Ciliary transition zone proteins coordinate ciliary protein composition and ectosome shedding

Abstract

The transition zone (TZ) of the cilium/flagellum serves as a diffusion barrier that controls the entry/exit of ciliary proteins. Mutations of the TZ proteins disrupt barrier function and lead to multiple human diseases. However, the systematic regulation of ciliary composition and signaling-related processes by different TZ proteins is not completely understood. Here, we reveal that loss of TCTN1 in Chlamydomonas reinhardtii disrupts the assembly of wedge-shaped structures in the TZ. Proteomic analysis of cilia from WT and three TZ mutants, tctn1, cep290, and nphp4, shows a unique role of each TZ subunit in the regulation of ciliary composition, explaining the phenotypic diversity of different TZ mutants. Interestingly, we find that defects in the TZ impair the formation and biological activity of ciliary ectosomes. Collectively, our findings provide systematic insights into the regulation of ciliary composition by TZ proteins and reveal a link between the TZ and ciliary ectosomes.

Fig. 1. Characterization of the C. reinhardtii tctn1 mutant.

a Diagram of the gene structure of TCTN1 (Cre03.g181450) with the aphVIII DNA insertional site. b Diagram of the domain structure of the TCTN1 protein. The TCTN1 protein (679 aa) contains the DUF1619 domain (78–418 aa) with unknown function. The red arrowhead marks the insertional site. c DIC images showing the ciliary phenotypes of WT, mutant, and rescued cells (A18, C19). Scale bar, 5 μm. d Immunostaining images depicting the very short cilium within the palmelloid of tctn1. The red signals (by anti-α-tubulin antibody) mark the cilia and the blue signals (by DAPI) mark the nucleus. Scale bar, 5 μm. e DIC images depicting the ciliary phenotype of tctn1 after treatment with autolysin. The mother cell walls are indicated by white arrowhead and the ciliary bugles are indicated by black arrowheads. Scale bar, 5 μm. f Scatter plot showing the elongation of cilia in e. Statistical significance was determined with an unpaired t test. ****P < 0.0001 by two tailed. g tctn1 exhibited shorter cilia after hatching with autolysin and slower kinetics of ciliary assembly. The arrow indicates the time point of autolysin or pH shock treatment. h tctn1 exhibited faster kinetics of ciliary disassembly. The arrow indicates the time point of Nappi treatment to induce ciliary shortening. i Immunostaining showing the localization of TCTN1 in the TZ. Cells were immunostained with HA (green) and α-tubulin (red, left) or centrin (red, right) antibodies. The nucleus was stained with DAPI (blue). The arrowheads indicate the TZ. The dotted boxes indicate the regions of higher magnification views. Scale bar, 5 μm. j Immunoblot analysis of the localization of TCTN1-HA in the rescued cell line. 1× (cilia) represents an equal proportion of cilia to that of the cell body (two cilia per cell body). 50× (cilia) represents equal cilia and cell body proteins. WC whole cell, CB cell body. Ciliary lengths are shown as the mean ± SD of 50 cilia. Source data are provided as a Source Data file.

Fig. 2. Loss of TCTN1 causes ultrastructural defects of the ciliary membrane and the TZ.

a EM images showing ciliary bulges with electron-dense material (white arrowheads) in a portion of tctn1 cells. Scale bar, 200 nm. b, c Longitudinal sections (b) and cross sections (c) through the TZ (b, brackets) of WT, tctn1 and rescued cells. The wedge-shaped structures (b, black arrowheads) and Y-links (c, black arrowheads) were indicated. Quantification of the presence of wedge-shaped structures in the longitudinal section (n = 20, number of wedge-shaped structures) and Y-links in the cross section (n = 17 for WT, n = 23 for tctn1, number represents the thin sections counted.). The presence of at least one Y-link in the thin section is considered as normal. Scale bar, 200 nm. d Scatter plot depicting the distances between the “H” structure and the ciliary membrane in WT, tctn1, and rescued cells. Data are the mean ± SD (n = 20). Statistical significance was determined with an unpaired t test. n.s., not significant; **P < 0.01 by two tailed. e, f TEM images of longitudinal (e) and cross (f) sections of the cilia from WT and tctn1 cells. Glycocalyx on the surface of ciliary membrane was indicated with black arrowheads. The high magnification regions were indicated by dotted boxes. OD outer doublet microtubule, mb ciliary membrane, g glycocalyx. Scale bar, 200 nm. Source data are provided as a Source Data file.

