Full text links CiteDisplay options Display options Format AbstractPubMedPMID
Abstract
Motile cilia have a '9+2' structure containing nine doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA projections remain largely unknown. Here, we combined genetic, proteomic and cryo-electron tomographic approaches to compare the CA of wild-type Chlamydomonas reinhardtii with those of three CA mutants. Our results show that two proteins, FAP42 and FAP246, are localized to the L-shaped C1b projection of the CA, where they interact with the candidate CA protein FAP413. FAP42 is a large protein that forms the peripheral 'beam' of the C1b projection, and the FAP246-FAP413 subcomplex serves as the 'bracket' between the beam (FAP42) and the C1b 'pillar' that attaches the projection to the C1 microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of the C1b, C1f and C2b projections, and loss of these proteins leads to ciliary motility defects.
Keywords: Axoneme; Central pair complex; Cryo-electron tomography; Flagella; Subtomogram averaging.
Competing interests The authors declare no competing or financial interests.
Figures
Fig. 1.
Known structure and proteome of…
Fig. 1.
Known structure and proteome of the CA, and motility phenotypes of Chlamydomonas CA… Fig. 1. Known structure and proteome of the CA, and motility phenotypes of Chlamydomonas CA mutants. (A) Schematic of a Chlamydomonas flagellum in cross-sectional view, showing the DMTs with IDAs and ODAs, N-DRCs, and RSs that project toward the CA. The cross-section is viewed from the proximal end (flagellar base). Box indicates the CA region shown in B. (B) Isosurface rendering of the averaged Chlamydomonas CA in cross-sectional view (from the proximal end) and predicted locations of CA proteins based on previous proteomic studies (Zhao et al., 2019). Naming and coloring of CA projections are adapted from Carbajal-Gonzalez et al. (2013) and Fu et al. (2019) using our data. (C) Average swimming velocities of WT Chlamydomonas and the three CA mutants fap246, fap413 and fap42. Mean±s.d. of n=20 cells. **P<0.01 (two-tailed, paired Student's t-test). (D–F) Swimming trajectories of Chlamydomonas WT and CA mutants fap246 and fap42. Each colored line represents the swimming path of one cell recorded for 2 s. Scale bar: 100 µm.
Fig. 2.
Cryo-ET and subtomogram averaging of…
Fig. 2.
Cryo-ET and subtomogram averaging of the fap246 CA reveals defects in the C1b… Fig. 2. Cryo-ET and subtomogram averaging of the fap246 CA reveals defects in the C1b projection, and 3D classification of the fap413 CA reveals partial loss of the bracket density. (A–H) Tomographic slices (A,B,E and F) and isosurface renderings (C,D,G and H; color-coded as in Fig. 1B) of the averaged CA repeats of WT (A–D) and fap246 (E–H) in cross-sectional (A,C,E and G; viewed from the proximal end) and longitudinal (B,D,F and H) views show that the C1b inner and outer bracket (ib and ob, respectively) densities are present in WT but missing in fap246 (white arrowheads). The thin white line in A indicates the position of the slices shown in B and F. In longitudinal slices, the proximal side is on the left. Inset, schematic of the C1b projection shows pillar, bracket and beam regions. Scale bar: 10 nm (shown in F, valid for A,B,E and F). (I–K) 3D classification focused on the bracket region of the fap413 CA average revealed three distinct classes (percentages of repeats are indicated for each class). Cross-sectional views (viewed from the proximal end) of the class averages show the presence (red arrowhead in I), reduction (pink arrowhead in J) and loss (white arrowhead in K) of the inner bracket density. Scale bar: 10 nm (shown in K, valid for I–K). In A,C,I, numbers indicate the protofilament numbers of C1.
Fig. 3.
Cryo-ET and subtomogram averaging localizes…
Fig. 3.
Cryo-ET and subtomogram averaging localizes FAP42 to the C1b projection. (A–J) Tomographic slices… Fig. 3. Cryo-ET and subtomogram averaging localizes FAP42 to the C1b projection. (A–J) Tomographic slices (A–C and F–H) and isosurface renderings (D,E,I and J; color-coded as in Fig. 1B) of the averaged CA repeats of WT (A–E) and fap42 (F–J) in cross-sectional (A,D,F and I; viewed from the proximal end) and longitudinal views (B,C,E,G,H and J) show that the beam and outer bracket are present in the WT CA repeats (blue and red arrowheads, respectively), but are missing in fap42 (white arrowheads). The thin lines in A indicate the position of the slices shown in B and G (white line) and in C and H (blue line). In longitudinal slices, the proximal side is on the left. ib, inner bracket; pp, peripheral part of the pillar. Scale bar: 10 nm (shown in H, valid for all EM images).
Fig. 4.
3D classification reveals structural heterogeneity…
Fig. 4.
