CCDC61 Is an Evolutionally Conserved Protein Paralogous to SAS6

(A) Domain architectures of the XRCC4 superfamily members. Low complexity regions are drawn by lines.

(B) A phylogenetic tree of CCDC61 orthologs. Accession numbers of the corresponding amino acid sequences are provided in Table S1. Numbers are bootstrap values.

(C) Crystal structure of hCCDC61¹⁻¹⁴³. The structure is presented using a cartoon representation and a rainbow color scheme from the N terminus (N; blue) to the C terminus (C; red). Missing loops are drawn with dotted lines.

(D) Crystal structures of the XRCC4 superfamily members SAS6, XRCC4, XLF, and PAXX (PDB: 2Y3W [van Breugel et al., 2011], 1IK9 [Sibanda et al., 2001], 2QM4 [Li et al., 2008], and 3WTD [Ochi et al., 2015], respectively).

See also Figures S1, S7 and Table S1.

CCDC61 Forms Linear Filaments via Homodimerization Mediated by the Head and Coiled-Coil Domains

(A) Crystal structure of the head-to-head homodimer of hCCDC61¹⁻¹⁴³. Missing loops are drawn with dotted lines. Key residues of the interaction interface are indicated by (i) and (ii), of which magnified views are shown in the square boxes on the right. Asterisk indicates the locations of the F128 residues. Dotted lines in panel (ii) indicate hydrogen bonds. Head-to-head dimers of SAS6 and XRCC4/XLF (PDB: 2Y3V [van Breugel et al., 2011] and 3W03 [Wu et al., 2011]) are shown at the bottom.

(B) AUC results showing that hCCDC61¹⁻¹⁴³ forms homodimers in solution.

(C) Crystal structure of the zCCDC61¹⁻¹⁷⁰ tetramer. On the right, straight arrows indicate the N-to-C direction of the coiled-coil domains. The angle between the arrows is 120°.

(D) CCDC61 forms higher-order oligomers in solution. Size-exclusion chromatography with multi-angle light scattering analysis of His₆-lipoyl-zCCDC61¹⁻¹⁷⁰ (red) and His₆-lipoyl-zCCDC61^{1−170; F129E/D130A} (blue) using a Superdex S200 column at room temperature. Protein concentrations (before injection onto the column) were 1, 6.5, and 65 mg/ml (lightest to darkest red, respectively) and 1, 6.8, and 73 mg/ml (lightest to darkest blue, respectively). The minimum and maximum refractive index values of each chromatography profile were normalized to 0 and 1, respectively.

See also Figures S2 and S3.

CCDC61 Binds Microtubules

(A) Fluorescent images of RPE-1 cells, transiently overexpressing GFP-hCCDC61 or hCCDC61^F128E/D129A, showing the different CCDC61 localization patterns observed under these conditions. Bar graphs show the percentage of GFP-positive cells containing clusters-only “C” versus filament-containing cells “F” (n = 279 for GFP-hCCDC61 and n = 468 for GFP-hCCDC61^F128E/D129A counted from three biological replicates). Error bars are standard deviations. Positions of blow-up images labeled with 1 (filament-containing cell) and 2 (cluster-only cell) are indicated with white-dotted squares in the top panels. Scale bars, 20 μm.

(B) Transiently overexpressed hCCDC61 colocalizes with microtubules in cells. Immunofluorescent images of RPE-1 cells transiently overexpressing GFP-hCCDC61, GFP-hCCDC61^F128E/D129A, GFP-hCCDC61^{144−287−NES}, and GFP-hCCDC61^288−512. Anti-GFP staining is shown in green, microtubule staining in red. Magnified views of the regions indicated by the white-dotted squares in the merged images are shown either below (GFP-hCCDC61 and GFP-hCCDC61^F128E/D129A) or as insets (GFP-hCCDC61^{144−287−NES} and GFP-hCCDC61^288−512). Displayed are representative images acquired from a total of 14, 8, 10, and 11 different RPE-1 cells for GFP-hCCDC61, GFP-hCCDC61^F128E/D129A, GFP-hCCDC61^{144−287−NES}, and GFP-hCCDC61^288−512, respectively. Scale bars, 10 μm.

(C) Coiled-coil and C-terminal regions of hCCDC61 bind microtubules in vitro. Coomassie-stained SDS-PAGE gel showing a co-pelleting assay of taxol-stabilized microtubules with the head domain (1–143), PAXX-fused coiled-coil domain (144–287), or the C-terminal region (288–512) of hCCDC61. S and P indicate supernatant and pellet fraction, respectively.

