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. 2012 Dec 24;199(7):1083-101.
doi: 10.1083/jcb.201202126. Epub 2012 Dec 17.

Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis

Kerstin N Schmidt  1 , Stefanie Kuhns, Annett Neuner, Birgit Hub, Hanswalter Zentgraf, Gislene Pereira
Affiliations

Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis

Kerstin N Schmidt et al. J Cell Biol. .

Abstract

Cilia formation is a multi-step process that starts with the docking of a vesicle at the distal part of the mother centriole. This step marks the conversion of the mother centriole into the basal body, from which axonemal microtubules extend to form the ciliary compartment. How vesicles are stably attached to the mother centriole to initiate ciliary membrane biogenesis is unknown. Here, we investigate the molecular role of the mother centriolar component Cep164 in ciliogenesis. We show that Cep164 was indispensable for the docking of vesicles at the mother centriole. Using biochemical and functional assays, we identified the components of the vesicular transport machinery, the GEF Rabin8 and the GTPase Rab8, as interacting partners of Cep164. We propose that Cep164 is targeted to the apical domain of the mother centriole to provide the molecular link between the mother centriole and the membrane biogenesis machinery that initiates cilia formation.

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Figures

Figure 1.
Figure 1.
Cep164 centrosomal localization is cell cycle dependent independently of microtubules. (A) RPE1 cells were stained for Cep164 (anti-Cep164-N), γ-tubulin, and DNA. Centrosomes of representative cells in G1 (arrowheads) and mitosis (arrows) are indicated. (B) Quantification of A. A.U., arbitrary units. (C and D) Western blot analysis of Cep164 protein levels during the cell cycle. (C) RPE1 cells were released from double thymidine block (lanes 1–6) or from double thymidine block followed by nocodazole arrest (lanes 7–9) and harvested at the indicated time points. In D, cycling cells (t = 0) were serum starved as indicated. Plk1 is up-regulated during mitosis and served as mitotic marker. Actin served as a loading control. Low and high exposures are shown in D. The percentage of ciliated cells in D was based on polyglutamylated tubulin staining. Data are means ± SD. (E) Microtubule regrowth assay in RPE1 cells. Staining for α-tubulin in the left panels indicates depolymerization and repolymerization of microtubules. Representative cells are shown for the indicated time points. Regions inside the white boxes are shown at higher magnification in the top right corner of the corresponding panels.
Figure 2.
Figure 2.
Localization of Cep164 at the centrosome depends on its C terminus. (A) Schematic representation of Cep164 constructs used in this study. Numbers indicate amino acid positions. (B) Western blot analysis of the indicated GFP-Cep164 truncations. Specific protein bands are indicated with red asterisks. (C and D) RPE1 cells were transiently transfected with the indicated GFP-Cep164 constructs for 24 h and stained for γ-tubulin and DNA in C or for centrobin and γ-tubulin in D. (E) RPE1 cells expressing low levels of GFP-Cep164-C were control depleted or depleted for Cep164 for 24 h with serum followed by 48 h without serum. Cells were fixed and stained for Cep164, γ-tubulin, and acetylated tubulin. (F) RPE1 cells were transiently transfected with the indicated GFP constructs as in C and stained for centrobin and γ-tubulin. (G) Quantification for F. The left panels of C–F show merged images. Regions within the white boxes are shown at higher magnification to the right.
Figure 3.
Figure 3.
