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. 2007 Feb 12;176(4):473-82.
doi: 10.1083/jcb.200611115.

Chlamydomonas Reinhardtii Hydin Is a Central Pair Protein Required for Flagellar Motility

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Free PMC article

Chlamydomonas Reinhardtii Hydin Is a Central Pair Protein Required for Flagellar Motility

Karl-Ferdinand Lechtreck et al. J Cell Biol. .
Free PMC article

Abstract

Mutations in Hydin cause hydrocephalus in mice, and HYDIN is a strong candidate for causing hydrocephalus in humans. The gene is conserved in ciliated species, including Chlamydomonas reinhardtii. An antibody raised against C. reinhardtii hydin was specific for an approximately 540-kD flagellar protein that is missing from axonemes of strains that lack the central pair (CP). The antibody specifically decorated the C2 microtubule of the CP apparatus. An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes. Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1). In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity. Hydrocephalus caused by mutations in hydin likely involves the malfunctioning of cilia because of a defect in the CP.

Figures

Figure 1.
Figure 1.
HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3, which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3, and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 (Cole et al., 1998) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼106 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes (Hou et al., 2004). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.
Figure 2.
Figure 2.
Hydin localizes to the CP. (A) Immunofluorescence microscopy of isolated axonemes (a–c) and axonemes treated with ATP and trypsin to induce partial (d–f) or complete (g–i) extrusion of the CP. Staining with anti-acetylated tubulin (a, d, and g) reveals the CP as a thin microtubular bundle projecting (arrowheads in d and f) or completely extruded (arrowheads in g and i) from the axonemes. Anti-hydin staining (b, e, and h) colocalized with the thin microtubular structures (arrowheads in f and i) and was absent from regions of axonemes now vacated by the CP (f) as well as entire axonemes from which the CP was completely extruded (i). Because of the prolonged incubation with protease, signal strength had declined in panels h and i. Bar, 5 μm. (B) Whole mount immunogold EM of extruded CPs. (a) Negatively stained axonemes with protruding CPs (arrowheads) showing the preparation used for immunogold labeling experiments. (b and c) 15-nm gold complexes (arrowheads) labeling hydin were observed mostly along the C2 microtubule on the concave side of the arc formed by the CP. The bracket shows increased label density on disintegrated CP. (d and e) In contrast, gold particles labeling the C1 proteins CPC1 (d) and PF6 (e) were predominately located on the convex side of the CP. Immunostaining with anti-PF6 was performed as described by Bernstein et al. (1994). Bars: (a) 2 μm; (b–d) 500 nm; (e) 350 nm.
Figure 3.
Figure 3.
Hydin is greatly reduced in flagella of CP mutants. (A) Immunofluorescence microscopy of detached flagella. Wild-type (CC124; a and b), pf15a (c and d), and pf18 (e and f) cells were allowed to settle onto polyethylenimine-treated slides that were then submerged into −20°C methanol. This procedure frequently caused the flagella to detach, and the cell bodies were subsequently washed off. (top) Anti-hydin signal; (bottom) merged images of anti-hydin and anti-acetylated tubulin. The hydin signal was nearly undetectable in flagella of pf15a and pf18. Bar, 5 μm. (B) Western blot of isolated axonemes of CC124 (wild type), pf15a, and pf18. The blot was probed with anti-hydin and an antibody to the outer dynein arm intermediate chain IC2 (King and Witman, 1990) as a loading control.
Figure 4.
Figure 4.
HY3 knockdown affects flagellar assembly. (A) HY3 mRNA is reduced in HY3 RNAi cells. Quantitative PCR was used to determine the amount of HY3 message in several transformants. The data are shown as a percentage of wild type for strains hyN3, hyN4, hyN11, hyD2, hyS1, and hyS2 and are based on one (hyS1), two (hyD2 and hyS2), or three independent isolates of total RNA. (B and C) HY3 RNAi cells have short flagella or lack flagella. Data are based on immunofluorescence images obtained with anti-acetylated tubulin. (B) Frequency of stumpy flagella (<1 μm) and bald cells in control (CC3395) and the HY3 RNAi strains hyN3 and hyN4. (C) Flagellated cells of strains hyN3 and hyN4 have shorter flagella than those of the control strain CC3395. Only flagella >1 μm were measured.
Figure 5.
Figure 5.
The flagella of HY3 RNAi strains arrest in the hands-up or hands-down position. (A) DIC image of living cells of hyN3 showing the resting positions of the two flagella of each cell. Arrowheads indicate the hands-down flagellum positioned along the cell body. (B) Residual flagellar movement of a hyS1 cell documented by DIC microscopy. The hands-down flagellum (arrowheads) is relatively inactive. Frame numbers are indicated and approximately equal the time in seconds. The complete sequence is shown in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200611115/DC1). (C) Methanol-fixed hyN3 cells were double labeled with anti-hydin (red) and anti-acetylated tubulin (green). Frequently, residual hydin accumulated in one of the two flagella (arrowheads). Bars, 5 μm.
Figure 6.
Figure 6.
Flagella of HY3 RNAi cells lack the C2b projection and part of the C2c projection. (A–H) Axonemal cross sections of wild type (A and G; CC3395) and the HY3 RNAi strains hyN4 (B, D, and H), hyN3 (C), and hyD2 (E and F). (G and H) Detail from A and B, respectively. (I and J) Image averages based on six wild-type (I) and six hyN4 images (J). (K and L) Schematic representations of the CP apparatus of wild type (K) and HY3 RNAi cells (L). Similar defects were observed in all four hydin RNAi strains analyzed by EM. In H, J, and L, the defects in the CP of the hydin RNAi cells are marked with arrowheads (absence of projection C2b) and arrows (absence of density of C2c). Arrowheads in E point out defective and missing doublets; note the presence of dynein arms on some of the singlet microtubules in E. Black dots indicate doublet No. 1 lacking the outer dynein arm. Bar, 0.1 μm.
Figure 7.
Figure 7.
Hydin interacts with KLP1 and the CPC1 complex. (A) Western blots of isolated axonemes from wild-type, hyN3, and hyN4 probed with antibodies to the CP proteins hydin, PF6, FAP101, CPC1, and KLP1. Antibodies to α-tubulin and IC2 were used as loading controls. Both hydin and KLP1 were significantly reduced in the axonemes of hydin RNAi cells. (B) Western blot comparing the amounts of hydin, PF6, CPC1, KLP1, IC2, and IFT139 in the detergent-soluble flagellar membrane-plus-matrix fraction (M&M), axonemes (Ax), residual axonemes after 0.6 M KCl extraction (KCl-P), and the KCl extract of axonemes (KCl-S). Protein from equivalent numbers of flagella were loaded for each sample. (C) Western blot comparing the solubility of hydin, PF6, CPC1, and KLP1 in wild-type and cpc1 flagella. Protein from equal numbers of flagella was loaded for each sample. (D) Schematic representation of the CP apparatus. In this model, hydin is an essential component of the C2b projection. It extends to the C2a projection, where it stabilizes the association of KLP1 with the C2 microtubule, and extends in the other direction to interact with the CPC1 complex of the C1 microtubule.

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