Fig 1.
Cryo-ET structure of Tetrahymena N-DRC.
(A) Schematic representation of a Tetrahymena cell and a cross-section of the cilium (marked with a red rectangle on the diagram of Tetrahymena) showing its ultrastructural organization. (A’, A”) Schematic representation of a microtubule doublet cross-section (A’) and a schematic of a longitudinal view of a microtubule doublet with the 96-nm axonemal repeat and its main protein complexes (A”): ODAs (yellow), two-headed (f/I1) and single-headed (a, b, c, e, d, g) IDAs (pink), radial spokes (cyan), N-DRC (navy blue), T/TH (dark green), MIA-like complex (light green) and Ccdc96/Ccdc113 complex (orange); (prepared based on cryo-ET images [6,7]). (B) Cryo-tomographic slice through the averaged volume of the wild-type 96-nm repeat showing the position of the N-DRC. (C) Corresponding isosurface rendering of the 96-nm repeat average with the N-DRC colored in navy blue. (D) The segmented N-DRC structure as seen from the central pair complex (left) and from the opposite (~180 degrees rotation) side (right). The yellow spheres represent the individual connections (1–18) to neighboring axonemal components. The putative position of some of the N-DRC proteins (DRC1 yellow, DRC2 green, DRC3 bright green, DRC4 pink) was estimated by comparing our structure with published ones from both Tetrahymena and Chlamydomonas models [17,24,28,29]. Connections with the Ccdc113/Ccdc96 complex were identified by comparison of the 96 nm repeat structure from WT with the structures of CCDC113-KO and CCDC96-coDel mutant cells. The identification of other axonemal protein complexes that connect with the N-DRC, potentially directly or through yet unidentified proteins, was derived by comparisons of our structure with previously published structures of the 96 nm repeat in different species [4–10,17,24,28,29]. (E) N-DRC (navy blue) connections (yellow spheres) shown in the context of the axoneme, (IDAs (pink), radial spokes (cyan), MIA-like complex (light green), and Ccdc96/Ccdc113 complex (orange). List of connections: nBMT-PF9, protofilament 9 of the neighboring B-tubule; B-MT-PF11, protofilament 11 of the B-tubule; Ccdc113/Ccdc96, Ccdc113/Ccdc96 complex; RS3, radial spoke 3; A-MT-PF2, protofilament 2 of the A-tubule; A-MT-PF3, protofilament 3 of the A-tubule; RS2, radial spoke 2; IDA g (e, c, e), inner dynein arms g (e, c, e, respectively); udL1, undefined linker 1; udL2, unidentified linker 2. Abbreviations: a, b, c, e, d, g–single-headed inner dynein arms; fα, fβ–dynein heavy chains of two-headed inner dynein arm IDA f/I1; IC/LC–intermediate and light chains of the IDA f/I1, MIP1a, MIP1b –microtubule inner proteins 1a and 1b; MIA–modifier of inner arms, N-DRC–nexin-dynein regulatory complex; RS1, RS2, RS3 –radial spokes 1, 2 and 3, T/TH–tether/tetherhead complex.
Fig 2.
Ccdc113 and Ccdc96 localize in cilia but not at the ciliary tips.
(A) Western blot of the ciliary proteins isolated either from wild-type cells (WT) or cells expressing HA-tagged Ccdc113 under the control of a native promoter (native level, nat, with 3HA) or the MTT1 promoter (overexpression, oex, with single HA), or rescued cells (res, 3HA). Note the presence of two co-migrating bands (arrows), suggesting post-translational modifications of Ccdc113 protein (see also S4A Fig). A star indicates a band which is non-specifically recognized by the secondary antibodies (serving as a loading control). (B-G) Immunofluorescence confocal images of Tetrahymena cells expressing Ccdc113-3HA at the native level, double labeled with anti-HA (B, B’ and E) and either anti-α-tubulin (C, C’) or anti-centrin (F) antibodies. (D, D’ and G) Merged images. Note the absence of Ccdc113 at the cilia tips. (B’, C’, D’) The magnified cilia marked with white insets on B, C, and D. Scale bar = 10 μm. (H) Western blot of ciliary proteins isolated from WT cells and cells expressing Ccdc96-2V5 under the control of its native promoter. Arrow indicates the position of the Ccdc96-2V5 protein. (I-K”) Immunofluorescence confocal images of Tetrahymena cells expressing Ccdc96-2V5 at the native level, double labeled with anti-V5 (I, I’) and anti-α-tubulin (J, J’) antibodies showing that Ccdc96 protein is distributed along the entire cilium except for the tip. (K-K”) Merged image. (I’, J’, K’, K”) Magnified cilia marked with white inset on I, J and K. (K”) Note the shift in the merged red and green channels enabling better visualization of the region where Ccdc96-2V5 localizes. Scale bar = 10 μm.
