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. 2014 May;25(9):1472-83.
doi: 10.1091/mbc.E13-08-0464. Epub 2014 Feb 26.

FAP20 Is an Inner Junction Protein of Doublet Microtubules Essential for Both the Planar Asymmetrical Waveform and Stability of Flagella in Chlamydomonas

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FAP20 Is an Inner Junction Protein of Doublet Microtubules Essential for Both the Planar Asymmetrical Waveform and Stability of Flagella in Chlamydomonas

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

The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo-electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform.

Figures

FIGURE 1:
FIGURE 1:
Four mutant alleles of dmj1. (A) The mutation sites of four different dmj1 alleles are indicated on the exon/intron structure of the FAP20 gene (Cre07.g351650.t1.3 in Phytozome v9.1; www.phytozome.net/). The details of the mutations are described in Supplemental Figure S1. (B) FAP20 protein is mainly present in flagella. Whole-cell (WC), cell body without flagella (CB), and flagella (Fla) samples were analyzed by Western blotting with anti-FAP20 antibody. The WC and CB lanes contain the same number of cells. Chlamydomonas cells have two flagella; thus twice as many flagella as cell bodies were loaded in the Fla lane. (C) FAP20 protein is associated with the axoneme. Flagella (Fla), membrane and matrix (M+M), and axonemal (Axo) samples were analyzed by Western blotting with anti-FAP20 antibody. (D) Western blot analysis of axonemes from the FAP20 mutants and rescued strains with FAP20 antibody. The axonemes of dmj1-1, dmj1-2, and RL-11 completely lack the FAP20 protein. The axoneme of dmj1-3 contains reduced amount of a truncated FAP20 protein. The axonemes of the rescued strains contain wild-type amounts of the GFP-, BCCP-, and 3xHA-tagged FAP20 proteins. Coomassie-stained bands of tubulins were used as a loading control.
FIGURE 2:
FIGURE 2:
FAP20 is essential for both asymmetrical waveform and stability of flagella. (A) FAP20 mutants produce wild-type length of the flagella (n = 33). (B) Introduction of the tagged FAP20 constructs rescued the motility defect of the FAP20 mutant (n = 30). Error bars represent SD. (C) Waveforms from representative cells of wild type and each mutant allele as traced from recorded videos. Time between frames 1 and 10 is 0.02, 0.02, 0.033, 0.02, and 0.01 s, respectively, for WT (asymmetrical), fap20ΔC (intermediate type), fap20null, mbo2, and WT (symmetrical). (D) The axoneme of fap20null strain was unstable and liable to split or fray during beating. Demembranated axonemes of wild type and fap20null were reactivated with 0.1 mM ATP in presence of protease inhibitors. Bar, 10 μm. (E) Quantitative analysis of the disintegration of axonemes. Values in parentheses represent number of axonemes used for analyses.
FIGURE 3:
FIGURE 3:
Ultrastructural defects of the fap20null axoneme. Cross sections of Epon-embedded axonemes of wild type, fap20null, and rescued strains. The 1–2 bridge (asterisks) was used to determine the position of DMT1. The beak structures contained in DMT1, 5, and 6 are indicated with arrowheads. The beak structures were completely missing in the axoneme of fap20null and restored in those of rescued strains. Bar, 50 nm. See Table 1 for quantitative analysis of the beak loss.
FIGURE 4:
FIGURE 4:
FAP20 localizes along the entire length of flagellum and is present in all nine DMTs. (A) Immunofluorescence microscopy of nucleoflagellar apparatus (NFA) shows that FAP20 is evenly distributed along the entire length of wild-type flagella, but there is no signal in fap20null flagella. Acetylated α-tubulin was used to show the position of flagella. (B) Fluorescence image of the rescued strain FAP20-GFP. The FAP20-GFP signal is evenly distributed along the entire length of flagella. (C) The axonemes of sup-pf1 FAP20-GFP strain, frayed by treatment with ATP and a protease, show that FAP20-GFP localizes to all nine DMTs. The base of the axoneme is resistant to the protease and remains connected (asterisk). (D) Confocal images of a deflagellated FAP20-GFP cell and a wild-type cell with flagella. The FAP20-GFP signal remains on the basal bodies as two discrete dots after deflagellation. There is no GFP signal on the wild-type flagella and the basal bodies. Horseshoe-shaped fluorescence in the cell bodies (B and D) is autofluorescence of the chloroplast. Bars, 10 μm.
FIGURE 5:
FIGURE 5:
FAP20 is tightly associated with the DMT. (A) Western blot analysis of axonemes from a variety of flagellar mutants with FAP20 antibody. Coomassie-stained bands of tubulins were used as a loading control. Structures missing in each mutant are described in the text and Supplemental Table S1. (B) Western analysis of axonemes from wild type and FAP20 mutants with antibodies against a variety of axonemal proteins. Two DMT components, tektin and PACRG, are reduced in the dmj1 axonemes. (C) Biochemical fractionation of the axonemes. The wild-type axonemes were extracted with 0.6 M KCl or various concentrations of Sarkosyl. Supernatant (S) and precipitate (P) after centrifugation were analyzed by Western blot with Rib72, tektin, PACRG, and FAP20 antibodies. Tektin, PACRG, and FAP20 exhibited a similar pattern of extraction. Rib72, a component of the PF ribbon that remains after 0.7% Sarkosyl extraction, was used as a control. Representative structures that remain in precipitates under each extraction condition are illustrated below the blots.
FIGURE 6:
FIGURE 6:
FAP20 is an IJ protein. (A–D) The fap20null axoneme is defective in the IJ between A- and B-tubules. Images of DMTs in cross sections of Epon-embedded axonemes were averaged. DMT1, 5, and 6 were excluded from the analyses. The number of DMT images used for the averaging is 22, 22, 65, and 34, respectively, for wild type, fap20null, FAP20-GFP, and FAP20-BCCP. IDAs, inner dynein arms; IJ, inner junction (arrows); ODA, outer dynein arm; RS, radial spoke. (A) The wild-type B-tubule is composed of 10 tubulin PFs and one smaller, nontubulin subunit at the IJ (B) The DMT of the fap20null clearly lacks the nontubulin subunit. (C, D) The nontubulin subunits are restored in the rescued strains. Bar, 20 nm. (E, F) FAP20 localized to the inner junction (IJ) of the DMT. (E, F) Wild-type and FAP20-BCCP axonemes were labeled by an enhanced streptavidin-label method. The position of the streptavidin–cytochrome C label (orange) in the DMT was visualized by comparing averaged subtomograms of the two strains. (E) Cross-sectional view from the base of the axoneme. (F) Lateral view from a direction indicated by an arrow in E. Bars, 25 nm. (G) Model of FAP20 localization and functions. FAP20 localizes to the IJ between A- and B-tubules and stabilizes the DMT. N-DRC components, DRC1/2, or its associated proteins may extend to the IJ and be anchored by FAP20. In DMT1, 5, and 6, FAP20 may work as a scaffold for intratubular proteins to produce the beak structures.
FIGURE 7:
FIGURE 7:
FAP20 is assembled into the flagella from base to tip. FAP20-GFP gametes were mated with both fap20null (A) and wild-type (B) gametes and dikaryons observed for the emergence of FAP20-GFP in the unlabeled flagella. Bright-field (BF) and GFP fluorescence images of dikaryons were recorded at 15 and 60 min after mixing. (A) FAP20-GFP fluorescence first appeared at the base of the fap20null flagella and gradually extended to the tip. (B) FAP20-GFP was not incorporated into wild-type flagella. Arrows indicate positions of flagellar tips.

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