XB-ART-52451Elife. April 28, 2016; 5
Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis.
Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.
PubMed ID: 27623009
PMC ID: PMC5045295
Article link: Elife.
Grant support: K99 HL127275 NHLBI NIH HHS , R01 GM042341 NIGMS NIH HHS , R01 GM076507 NIGMS NIH HHS , S10 OD018136 NIH HHS , P30 DK072517 NIDDK NIH HHS , S10 OD018136 NIH HHS , P30 DK072517 NIDDK NIH HHS , R01 GM042341 NIGMS NIH HHS , K99 HL127275 NHLBI NIH HHS , R01 GM076507 NIGMS NIH HHS , S10 OD018136 NIH HHS , P30 DK072517 NIDDK NIH HHS , R01 GM042341 NIGMS NIH HHS , K99 HL127275 NHLBI NIH HHS , R01 GM076507 NIGMS NIH HHS , P30 CA014195 NCI NIH HHS
Genes referenced: ccp110 cfp foxj1.2 gnl3 herpud1 nkx2-2 pax6 pitx2 pxn tub
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|Figure 3. Cp110 localizes to cilia-forming basal bodies in MCCs.(A–D) Cp110 localizes to cilia-forming basal bodies in Xenopus epidermal (A, C) and human airway epithelial cell (HAEC) (B, D) MCCs. (A) gfp-cp110 (green) was expressed at levels permitting ciliogenesis, together with centrin4-cfp (basal bodies, blue). Immunofluorescent staining (Ac.-α-tub., red) was used to visualize cilia. (B) Immunofluorescent staining for endogenous Cp110 (red), cilia (Ac.-α-tub.; blue) and Actin (green) in MCCs (n donors = 1, n MCCs = 4). Yellow arrows in A’ and B’ indicate the base of cilia. (C) Apical view (top) of individual MCC co-injected with gfp-cp110 (green), centrin4-cfp (blue) and clamp-rfp (red) to visualize Cp110, basal bodies and rootlets, respectively. Localization of basal bodies to the apical membrane is shown in lateral projection (bottom). n embryos/MCCs: (4/18). (C') High-magnification analysis of GFP-Cp110 (green, indicated by yellow arrows and green circle) binding to an individual basal body from the MCC shown in (C) (basal body and rootlet are indicated). Inset shows rootlet domain (dashed box) with increased brightness. (D) Lateral projection of MCC stained for endogenous Cp110 (green) and cilia (Ac.-α-tub.; red). n donors = 1, n MCCs = 12 (same samples as in Figure 3—figure supplement 1C) (E–F) Endogenous Cp110 (green) and Centrin 1 (red) staining shows Cp110 adjacent to MCC basal bodies by confocal microscopy (E) and 3D-SIM imaging (F). n donors = 1, n MCCs = 3 each for confocal and 3D-SIM.DOI: http://dx.doi.org/10.7554/eLife.17557.010Figure 3—figure supplement 1. Cp110 localizes to cilia-forming basal bodies in MCCs.(A–B) GFP-Cp110 localization to basal bodies in Xenopus MCCs. (A) GFP-Cp110 (green) localizes to basal bodies (Centrin4-CFP, blue) prior to apical docking, during the stages of apical basal body transport. Actin staining shown in red. n = 2 embryos, 18 MCCs. (B) GFP-Cp110 (green) shows asymmetric localization to basal bodies/rootlets (Clamp-RFP, red) along the anterior-posterior axis. n = 3 embryos, 30 MCCs. (C) Immunofluorescent staining for Cp110 (green) and cilia (Acetylated-α-tubulin, red) shows Cp110 localization at the level of basal bodies and at the lateral membrane (white arrows) in human HAEC MCCs. Three levels along apical-basal axis are shown (top, apical ciliary tuft level; middle, apical MCC membrane level; bottom, cytoplasmic level). n = 1 donor, 12 MCCs. (D–E) Mouse trachea staining for Cp110 (green), cilia (Acetylated-α-tubulin, red) and nuclei (DAPI, blue). (n = 4). (D) Magnified view of MCCs. (E) Greater area view of mouse trachea with multiple MCCs. (E’) Negative control immunofluorescent staining as described in (E), but without the use of primary anti-Cp110 antibody. (n = 1).DOI: http://dx.doi.org/10.7554/eLife.17557.011|