Fig 3.
Ccdc113 and Ccdc96 are required for normal ciliary beating.
(A-C) Swimming paths of (A) WT, (B) CCDC113-KO and (C) CCDC113-KO rescued cells recorded for 3.2 s using a video camera. The trajectories are indicated by colored lines; note that the drawn lines were shifted to be positioned parallel to the cell paths and make the original paths visible. Dots are most likely immotile dividing cells. Very short trajectories may represent cells that slowed down in order to change the direction of swimming. Bar = 400 μm. (D) Graph representing the average distance swum by WT, CCDC113-KO mutant (KO) and CCDC113-KO rescued (res) cells expressing Ccdc113-3HA, normalized to WT values. Error bars represent standard error. (E-F) Trajectories of swimming CCDC96-coDel mutant (E) and CCDC96-coDel rescued cells (F) recorded for 3.2 s using a video camera. Trajectories are marked with colored lines. Note that no selection pressure to increase the number of wild-type copies of CCDC96 was applied in rescued mutant and thus some cells have more copies and swam almost as WT cells while others (with low copy number) swam similarly to mutant cells. Bar = 400 μm. (G) Graph representing the average distance swum by WT, CCDC96-coDel mutants (coDel) and CCDC96-coDel rescued (res) cells normalized to the WT value. Error bars represent standard error. (H) Trajectories of swimming DRC3-coDel mutant cells marked with colored lines. Note that DRC3-coDel trajectories are straight while those of CCDC113-KO and CCDC96-coDel mutants are wavy and kinky, suggesting frequent changes in swimming direction. Bar = 400 μm. (I) Drawings representing examples of the observed subsequent positions of a cilium of WT and mutant cells (CCDC113-KO, CCDC96-coDel, DRC3-coDel) during the power (red) and recovery (blue) stroke. The position of the cilium marked in purple represents a cilium that is still bending (as in the recovery stroke) but already lifting up (as in the power stroke). Drawings were prepared using the individual frames extracted from digitized videos of WT and mutant Tetrahymena. Traced cilia were positioned at the middle dorsal region of the cell. (J) Graph representing cilia beating frequency of WT and mutant cells. T-test denotes P value < 0.001. (K) The analyses of the ciliary amplitude. The schematic representation of all recorded consecutive positions of the cilium during the power and recovery stroke. The amplitude was measured as the angle between two most angled positions of the cilium (calculated as the angle of the triangle marked by the intersection points of the cilium with the measuring line and cilium base. (L) Graph representing cilia beating amplitude of WT and mutant cells. T-test denotes P value < 0.001 for CCDC113-KO and DRC3-coDel, and 0.05 for CCDC96-coDel. Numerical data are in S10 Table.
Fig 4.
Ccdc113 and Ccdc96 form a complex positioned between N-DRC, RS3, and IDA g.
(A) Slice through a subvolume extracted from a filtered tomogram showing the 96-nm repeat (square). (B) 3D isosurface rendering of 96-nm repeat average obtained from wild-type Tetrahymena axonemes. (C) 3D isosurface rendering of 96-nm repeat average from CCDC96-coDel axonemes. (D, H, L) show the isosurface rendering structure of the Ccdc96/Ccdc113 complex (in orange) in wild-type axonemes. (D) Cross-sectional view. (H) Longitudinal view from the position of the neighboring MTd. (L) Longitudinal view from the position of the central pair (E, I, M) show tomographic slices through D, H, and L, respectively, at the positions indicated by the magenta, green and blue squared and lines in D, H, and L. (F, J, N) show the corresponding tomographic slices through the reconstruction of the CCDC96-coDel mutant, and the absence of the Ccdc96/Ccdc113 complex density. (G, K, O) show the corresponding tomographic slices through the reconstruction of the CCDC113-KO mutant, and, also in this case, the absence of the Ccdc96/Ccdc113 complex density. (E-G, I-K, M-O) Each tomographic digital section is accompanied by a corresponding segmented copy (bottom panels) with the Ccdc96/Ccdc113 complex colored in orange (and orange arrowheads), the N-DRC in navy blue, the IDA in pink and the radial spokes in cyan. Labels: RS1 to RS3–radial spokes 1 to 3; IDA a, b, c, d, e, g–inner dynein arms a, b, c, d, e, g; IC/LC–intermediate chain/light chain of the inner dynein arm l1/f, fα, fβ-heavy chains α, β of inner dynein l1/f; N-DRC–nexin-dynein regulatory complex; ODA–outer dynein arms. IDAs are named according to [7].
