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. 2013 Nov 1;126(Pt 21):5018-29.
doi: 10.1242/jcs.133439. Epub 2013 Aug 22.

Centrosomal Protein CEP104 (Chlamydomonas FAP256) Moves to the Ciliary Tip During Ciliary Assembly

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Free PMC article

Centrosomal Protein CEP104 (Chlamydomonas FAP256) Moves to the Ciliary Tip During Ciliary Assembly

Trinadh V Satish Tammana et al. J Cell Sci. .
Free PMC article

Abstract

The ciliary tip has been implicated in ciliary assembly and disassembly, and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be approximately twice as concentrated in half-length compared with full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly. Similarly, its vertebrate counterpart, CEP104, localizes on the distal ends of both centrioles of nondividing cells until the mother centriole forms a cilium and then localizes at the tip of the elongating cilium. A null mutant of FAP256 in Chlamydomonas and RNAi in vertebrate cells showed that FAP256/CEP104 is required for ciliogenesis in a high percentage of cells. In those cells that could form cilia, there were structural deformities at the ciliary tips.

Keywords: CEP104; Centrosomal proteins; Ciliary tip; Ciliogenesis; FAP256; Flagellar assembly/disassembly.

Figures

Fig. 1.
Fig. 1.
Flagellar tip and basal body localization of FAP256 in Chlamydomonas cells with full-length, assembling and resorbing flagella. (A) Wild-type Chlamydomonas cells were fixed and stained with antibodies against acetylated α-tubulin (green, ciliary axonemal marker) and FAP256 (red). Insets show the tip region of one of the flagella stained with FAP256 at the apical end. (B,C) Chlamydomonas cells were fixed at different stages of flagellar growth and stained for the axoneme and FAP256. (D,E) Wild-type cells with full-length flagella were induced to resorb flagella and processed for IFM. Arrowheads indicate flagellar tips and arrows show basal body staining of FAP256. Scale bar: 5 µm.
Fig. 2.
Fig. 2.
Loss of FAP256 in the Chlamydomonas insertional mutant Roc22. (A) Chlamydomonas wild-type (CBR34mt+) and Roc22 mutant cells were fixed and stained with antibodies against acetylated α-tubulin (green, ciliary axonemal marker) and FAP256 (red). Loss of FAP256 staining at the flagellar tip (arrowheads) and the basal body region (arrows) can be seen in Roc22 mutants. Insets show the tip region of one the flagella stained with FAP256 at the apical end. Scale bar: 5 µm. (B) Western blots showing the absence of FAP256 in Roc22 insertional mutant. Equal amounts of lysates from whole cell and isolated flagella of Chlamydomonas wild-type (CBR34mt+) and Roc22 mutants were probed with antibodies against FAP256 and intermediate chain IC69 (loading control). (C) Roc22 mutants fail to regenerate flagella upon deflagellation. Roc22 mutants (gray) as well as wild-type cells (CBR34mt+, black) were deflagellated by pH shock and allowed to regenerate flagella. Percentage of nonflagellated cells is shown at various time points (minutes). ∼200 cells were counted for each experiment. Values shown are mean ± s.d. from three independent experiments. (D) Flagellar lengths were measured in Roc22 cells that regenerated flagella at various time points and mean flagellar lengths are plotted. Flagella of Roc22 cells were shorter than those of wild-type cells. ∼100 flagella were counted at each time point and P-values at various time points were calculated by unpaired t-test (30 minutes, P = 0.0006; 60, 90, 120 and 180 minutes, P = 0.0001).
Fig. 3.
Fig. 3.
FAP256 localizes at the tip of the central-pair and outer-doublet MTs in splayed axonemes. (A) Chlamydomonas flagellar axonemes were splayed and processed for IFM using antibodies against acetylated α-tubulin (green) and FAP256 (red). Arrows indicate the tip localization of FAP256 in splayed axonemes. Scale bar: 5 µm. (B) Immunogold labeling of FAP256 on splayed axonemes stained with 2% uranyl acetate. Representative micrographs showing gold particles at the tip of the splayed axoneme (arrows). Inset shows enlarged flagellar tip region. Arrows show the gold particles labeling individual outer-doublet MTs and arrowheads indicate central-pair MTs of the splayed axoneme.
Fig. 4.
Fig. 4.
