-tubulin (middle), or polyglycylated tubulin (bottom). Note that the levels of the anti-FAP91/CaM-IP2 band are strongly reduced in the strains lacking the CSC protein homologues, indicating that the antibodies are specific to the Tetrahymena FAP91 homologue. Furthermore, the levels of the FAP91 homologue are strongly reduced in the strain lacking FAP206, indicating that axonemal assembly of FAP91 and likely other CSC components depends on the presence of FAP206. Images of the entire blots are shown in Supplemental Figure S1. (B) Western blot of axonemes of wild type and FAP206-KO probed with anti-FAP91 and 12G10 anti–α-tubulin antibodies. (C) Silver stained SDS–PAGE gels showing an immunoprecipitate obtained from the radial spoke-enriched supernatants of C. reinhardtii axonemes. The positions of known CSC components and major radial spoke proteins are marked. The two bands (boxed area) that migrated with an apparent molecular weight of 80 kDa were found to contain the Chlamydomonas FAP206 homologue (CHLREDRAFT_171124). (D) Immunoprecipitates obtained with the anti-FAP91/CaM-IP2 antibodies from the radial spoke–enriched supernatant of Chlamydomonas axonemes that were either wild type or spokeless pf14 mutant. Note the absence of the FAP206 bands, indicating that the CSC-FAP206 interaction requires RSP3 as an intermediate.

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References

    1. Andrivon C. Inhibition of ciliary movements by Ni-2+ions in triton-extracted models of Paramecium caudatum. Arch Int Physiol Biochim. 1974;82:843–852. - PubMed
    1. Angus SP, Edelmann RE, Pennock DG. Targeted gene knockout of inner arm 1 in Tetrahymena thermophila. Eur J Cell Biol. 2001;80:486-497. - PubMed
    1. Barber CF, Heuser T, Carbajal-Gonzalez BI, Botchkarev VV, Jr, Nicastro D. Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella. Mol Biol Cell. 2012;23:111–120. - PMC - PubMed
    1. Brokaw CJ, Kamiya R. Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil Cytoskeleton. 1987;8:68–75. - PubMed
    1. Bui KH, Sakakibara H, Movassagh T, Oiwa K, Ishikawa T. Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella. J Cell Biol. 2009;186:437–446. - PMC - PubMed

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