Overexpression of Cep164 truncations acts dominantly negative on ciliogenesis. (A) GFP-Cep164 truncations were transiently overexpressed in RPE1 cells for 24 h, serum starved for another 24 h, and stained for γ-tubulin and acetylated tubulin and DNA. (B) Quantification of A. Data are means ± SD. (C) RPE1 cells with the indicated constructs were treated as in A. Cells in G0 or G1 were determined by absent or nucleolar Ki67 staining. n > 100 from a single experiment. (D) GFP-Cep164 truncations were overexpressed in RPE1 cells for 24 h and stained for γ-tubulin. Endogenous Cep164 was visualized with anti–Cep164-M (top two panels) or anti–Cep164-N (bottom three panels). The top panels on the right (a) show untransfected cells, whereas the bottom panels (b) show transfected cells. (E) Quantification of D. Data are means ± SD. (F) GFP-Cep164-N fusion proteins were overexpressed in RPE1 cells as described in A. Cells were stained for γ-tubulin and acetylated tubulin and DNA. WT, wild type. (G) Quantification of F. Data are means ± SD. Merged images are shown in the left panels of A and D. Regions within the white boxes in A, D, and F are shown at higher magnification to the right in A and D and in the top right corner in F. Please note that overexpression in A and D was high, whereas overexpression in Fig. 2 E was low.
Figure 4.
Figure 4.
Vesicular docking to the M-centriole is impaired in Cep164-depleted cells. (A) RPE1 cells were treated with the indicated siRNAs for 48 h and serum starved for another 24 h. Ciliated cells were counted based on polyglutamylated tubulin staining. Data are means ± SD. (B) Western blot analysis of cells treated as in A. (C) RPE1 cells transfected with the indicated constructs for 30 h were subsequently treated with control or Cep164_1 siRNA for 24 h before serum starvation for another 24 h. Cells were stained for FLAG, Cep164, γ-tubulin, and acetylated tubulin. (D) Quantification of C. Data are means ± SD. (E) Western Blot of cells treated as in C. (F) Scheme illustrating the different stages of ciliogenesis (Molla-Herman et al., 2010; Ghossoub et al., 2011). The M-centriole with distal (DA) and subdistal appendages (SDA) is shown (0). During early stages, primary ciliary vesicles (PCVs) dock at the M-centriole (1), while nearby secondary vesicles (V) are subjected to later fusion with the PCVs. Accumulation of electron-dense material at the distal end of the M-centriole builds the so-called ciliary bud (CB), which invaginates the PCV (2). The axonemal shaft (AS) grows out and the ciliary pocket (CiPo) forms (3). Last, the cilia membrane fuses with the apical plasma membrane (PM) (4). M-centriole and axonemal shaft are illustrated in black, and membrane structures are shown in red. (G–K) Electron micrographs showing serial sections of RPE1 cells serum starved after control (G and H) or Cep164 depletion (I–K). CP, coated pit; MT, microtubules. (L) Quantifications for G–K. KD, knockdown.
Figure 5.
Figure 5.
Cep164 is involved in recruiting the distal appendage proteins NPHP1 and IFT88 to the M-centriole. (A) RPE1 cells stably expressing LAP-CSAP were treated with control or Cep164 siRNA for 24 h and serum starved for 48 h. Cells were stained for NPHP1 and Cep164. LAP-CSAP localizes to both centrioles and the axoneme and served as a centriolar and ciliary marker (Backer et al., 2012). (B) RPE1 cells, treated as in A, were stained for IFT88, Cep164, and polyglutamylated tubulin. In control depleted cells, IFT88 decorates the M-centriole of nonciliated cells and additionally the axoneme of ciliated cells. (C–E) Box-and-whisker plots showing NPHP1 (C), IFT88 (D), and Cep164 (E) signal intensity at the centrosome in RPE1 cells treated with the indicated siRNAs for 48 h and serum starved for another 24 h. (F) Western blot analysis of the experiment shown in C–E. Please note that the top and bottom panels are identical to Fig. 4 B, as these experiments were performed together. (G and H) Box-and-whisker plots show the relative signal intensity of NPHP1 (G) and IFT88 (H) at centrosomes in control and Cep164-depleted RPE1 cells expressing GFP or FLAG-Cep164-R1. Cells were treated as in Fig. 4 C. Knockdown efficiency was confirmed by Western blot analysis (Fig. 4 E). The left panels of A and B show merged images. Regions within the white boxes are shown at higher magnification to the right. A.U., arbitrary units.
Figure 6.
Figure 6.