Fig. 3. TCTN1 is located between CEP290 and NPHP4 independently.

a–d Immunostaining images and graphs depicting the colocalization of TCTN1 and NPHP4/CEP290. The rescued cells expressing TCTN1-HA were immunostained with anti-acetylated α-tubulin (ac-tubulin, red), anti-HA (green), and anti-CEP290 (a, magenta) or anti-NPHP4 (c, magenta) antibodies. The nucleus was stained with DAPI (blue). The arrowheads and arrows indicate the localization of TCTN1-HA and CEP290 (a)/NPHP4 (c), respectively. The scan plots of the rectangular gray value in the merged image show the relative intensities (arbitrary units, A.U.) of the indicated proteins in the TZ. Scale bar, 1 μm. e TEM imaging showing the longitudinal sections of TCTN1–HA transition zones labeled with anti-HA (10-nm gold, red arrowheads). Scale bar, 0.1 μm. f Schematic of the localization of TCTN1, CEP290, and NPHP4 at the TZ. g Immunostaining depicting the TZ localization of TCTN1, NPHP4, and CEP290 in WT, tctn1, cep290, and nphp4 cells. Cells as indicated were immunostained with anti-acetylated α-tubulin (ac-tubulin, red), anti-TCTN1/CEP290/NPHP4 (green) antibodies. The nucleus was stained with DAPI (blue). Scale bar, 1 μm.

Fig. 4. Loss of TCTN1 attenuates the ciliary gating role of the TZ.

a SDS–PAGE and silver staining of isolated whole cilia or fractionations of cilia from WT, tctn1, and rescued cells expressing TCTN1-HA. Arrows indicate the differences among samples. Res rescued cells, Cilia ciliary samples. M + M, membrane and matrix samples of cilia. Axo axonemal samples of cilia. b Volcano plot displaying the differentially expressed proteins in cilia. The red and blue dots represent the upregulated and downregulated proteins in cilia of the tctn1 mutant. The nonaxial vertical lines indicate fold change of tctn1/WT < 0.5 and >2. The nonaxial horizontal line represents P value of 0.05. Statistical significance was two-sided without adjustments. c Bar–scatter graph depicting the fold changes in the representative proteins in cilia between WT and tctn1 in b. Data are the mean ± SD (n = 3). d Immunostaining images displaying the enrichment of IFT122 particles in the cilium of tctn1. Cells were immunostained with anti-IFT122 (green) and anti-acetylated α-tubulin (ac-tubulin, red) antibodies. The arrowheads indicate the accumulations of IFT122 in the cilium. Scale bar, 5 μm. e Immunoblot and statistical analysis of the isolated cilia from WT, tctn1, and rescued cells. α-tubulin was used as a loading control. The graph showing the gray value of the immunoreactive bands was prepared using the mean ± SEM (n = 3). Statistical significance to WT group was determined using two-way ANOVA with Dunnett’s test. n.s., not significant. ***P < 0.001; ****P < 0.0001. f Immunoblot of cilia isolated from WT, tctn1, and rescued cells. The antibodies against PSBC and PSAD were used for the confirmation of proteomics results from b. g Immunostaining images showing enrichment of PSBC, PSAD in tctn1. Cells were immunostained with anti-PSBC (green, left), or anti-PSAD (green, right) and anti-acetylated α-tubulin (ac-tubulin, red) antibodies. The arrowheads indicate the accumulation of photosystem proteins in the cilium. Scale bar, 5 μm. Source data are provided as a Source Data file.