The tips of flagella from Roc22 differ dramatically from those of wild-type cells. (A,B) In the tip of wild-type flagella the central-pair MTs are capped by an end plate (arrows). (C–E) In Roc22 flagella, the endplate is less distinct or absent (an endplate might be present in E, arrowheads). Furthermore, in Roc22, the central pair MTs can be of unequal length (D, arrowheads), nearly reaching the overlying membrane (C–E, arrowheads). In wild-type flagella, the outer-doublet MTs end proximal to the central-pair endplate (* in A,B); in the tip, they are devoid of radial spokes so that the matrix appears empty. In Roc22, the outer-doublet microtubules can be nearly as long or longer (* in C,D) than the central pair, and radial spokes connect them to the central pair almost to the end of the flagellum (>,< in C). The narrowing of the flagellum seen in the tips of the wild type is not apparent in Roc22 (compare B and C). The flagellar tips of Roc22 are more blunt than in the wild type, which often end with a cone-shaped bulge (most distinct in B).
Fig. 5.
Fig. 5.
Tip localization of CEP104 in the primary cilia of RPE1 cells and motile cilia of human tracheal epithelial cells. (A) Confluent RPE1 cells were serum starved to induce cilia formation and stained with antibodies against Arl13B (ciliary marker, red, bracket), γ-tubulin (centrosomal marker, also in red) and CEP104 (green). Arrows indicate ciliary tips and arrowheads show daughter centriole localization of CEP104. (B) Frozen sections of human trachea were processed for IFM using antibodies against β-tubulin, to label tracheal cilia (red, bracket) and CEP104 (green). A ridge of cilia on the tracheal epithelium is shown at higher magnification in the inset. Arrows indicate the staining of ciliary tips with CEP104. Faint cytosolic staining of CEP104 can also be seen in these cells. Nuclei are stained with DAPI (cyan). Scale bars: 5 µm.
Fig. 6.
Fig. 6.
CEP104 from the mother centriole moves to the ciliary tip during ciliary assembly. Confluent RPE1 cells were serum starved and fixed at various time intervals during cilia assembly. Cells were processed for IFM using antibodies against Arl13B (ciliary marker, red), γ-tubulin (centrosomal marker, also in red) and CEP104 (green). (A) CEP104 (arrows) localizes to both mother and daughter centrioles in non-ciliated RPE1 cells. (B–D) Ciliary tip and daughter centriole staining of CEP104 (green) during various stages of ciliary growth. Loss of CEP104 at the mother centriole and tip localization can be seen from a very early stage of ciliary growth (B). Arrowheads indicate ciliary tip and arrows show daughter centriole localization of CEP104. Nuclei are stained with DAPI (cyan). Approximately 200 cells were analyzed at each time point. Scale bars: 5 µm.
Fig. 7.
Fig. 7.
Localization of CEP104 at the tip during ciliary disassembly. Cells with fully grown cilia were induced for cilia disassembly by adding the serum and fixing at various time intervals after serum addition. Cells were stained with Arl13B (ciliary marker, red), γ-tubulin (centrosomal marker, also in red) and CEP104 (green). (A,B) Tip localization of CEP104 (arrowheads) can be seen in full-length and disassembling cilia. (C) By 24 hours of serum addition, most cells in the culture had lost their cilia and CEP104 staining was seen on both mother and daughter centrioles. Arrows indicate centriole localization of CEP104. Nuclei are stained with DAPI (cyan). Approximately 200 cells were analyzed at each time point. Scale bars: 5 µm.
Fig. 8.
Fig. 8.
Depletion of CEP104 in RPE1 cells impairs cilia formation. (A,B) Both non-targeting (mock) and CEP104-siRNA-treated samples were processed for IFM using antibodies against Arl13B (ciliary marker, red) and CEP104 (green). Depletion of CEP104 at the centrosomal region (arrows) in non-ciliated cells (A) and ciliary tip (arrowheads) in ciliated cells (B) was seen in CEP104-siRNA-treated cells when compared with mock-transfected cells. Nuclei were visualized by DAPI staining (cyan). Scale bars: 5 µm. (C) Percentage of full-length cilia, no cilia and short cilia bearing cells in mock (black) and CEP104-siRNA-treated (gray) populations were plotted from immunofluorescence images. ∼200 cells were counted for each experiment. Values shown are mean ± s.d. from three independent experiments. (D) Western blots showing the depletion of CEP104 in RPE1 cells treated with CEP104 siRNA. Equal amount of cell lysates from mock treated, siRNA-treated and untreated RPE1 cells serum starved for 24 hours were probed with mouse anti-CEP104 antibodies. Antibodies against α-tubulin were used as a loading control.

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