The C terminus of Cep164 interacts with Rabin8 and Rab8. (A) Yeast strains expressing the indicated gene fusions were grown on selective plates lacking either leucine and tryptophan (−LW) or leucine, tryptophan, histidine, and adenine (−LWHA). Growth on −LWHA plates indicates interaction. Duplicates are shown. Negative (Lamin C and T7-T-antigen) and positive (p53 and T7-T-antigen) controls were included. Colonies from noninteractors appear red on −LW plates because cells deficient for ADE2 expression accumulate a red pigment. (B–E) HEK293T cells were transiently cotransfected with the indicated constructs. Immunoprecipitations (IP) were performed using anti-FLAG agarose and interacting proteins were detected by Western blot. Arl6-GFP in D, a small GTPase with a role in ciliogenesis (Wiens et al., 2010), served as negative control.
Figure 7.
Figure 7.
Cep164-C interacts with Rabin8, but not with Rab8, in vitro. (A–C) In vitro binding assays using bacterially purified proteins. (A) MBP or MBP-Cep164-C beads were incubated with purified 6His-Rabin8. (B) MBP, MBP-Rabin8, or MBP-Cep164-C beads were incubated with NusA-Rab11a-6His. (C) MBP-fusion proteins (immobilized on beads) were incubated with purified NusA-Rab8a-T22N-6His. Proteins were visualized by Western blot. (D) Coomassie blue–stained gel of proteins used in A–C. The asterisks indicate unspecific protein bands.
Figure 8.
Figure 8.
Rab8 centrosomal/ciliary localization requires Cep164. (A) RPE1 cells stably overexpressing GFP-Rab8a were treated with control or Cep164 siRNA for 48 h, serum starved for 24 h, and stained for Cep164, γ-tubulin, and acetylated tubulin. Representative fluorescence micrographs are shown for the localization patterns of GFP-Rab8a (a–e). The left panels show merged images. Regions within the white boxes are shown at higher magnification to the right. (B) Quantification of cells treated with the indicated siRNAs as described in A. Only cells with complete loss of Cep164 were considered. (C) Centrosomal/ciliary localization of Cherry-Rab8a in serum-starved RPE1 cells expressing GFP or FLAG-Cep164-R1 for 30 h. Cells were then transfected with the indicated siRNAs for 30 h and serum starved for 18 h. Data are means ± SD. (D) Box-and-whisker plots showing endogenous Cep164 signal intensity at the centrosome of cells treated as in C, confirming efficient depletion of Cep164. Please note that the efficiency of Cep164 knockdown in RPE1 cells co-overexpressing Cherry-Rab8a and FLAG-Cep164-R1 was estimated based on the quantification of endogenous Cep164 centrosomal signal from cells not expressing FLAG-Cep164-R1 within the same population (FLAG-negative cells). A.U., arbitrary units.
Figure 9.
Figure 9.
Overproduction of Rab8a rescues Cep164-dependent ciliogenesis defects. (A) The indicated GFP constructs were transiently overexpressed in RPE1 cells for 24 h. Cells were subjected to control or Cep164 depletion for 48 h and serum starved for 24 h. Cep164, γ-tubulin, and acetylated tubulin staining are shown. Equal knockdown efficiencies were confirmed by measuring Cep164 signal intensity (Student’s t test analysis). The left panels show merged images. Regions within the white boxes are shown at higher magnification to the right. (B) Graph shows fold increase in the percentage of ciliated cells processed as in A. Only medium depleted cells, in which 10–60% of initial Cep164 staining remained at the centrosome, were considered (see Fig. S5 for details). A single representative experiment out of three repeats is shown. For the experiment shown, 40–45 cells were observed per cell line.
Figure 10.
Figure 10.
A model of Cep164 function during ciliogenesis. At the end of mitosis, the amount of Cep164 that associates with the M-centriole increases, reaching a maximum in interphase. Upon induction of ciliogenesis, centrosomal Cep164 is needed for the recruitment of Rab8 and docking of vesicles at the M-centriole to initiate the formation of the cilium (see Discussion for details).
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