Fig. 5. Proteins differentially regulated in cilia by different TZ proteins.

a–d Heatmap of representative groups of proteins identified from ciliary proteomics analysis of cilia from WT, tctn1, cep290, and nphp4 cells (two replicates for each strain, column). Representative proteins were selected from the mass spectrometry data in Supplementary Fig. 6a and grouped into IFT and BBSome (a, top), IFT motor (a, bottom), Axoneme (b), Ciliary membrane (c), Golgi (d, left), Chloroplast (d, middle), and Mitochondrion (d, right) proteins. The Z–score is shown by color key in the heatmaps. e, f Immunostaining images displaying the enrichment of photosystem proteins (PSBC and PSAD) in TZ mutants (cep290 and nphp4). WT and TZ mutants were immunostained with anti-PSBC (e, green), or anti-PSAD (f, green) and anti-acetylated α-tubulin (ac-tubulin, red) antibodies. The non-ciliary proteins were transported and accumulated in cilia of TZ mutant cells. The arrowheads indicate the accumulations of photosystem proteins in the cilium. Scale bar, 5 μm. g Bar–scatter graphs show the ciliary fluorescence intensity (arbitrary units, A.U.) of PSBC or PSAD. Data are the mean ± SD (n = 20). Statistical significance was determined with an unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001 by two tailed. Source data are provided as a Source Data file.

Fig. 6. The formation of ciliary ectosomes is regulated by the integrity of the TZ during gamete mating events.

a Heatmap of representative ectosome-related proteins identified from ciliary proteomics analysis of cilia from WT, tctn1, cep290, and nphp4 cells (two replicates for each strain, column). Representative proteins were selected from the mass spectrometry data in Supplementary Fig. 6a. The Z–score is shown by color key in the heatmap. b Negative stain transmission electron micrographs of purified ciliary ectosomes (black arrows) from gametes in the mating process. 21gr (WT), ectosomes from mating supernatant of 21gr × 6145c; tctn1, ectosomes from mating supernatant of tctn1 × 6145c; cep290, ectosomes from mating supernatant of cep290 × 6145c; nphp4, ectosomes from mating supernatant of nphp4 × 6145c. Scale bar, 500 nm. c Scatter plot showing the diameters of ectosomes shedded from the cilia of different mating gametes. The sizes of the ectosomes are the mean ± SD (n = 500) described in b. Statistical significance was determined with an unpaired t test. ****P < 0.0001 by two tailed. d Graph showing the distribution of ciliary ectosomes of different sizes purified from 21gr × 6145c, tctn1 × 6145c, cep290 × 6145c, and nphp4 × 6145 described in b. Source data are provided as a Source Data file.

Fig. 7. The activity and protein composition of ciliary ectosomes shedding from gametes of different TZ mutants are different.

a Bar–scatter graph depicting the agglutinating gametes number (%) from 6145c (mt-) mixed with the supernatant (S20), the final supernatant (Sup), and ectosome pellet (Eco) from gamete mating culture (21gr × 6145c). b PH images showing the agglutination of gametic 6145c (mt-) induced by mixing with the ectosomes (Eco). Gametic 6145c alone, vegetative 6145c with Eco, and 21gr × 6145c were showed as the negative and positive controls, respectively. The black arrows indicate the agglutination of gametic cells. G gametic cells, V vegetative cells. Scale bar, 20 μm. c Bar–scatter graph displaying the agglutination efficiency (normalized to WT) of gametic 6145c (mt-) mixed with the ectosome pellet (Eco) from gamete mating cultures (21gr × 6145c, tctn1 × 6145c, cep290 × 6145c, nphp4 × 6145c). The group of vegetative 6145c with Eco was as the negative control (Con). d Silver-stained SDS–PAGE gel showing protein variations in ectosomes isolated from the plus mating type gamete (21gr, tctn1, cep290, nphp4) with minus mating type gamete (6145c). The indicated ciliary ectosomes purified from the same amount of cells were loaded with equal number of mating gametes, separated on 4–20% SDS–PAGE and then visualized by silver staining. The positions of standard proteins and their molecular masses in kDa are indicated. Data are presented as the mean ± SD (n = 3, independent experiments) in this figure. Statistical significance was determined with an unpaired t test in this figure. **P < 0.01; ***P < 0.001; ****P < 0.0001 by two tailed. Source data are provided as a Source Data file.