3D classification reveals structural heterogeneity for the C1b and C1f projections in fap42… Fig. 4. 3D classification reveals structural heterogeneity for the C1b and C1f projections in fap42 but not in WT. (A–H) Tomographic slices of averaged CA repeats of WT (A–D) and fap42 (E–H) axonemes viewed in cross-sectional (A,E; viewed from the proximal end) and longitudinal orientations (B–D and F–H) show that, in addition to lacking the beam and outer bracket density, several C1b and C1f densities, including the inner bracket (red arrowheads), the peripheral part of the pillar (blue arrowheads) and the peripheral part of C1f (green arrowheads) are blurred in the averages of all CA repeats of fap42 axonemes compared to those of the WT. The thin lines in A indicate the position of the slices shown in B and F (red line), C and G (blue line), and D and H (green line). In longitudinal slices, the proximal side is on the left. Dashed boxes in F,G and H indicate regions shown as separate classes in J,M; K,N; and P,R,T, respectively. (A′) Schematic of the C1b (with pillar, FAP246–FAP413 bracket and FAP42 beam) and C1f projections. The red, blue and green colored regions indicate regions that are blurred in the average of all fap42 CA repeats, as shown in E–H. (I–T) 3D classification focused on the peripheral regions of C1b (I–N) and on C1f (O–T) reveals distinct classes (percentages of repeats are indicated for each class). Cross-sectional (I,L,O,Q and S; viewed from the proximal end) and longitudinal views (J,K,M,N,P,R and T). In longitudinal slices, the proximal side is on the left. The red, blue and green arrowheads indicate presence of densities, whereas the white arrowheads indicate missing densities. The turquoise dotted lines in R and T serve as references to show the positional differences of structures in different classes. Scale bars: 10 nm (in H, valid for A–H; in N, valid for I–N; in T, valid for O–T).
Fig. 5.
Summary model depicting the structural…
Fig. 5.
Summary model depicting the structural organization of the C1b projection and showing the… Fig. 5. Summary model depicting the structural organization of the C1b projection and showing the effects of partial loss of the FAP246–FAP413–FAP42 subcomplex on the CA structure. (A–C) The isosurface rendering (A) and schematic drawings (B,C) show part of the 3D structure of the averaged Chlamydomonas WT CA repeat viewed in longitudinal orientation (A,B; two 16 nm repeats are shown, with the proximal end to the left) and cross-sectional orientation (C; viewed from the proximal end). The views highlight the subunit organization of the C1b projection, including the FAP246–FAP413 bracket, with the longer ‘outer bracket’ (yellow) and the attached ‘inner bracket’ density (orange), and the FAP42 beam (green). In C, the pillar base and peripheral density are shown in dark and medium blue, respectively. The predicted locations of neighboring candidate proteins in the C1b, C1f and C2b projections are indicated in C, along with the C1 and C2 microtubules (numbers indicate protofilament numbers of C1 and C2). Dashed line in C indicates the position of the CA bridge. (D–F) Schematic drawings of the averaged 16 nm CA repeat of fap246 (D), fap42 (E) and fap413 (F) in cross-sectional view (viewed from the proximal end), showing the observed structural CA defects in the mutants compared to the WT CA. In fap246 the FAP246–FAP413 bracket is missing, and the pillar (base and peripheral density colored dark and medium blue, respectively) and the beam (green) densities show positional flexibility. In fap42 the beam (FAP42) and partial bracket (FAP413) are missing, and neighboring C1b and C1f densities show positional flexibility and partial reduction. In fap413 the bracket density (FAP413) is reduced. Compared to the WT structure, the C2b density (turquoise) in all three C1b mutants was reduced, suggesting partial protein loss. See this image and copyright information in PMC
Similar articles
Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a. Hou Y, Zhao L, Kubo T, Cheng X, McNeill N, Oda T, Witman GB.Hou Y, et al.J Cell Sci. 2021 Jun 15;134(12):jcs258540. doi: 10.1242/jcs.258540. Epub 2021 Jun 28.J Cell Sci. 2021.PMID: 33988244Free PMC article.
Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus. Fu G, Zhao L, Dymek E, Hou Y, Song K, Phan N, Shang Z, Smith EF, Witman GB, Nicastro D.Fu G, et al.J Cell Biol. 2019 Dec 2;218(12):4236-4251. doi: 10.1083/jcb.201906006. Epub 2019 Oct 31.J Cell Biol. 2019.PMID: 31672705Free PMC article.
Composition and function of the C1b/C1f region in the ciliary central apparatus. Joachimiak E, Osinka A, Farahat H, Świderska B, Sitkiewicz E, Poprzeczko M, Fabczak H, Wloga D.Joachimiak E, et al.Sci Rep. 2021 Jun 3;11(1):11760. doi: 10.1038/s41598-021-90996-9.Sci Rep. 2021.PMID: 34083607Free PMC article.
Composition and function of ciliary inner-dynein-arm subunits studied in Chlamydomonas reinhardtii. Yamamoto R, Hwang J, Ishikawa T, Kon T, Sale WS.Yamamoto R, et al.Cytoskeleton (Hoboken). 2021 Mar;78(3):77-96. doi: 10.1002/cm.21662. Epub 2021 Apr 28.Cytoskeleton (Hoboken). 2021.PMID: 33876572Free PMC article.Review.
Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a. Hou Y, Zhao L, Kubo T, Cheng X, McNeill N, Oda T, Witman GB.Hou Y, et al.J Cell Sci. 2021 Jun 15;134(12):jcs258540. doi: 10.1242/jcs.258540. Epub 2021 Jun 28.J Cell Sci. 2021.PMID: 33988244Free PMC article.
References
Afzelius, B. A. (2004). Cilia-related diseases. J. Pathol. 204, 470-477. 10.1002/path.1652 - DOI - PMC - PubMed
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. 10.1016/S0022-2836(05)80360-2 - DOI - PubMed
Braun, D. A. and Hildebrandt, F. (2017). Ciliopathies. Cold Spring Harb. Perspect. Biol. 9, a028191. 10.1101/cshperspect.a028191 - DOI - PMC - PubMed
Brown, J. M. and Witman, G. B. (2014). Cilia and Diseases. Bioscience 64, 1126-1137. 10.1093/biosci/biu174 - DOI - PMC - PubMed
Brown, J. M., Dipetrillo, C. G., Smith, E. F. and Witman, G. B. (2012). A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus. J. Cell Sci. 125, 3904-3913. 10.1242/jcs.107151 - DOI - PMC - PubMed