(D) The coiled-coil domain of hCCDC61 directly binds microtubules. Negative-stain EM micrographs of microtubules that show their decoration with a layer of PAXX-hCCDC61^144−287 that is not observed with the corresponding 5E mutant of CCDC61. Scale bars, 200 and 50 nm in the overview panels (left) and the magnified panels (right), respectively.

(E) Quantification of the widths of microtubules decorated by PAXX-hCCDC61^144−287 or in the presence of PAXX-hCCDC61^{144−287; 5E} from (D). Widths of five different positions of ten microtubules were measured for each construct. Each point (blue for PAXX-hCCDC61^144−287 and green for PAXX-hCCDC61^{144−287; 5E}) represents a measured width at each position. Error bars (standard deviations from the mean) are shown in black lines with flat arrow ends.

See also Figure S4.

CCDC61 Associates with Basal Bodies and Plays a Role in Ciliogenesis

(A) xCCDC61 associates with basal bodies and rootlets in multi-ciliated epidermal cells of Xenopus embryos. A fluorescent image of a Xenopus embryo expressing xCCDC61-RFP (red), the basal body component Centrin2-BFP (blue), and the rootlet component Clamp-GFP (green). Scale bar, 3 μm.

(B) Location of hCCDC61 at the periphery of basal bodies of primary cilia. Immunofluorescent image of an RPE-1 cell transiently overexpressing GFP-hCCDC61. Co-immunofluorescent staining was performed against GFP (green), basal bodies (γ-tubulin, red), and the ciliary axoneme (ARL13B, magenta). Scale bar, 1 μm.

(C) Ciliated cells of control and CCDC61-knockout RPE-1 cells. Immunofluorescent images show representative immunofluorescent images used for quantifications of ciliogenesis of primary cilia. Scale bar, 10 μm. The bar graph shows that ciliogenesis was delayed in the CCDC61 knockout cells. Data shown correspond to three biological replicates (total cell counts n = 1,181, 1,103, and 1,008 for control, clone 1 and clone 2 cells after 24-h serum starvation respectively, and n = 1,151, 1,046 and 1,242 for control, clone 1 and clone 2 after 48-h serum starvation, respectively). Percentages are relative to control cells. Bar graphs show mean ± standard deviation.

See also Figure S5.

Chlamydomonas VFL3 Protein Localizes to Basal Bodies and the Proximal Ends of Flagella

(A) Rescue of abnormal flagella numbers in vfl3 strains by wild-type VFL3. Bar chart showing flagella numbers observed in wild-type strains (CC-124 and CC125), mutant strains (vfl3-1 and vfl3-2), and the vfl3-1 and vfl3-2 strains expressing VFL3 constructs in Chlamydomonas. The numbers of cells “n” used for calculating ratio flagella numbers are shown on the right side of the chart. A χ² test was used to determine if the number of cells with zero flagella was significantly different. NS, not significant; ^∗∗∗p < 0.0001.

(B) Wild-type VFL3 protein localizes to Chlamydomonas basal bodies. In the first column, cells were stained with acetylated α-tubulin (red) for cilia and rootlet microtubules, anti-HA (green) for UNI2, and anti-GFP (magenta) for VFL3. Scale bar, 4 μm. Magnified views (4×) of the basal body regions (white boxes) are shown on the other three columns. Scale bars, 1 μm.

(C) Localization of VFL3 is affected in the 5E mutant. In the first column, cells were stained with acetylated α-tubulin (red) for cilia and rootlet microtubules, anti-HA (green) for wild-type and mutant VFL3, and anti-BLD10/CEP135 (magenta). Scale bar, 4 μm. Magnified views (4×) of the basal body regions (white boxes) are shown on the other three columns. Scale bar, 1 μm.

See also Figure S6.

Model of the Role of CCDC61 in Ciliary Function (in Chlamydomonas)

CCDC61 localizes to the basal body and forms filaments that bind to centriolar and/or non-centriolar microtubules. This facilitates striated fiber formation and the correct formation of basal body-associated structures, and therefore, results in the correct cilium number. A CCDC61 mutant that does not bind microtubules (MT-binding null mutant) still localizes to the basal body region. However, the mutant is incapable of facilitating striated fiber formation, leads to incorrect formation of basal body-associated structures, and therefore causes abnormal cilium numbers.