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. 2015 Sep 8;11(9):e1005508.
doi: 10.1371/journal.pgen.1005508. eCollection 2015 Sep.

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated With the Loss of Multiple Axonemal Structures

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Free PMC article

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated With the Loss of Multiple Axonemal Structures

Huawen Lin et al. PLoS Genet. .
Free PMC article

Abstract

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11. These alleles partially rescue the flagellar length defect, but not the motility defect. The suppressor does not restore the missing N-DRC and inner dynein arm proteins. In addition, the cnk11 mutations partially suppress the short flagella phenotype of N-DRC and axonemal dynein mutants, but do not suppress the motility defects. The tpg1 mutation in TTLL9, a tubulin polyglutamylase, partially suppresses the length phenotype in the same axonemal dynein mutants. In contrast to cnk11, tpg1 does not suppress the short flagella phenotype of pf7. The polyglutamylated tubulin in the proximal region that remains in the tpg1 mutant is reduced further in the pf7; tpg1 double mutant by immunofluorescence. CCDC40, which is needed for docking multiple other axonemal complexes, is needed for tubulin polyglutamylation in the proximal end of the flagella. The CCDC39 and CCDC40 proteins are likely to be involved in recruiting another tubulin glutamylase(s) to the flagella. Another difference between cnk11-1 and tpg1 mutants is that cnk11-1 cells show a faster turnover rate of tubulin at the flagellar tip than in wild-type flagella and tpg1 flagella show a slower rate. The double mutant shows a turnover rate similar to tpg1, which suggests the faster turnover rate in cnk11-1 flagella requires polyglutamylation. Thus, we hypothesize that many short flagella mutants in Chlamydomonas have increased instability of axonemal microtubules. Both CNK11 and tubulin polyglutamylation play roles in regulating the stability of axonemal microtubules.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Flagellar length of ccdc39/ccdc40 mutants can be partially rescued by the cnk11 suppressors.
At least 100 flagella from each strain were measured to determine the average flagellar length. Error bars represent standard deviation of the mean. (A) Flagellar length of various strains at 21°C. TG, transgene. *** indicates p<0.001 by t-test. (B) Flagellar length of various strains before (0 hr) and after (8 hr) temperature shift to 32°C.
Fig 2
Fig 2. Gene structure of CNK11 and its position on chromosome 7.
Blue arrows indicate the relative positions of changes found in individual mutants. Green boxes, 5’ UTR; black solid lines, introns; orange boxes, exons; purple box, 3’ UTR. Magenta solid lines, relative positions of PCR products along CNK11 and its neighboring gene Cre07.g339104 in cnk11-6. +, PCR products amplified in both wild-type and cnk11-6;-, PCR products amplified in wild-type but not in cnk11-6.
Fig 3
Fig 3. Reduced or absent N-DRC and other axonemal proteins are not restored in pf7; pf8 suppressors.
Ten micrograms of axonemes from various strains were used in the immunoblots. (A) Strains resuspended in nitrogen-free medium for 4 hours at 21°C. TG, transgene. (B) Cells were resuspended in nitrogen-free medium for 2 hours at 21°C. One-half of cells from each strain were switched to 32°C for 4 hours while the other half was maintained at 21°C for 4 hours. α-tubulin is included as a loading control.
Fig 4
Fig 4. The pf7 and pf8 mutants show splaying phenotype in isolated axonemes similar to splaying in N-DRC mutants.
(A) Fixed axonemes were stained with antibodies against LF5 (green) and acetylated α-tubulin (magenta). (B) Axonemes were scored as intact (blue), sliding (yellow), or splaying (green). At least 100 axonemes were score for each strain.
Fig 5
Fig 5. IFT velocities are not changed in the fla12 mutant.
Kymographs of IFT20::GFP in (A) FLA12 and (B) fla12 strains. Green and yellow arrows denote representative anterograde and retrograde tracks, respectively. Vertical scale bar, 2 μm; horizontal scale bar, 1 sec. (C) The anterograde IFT velocities were 1478 ± 41 nm/s (mean ± SEM) for FLA12 cells (green, n = 60) and 1460 ± 32 nm/s for fla12 cells (blue, n = 67). (D) The retrograde IFT velocities were 2074 ± 56 nm/s for FLA12 cells (green, n = 84) and 2023 ± 60 nm/s for fla12 cells (blue, n = 30).
Fig 6
Fig 6. The cnk11 mutants and paclitaxel can partially rescue flagellar shortness in motility mutants.
At least 100 flagella from each strain were measured to determine the average flagellar length. Error bars represent standard deviation of the mean. *** indicates p<0.001 by the t-test. (A) Flagellar length of various strains at 21°C. (B) Cells were either treated with autolysin only (blue) or treated with autolysin and 10 μm paclitaxel (green) for 30 minutes at 21°C before fixation.
Fig 7
Fig 7. The localization of polyglutamylated tubulin is affected by both tpg1 and pf7.
(A) Two micrograms of flagellar protein were used in immunoblots. α-tubulin is included as a loading control. (B) The nucleoflagellar apparatus (NFAP) from various strains was stained with polyglutamylated tubulin antibody (green) and acetylated α-tubulin (magenta). (C) The length of polyglutamylated tubulin (green) and flagella (indicated by acetylated α-tubulin, magenta). *** indicates p<0.001 by the t-test between the lengths of polyglutamylated tubulin in tpg1 and in pf7; tpg1.
Fig 8
Fig 8. The cnk11-1 mutant has increased tubulin turnover at the flagellar tip.
At least 100 flagella from each strain were measured to determine the average flagellar length. Error bars represent standard deviation of the mean. (A) Flagellar lengths of wild-type (CC-125, green circles) and cnk11-1 (blue triangles) cells before and after deflagellation by pH shock. (B) Cells that carry HA-α-tubulin were mated to cells of opposite mating type that have the corresponding genotype for 30, 60, and 90 minutes before fixation. Flagellar lengths were measured from the non HA-tagged parental strain in mating of wild-type x wild-type (CC-124, magenta), cnk11-1 x cnk11-1 (blue), tpg1 x tpg1 (purple), and cnk11-1; tpg1 x cnk11-1; tpg1 (black). The length of new HA-α-tubulin incorporation (green) was measured at the distal end of the flagella. Insert, a representative image of a cnk11-1 quadriflagellate cells (QFCs) 60 minutes after mating. (C) Flagellar lengths of wild-type (CC-125, green), cnk11-1 (blue), tpg1 (purple), and cnk11-1; tpg1 (black) before and 30 minutes after IBMX treatment. (D) A model of flagellar length regulation by paclitaxel (magenta), polyglutamylation (blue), and CNK11 (green). In short flagella mutants caused by multiple dynein deficiency, addition of paclitaxel and reduction of polyglutamylation, as well as blockage of CNK11, leads to longer flagella, presumably due to stabilized axonemal microtubules. In the CCDC39 and CCDC40 short flagella mutants, addition of paclitaxel and blockage of CNK11 lead to longer flagella. However, reduced polyglutamylation enhances the mutant phenotype and cause increased number of aflagellated cells.

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