Fig 5.
Interactions of the Ccdc96/Ccdc113 complex with neighboring axonemal structures.
(A, E, I) Positioning of the Ccdc96/Ccdc113 complex within the 96-nm repeat of WT cilia. (B, F, J) Detailed isosurface rendering of Ccdc96/Ccdc113 complex connections in the WT structure. (B) The wild-type structure of the Ccdc96/Ccdc113 complex shows three connections to protofilament A5 (MT1-MT3), one to the base of IDA g (IDAg1) and two connections to the N-DRC proximal lobe (NDRC1, NDRC2). (C, D) Remnant densities in the corresponding area in both mutant structures show only connections to MT1, NDRC2 and IDAg1. (F) Another view of the wild-type structure of the Ccdc96/Ccdc113 complex shows four connections to the N-DRC (NDRC1-NDRC4), two connections to the base of IDA g (IDAg1, IDAg2), one connection to the protofilament A3 (MT4), and one connection to the base of the radial spoke 3 (RS3). (G, H) Remnant densities in the corresponding area present in both mutant structures show only connections with NDRC2 and IDAg1. (J) View of the base of RS3 in the wild-type structure shows a connection to the basal part of N-DRC (NDRC4), two connections to protofilaments A2 and A3 (MT5, MT6) and a connection to the base of RS3. (K, L) Remnant densities in the corresponding area present in both mutant structures show a completely missing structure in this area.
Table 1.
Selected data obtained during mass-spectrometry analyses of the proteome of cilia isolated from wild-type and mutant cells.
Table 2.
Biotinylated proteins in cilia of cells expressing BirA*-tagged Ccdc113 or Ccdc96 identified using mass spectrometry.
Fig 6.
Ciliary localization of Ccdc96 requires the presence of Ccdc113.
(A-C) Immunofluorescence confocal images of (A) wild-type (WT), (B) CCDC113-KO and (C) CCDC96- coDel Tetrahymena cells expressing Fap57A-2V5 at native levels, labeled with anti-V5 antibodies showing that Fap57A localizes in cilia independently of Ccdc113 and Ccdc96. (D) Western blot of ciliary proteins isolated from WT and knockout cells expressing Fap57A-2V5 under the control of its native promoter. Star indicates the position of the Fap57A-2V5 protein. An additional band in one of the WT samples is a partly degraded Fap57A-2V5. In the case of a blot to the right, only 25 μg of proteins were loaded. (E-E3, F) Immunofluorescence confocal image (E) and Western blot (F) showing that Ccdc96-2V5 is not targeted to cilia in cells lacking Ccdc113 (E, E1) although it is present in cells (F). (G-G2) Immunofluorescence confocal images and Western blot (H) indicating that co-expression of Ccdc113-3HA with Ccdc96-2V5 restores ciliary localization of both proteins in CCDC113-KO mutants. (I-I3) Immunofluorescence confocal image showing that Ccdc113-2V5 is not targeted to cilia in cells lacking Ccdc96. (J-J4) Immunofluorescence confocal images (J-J2) and Western blot (J3-J4) indicating that co-expression of Ccdc113-2V5 with HA-Ccdc96 restores ciliary localization of both proteins in CCDC96-coDel mutants. (K) Immunofluorescence confocal image and Western blot showing that Ccdc113-3HA expressed at higher levels (under the control of the BTU2 promoter) is present in cilia in cells lacking Ccdc96 (CCDC96-coDel mutant).