Inner lumen proteins stabilize doublet microtubules in cilia and flagella.

Strains and culture conditions The Chlamydomonas strains used in this study are listed in Supplementary Table 3 . The double and triple mutants were constructed by standard methods 3 . All cells were cultured on Tris-acetate-phosphate (TAP) plates with 1.5% agar or in TAP medium. fap45 and fap52 mutants were isolated from a library of mutants generated by aphVIII gene insertion 31 . The mutants were backcrossed with wild type several times before use. For the PCR analyses, following primers were used; FAP45-F: 5′-GTGGCTTGAGCACCCTACTCACTT-3′; FAP45-R: 5′-CCGCTTGCTACCTCCAAATAAAGA-3′; FAP52-F: 5′-CGCCGACCTCTACATTTCTGAGTT-3′; FAP52-R: 5′-CTTTGACTCCAGGTCCCAGATGAT-3′; aphVIII -F: 5′-GTCGACTTGGAGGATCTGGACGA-3′. For the probe of the southern blot, the aphVIII coding sequence was amplified with following primers; 5′-ATGGACGATGCGTTGCGTGC-3′; 5′-TCAGAAGAACTCGTCCAACAGCCG-3′. The probe was PCR-labeled with biotin-16-dCTP and detected by Streptavidin-HRP.

Motility assay using the CLONA system

To assay the motilities of Chlamydomonas cells in TAP medium or TAP with ficoll, videos were recorded using a high-speed camera (EX-F1; Casio) attached on a dark-field light microscope (BX51; Olympus) at 600 fps. The videos were analyzed using the CLONA system 41 and the results were plotted using R Project software.

Antibodies

The antibodies used in this study are described in Supplementary Table 4 . Anti-FAP45 and anti-FAP52 antibodies were raised against full-length FAP45 and FAP52 proteins, respectively (Supplementary Fig. 2a ). The full-length cDNA of FAP45 or FAP52 was cloned between the EcoRI and BamHI sites of pMAL-c2x (New England Biolabs). In both constructs, a 6×His tag was inserted into the HindIII site of pMAL-c2x to enable purification in two steps. For affinity purification, each cDNA was also cloned between the NdeI and EcoRI sites of pColdI (Takara). Expression of the recombinant proteins was induced in Escherichia coli BL21(DE3) (TAKARA BIO Inc.) with 0.3 mM IPTG, and almost all of the expressed protein was solubilized from each construct. MBP-FAP45-6×His and MBP-FAP52-6×His were purified with amylose resin (New England Biolabs) and then further purified with Ni-NTA agarose (Qiagen). These purified proteins were used as antigens. Each antibody was affinity purified using polyvinylidene difluoride membranes blotted with FAP45-6×His or FAP52-6×His. For western blot analyses, we loaded 5 μg of isolated axonemes in each lane. Full blot images are shown in Supplementary Fig. 9 .

Chemical crosslinking of axonemes

Isolated axonemes of wild type, fap45 , and fap52 were treated with EDC (Thermo Scientific) in HMEK (30 mM Hepes–KOH, pH 7.4, 5 mM MgSO 4, 1 mM EGTA, 50 mM K-acetate) for 60 min at room temperature. After quenching the reaction, crosslinked axonemes were analyzed by SDS–PAGE and western blotting with anti-FAP45 and anti-FAP52 antibodies. The crosslinked products were immunoprecipitated with anti-FAP45 antibody following a previously described method 42 .

Mass spectrometry analysis

(LC tandem MS) of the precipitates was performed at the Proteomics and Mass Spectrometry Facility (University of Massachusetts Medical School).

Immunofluorescence microscopy Nucleoflagellar apparatus

(NFA) was prepared as previously described 43 . After fixation with 2% formaldehyde for 10 min at room temperature, NFAs were treated with cold methanol (−20 °C). The samples were immunostained as previously described 44 . Images were taken with a CCD camera (ORCA-R2; Hamamatsu Photonics) linked to a fluorescence microscope (IX70; Olympus).

Generation of BCCP-tagged strains

Fragments from the start codon to immediately before the stop codon in the FAP45 and FAP52 genes were amplified by genomic PCR and inserted into pIC2 plasmids 16 . In the FAP52 construct, a 3×HA tag was inserted into the C terminus of FAP52. Each construct was linearized and transformed into fap45 or fap52 cells by electroporation. Streptavidin-Alexa546 staining of the axonemes was performed as previously described 39 . Thin-section TEM Samples for thin-section TEM were prepared as previously described 45 except that we used 50 mM Na-phosphate, pH 7.0 instead of cacodylate buffer. The samples were observed using a transmission electron microscope (JEM-3100FEF; JEOL) equipped with a 4096 × 4096-pixel CMOS camera (TemCam-F416; TVIPS). All images were taken at 300 keV, with ~3 μm defocus, at a magnification of 40,000 and a pixel size of 2.5 Å.

Cryo-sample preparation

Purified axonemes in HMDEK buffer (30 mM Hepes–KOH (pH7.4), 5 mM MgSO 4 , 1 mM dithiothreitol, 1 mM EGTA, 50 mM potassium acetate, protease inhibitor cocktail (Nacalai Tesque)) were incubated with anti-beta-tubulin antibody (1:10,000 final; T0198, SIGMA) for 15 min on ice, followed by incubation with goat anti-mouse IgG (H+L) 15 nm gold (1:20 final; BB International) and 15 nm colloidal gold conjugated with BSA. The mixtures were loaded onto home-made holey carbon grids and plunged into liquid ethane at −180 °C using an automated plunge-freezing device (EM GP; Leica).

Image acquisition

Grids were transferred into the JEM-3100FEF using a high-tilt liquid nitrogen cryo-transfer holder (914; Gatan Inc.). Tilt series images were recorded at −180 °C using a K2 summit direct detector (Gatan) and the serialEM 46 . The angular range was ±60° with 2.0° increments. The total electron dose was limited to 100 e − /Å 2 and the nominal magnification was ×6000. An in-column energy filter was used with a slit width of 20 eV and a pixel size of 7.1 Å.

Image processing

Image processing for subtomogram averaging of DMT was carried out as previously described 33 , 34 . Tilt series images were aligned and back-projected to reconstruct 3D tomograms using IMOD 47 . Alignment and averaging of the subtomograms were performed using custom Ruby-Helix scripts 48 and PEET software 7 to average the 96-nm repeats of DMTs. UCSF Chimera was used for isosurface renderings 49 . FSC curves were shown in Supplementary Fig. 5f . HS-AFM observation Flagella were demembranated with 0.5% Nonidet P-40 in HMDEK (30 mM Hepes–KOH, 30 mM Hepes–KOH, pH 7.4, 5 mM MgSO 4 , 1 mM DTT, 1 mM EGTA, 50 mM K-acetate, protease inhibitor cocktail (Nacalai Tesque)), followed by centrifugation for 2 min. The axoneme pellets were suspended with ATP buffer (HMDEK containing 0.1 mM ATP, 1 mM ADP, and 0.5% polyethylene glycol (20,000) and incubated for 1 min at room temperature. Frayed axonemes were then diluted with HMDEK and placed on a mica stage. After 10 min incubation, residuals are washed with the HMDEK buffer. The AFM images were recorded using a home-built high-speed atomic force microscope based on tapping mode 50 – 52 at frame rates of 1–2 fps. All observations were performed in HMDEK buffer at room temperature. We selected DMTs of which the angle to the fast-scan axis is