|Figure 3—figure supplement 2. Cp110 localizes to cilia-forming basal bodies and ciliary tips of monociliated GRP cells.(A–D) GFP-Cp110 localizes to cilia-forming basal bodies in GRP cells injected with gfp-cp110 (green) and centrin4-cfp (basal body/daughter centriole, blue) and immunostained for cilia (Acetylated-α-tubulin, red). (A) Single GRP cilium of normal length (approximately 4 µm) with two GFP-Cp110 foci (yellow arrowheads; mother centriole/basal body and daughter centriole) overlapping with Centrin4-CFP at the base of the cilium. (n = 3). (B), Differential effects of gfp-cp110 expression in GRP cells. Basal bodies (blue) and cilia (red) in GRP cells expressing different amounts of GFP-Cp110 (green). (B’), Magnification of area depicted in (B) showing cilia of different length with different amounts of GFP signal at their base. (C) In some GRPs, a subset of cilia displayed GFP-Cp110 localization to the ciliary tip (boxes and yellow arrows). Three individual cases are shown in (C’–C’’’), where yellow arrows indicate ciliary tip. (n = 9 for B and C combined).DOI: http://dx.doi.org/10.7554/eLife.17557.012|
|Figure 3—figure supplement 3. Cep97 does not localize to cilia-forming basal bodies.(A–C) GFP-Cep97 localizes centrioles of epidermal cells in Xenopus, but does not localize to basal bodies in MCCs. (A) GFP-Cep97 (green) localizes to centrioles (Centrin4-CFP, blue; Clamp-RFP, red) of epidermal cells (inset). n = 3 embryos. (B) GFP-Cep97 (green) does not localize to basal bodies/rootlets in MCCs (Centrin4-CFP, blue; Clamp-RFP, red). n embryos/MCCs: 3/9. (C) Lateral projection of MCC shown in (B) shows primarily cytoplasmic localization of GFP-Cep97. (D) Overexpression of gfp-cep97 does not inhibit cilia in Xenopus MCCs. n embryos/MCCs: 3/15.DOI: http://dx.doi.org/10.7554/eLife.17557.013|
|Figure 3—figure supplement 4. Schematic depiction of Cp110 localization sites at centrioles, basal bodies and cilia.(A) Cp110 caps the distal ends of centrioles. (B) Cp110 localizes adjacent to the basal body at a posterior domain as well as to the tip of the rootlet. Additionally, Cp110 can localize to ciliary tips.DOI: http://dx.doi.org/10.7554/eLife.17557.014|
|Figure 4—figure supplement 2. Cp110 is required for ciliary adhesion complex formation in MCCs.(A-D) Cp110 is required for Vinculin (n embryos/MCCs: control (10/30), cp110MO (10/30)) and Paxillin (n embryos/MCCs: control (5/15), cp110MO (5/15)) localization to MCC basal bodies. Control and cp110 morphant embryos were injected with vinculin-gfp (A) and (C), green) or paxillin-gfp (B) and (D), green) together with centrin4-cfp (blue). Related to Figure 4C–D. (E) Full membranes of co-IP experiment shown in Figure 4B. In addition to FLAG-Cp110 (full length Cp110), a FLAG-Cp110-FSΔCCD1 was overexpressed, which misses the first coiled-coil domain, as well as the truncation of the cp110-fs clone (please compare to Figure 5D).DOI: http://dx.doi.org/10.7554/eLife.17557.017|
|Figure 4—figure supplement 3. Schematic representation of summary model of the roles of Cp110 in MCC ciliation.Cp110 levels in MCCs need to be precisely regulated for successful ciliogenesis and normal cilia function. Please see text for detailed description.DOI: http://dx.doi.org/10.7554/eLife.17557.018|
|Figure 5—figure supplement 2. Cp110 central domain deletion enhances centriolar and basal body phenotypes.(A–E) Related to Figure 5 and Figure 5—figure supplement 1. (A–B) gfp-cp110ΔCentral overexpression induces supernumerary centrioles, polynucleated cells and severe cytokinesis defects. (A) Controls and embryos injected with gfp-cp110ΔCentral (green) were analyzed for ciliation by immunofluorescent staining against Acetylated-α-tubulin (cilia, Ac.-α-tub., red) and nuclei (DAPI, blue). In uninjected control embryos, cell borders were visualized by Actin staining (green, left panel only). n embryos: control, 6; cp110MO, 5. (B) Embryos were injected with centrin4-cfp (centrioles, blue) and gfp-cp110ΔCentral (green). A central region of a non-MCC epidermal cell is shown. All GFP-Cp110ΔCentral foci overlap with Centrin4-CFP foci. (C–D) gfp-cp110ΔCentral overexpression induces increased numbers of basal bodies in MCCs, which frequently fail to separate. (C) Embryos were injected with centrin4-cfp (basal bodies, blue) and gfp-cp110ΔCentral (green), which caused strongly enlarged MCCs and aggregated basal bodies. (D) Magnification of basal body cluster from MCC shown in C. (E) Cp110 constructs generated in this study and their effect on epidermal cells. +, phenotype present; ++, strong phenotype; +++, very strong phenotype; -, phenotype not present.DOI: http://dx.doi.org/10.7554/eLife.17557.021|
|Figure 5—figure supplement 3. Schematic representation of Cp110 domains and their proposed function.Cp110 domains are depicted as described in Figure 5D. Proposed functions are indicated.DOI: http://dx.doi.org/10.7554/eLife.17557.022|
|Figure 6. Cp110 levels in MCCs are controlled by ciliary transcription factors and miR-34/449 microRNAs.(A) cp110 expression in MCCs is regulated through the MCC signaling/transcriptional cascade. Embryos were injected with Su(H)-dbm to stimulate MCC induction (green) or with Su(H)-dbm and dominant-negative multicilin (dn-mci) to prevent MCC induction (red). RNA-sequencing (RNA-seq) was performed at MCC specification stage (st. 16). Normalized counts are shown as bar graphs. n = 2. Related to Figure 6—figure supplement 1A. (B) cp110 expression is activated by ciliary transcription factors. Chromatin immunoprecipitation and DNA-sequencing (ChIP-seq; upper five lanes) and RNA-seq (bottom two lanes) at stage 16. Embryos were injected with Notch-icd to inhibit MCC induction or together with multicilin (mci) to induce MCCs. ChIP-seq using antibodies to mark active chromatin (Histone H3 lysine tri-methylation, H3K4me3; Histone H3 lysine acetylation, H3K27ac), E2F4 binding (E2F4), RFX2 binding (RFX2), and Foxj1 binding (Foxj1) are shown. A gene model is shown in bottom lane. ChIP-seq peaks are indicated by a yellow background. (C) cp110 levels at ciliogenesis stage (st. 25) are controlled by miR-34/449 miRNAs. For quantitative RT-PCR analysis (qPCR), manipulations were performed as described in (A) (green and red bars). Additionally, miR-34/449s were knocked down (miR-34/449MO, blue bar). The uninjected control was set to 1. n = 2. (D) miR-34/449 family members are regulated through the conserved MCC signaling/transcriptional cascade. qPCR analysis for miR-34/449 expression was performed as described (C). ND, not detected. n = 2. (E–F) Expression of miRNAs miR-34b/c and miR-449a-c is activated by ciliary transcription factors. ChIP-seq and RNA-seq was performed as described in (B). miRNA location in (E) is indicated by red box. miR-449a-c are expressed from cdc20b intron 2. Related to Figure 6—figure supplement 1B. The foxj1 expression analysis confirmed successful manipulation in (A, C) and (D). Error bars represent s.e.m. in (C) and (D).DOI: http://dx.doi.org/10.7554/eLife.17557.023Figure 6—figure supplement 1. Cp110 levels in MCCs are controlled by ciliary transcription factors and miR-34/449 microRNAs.(A) Related to Figure 6A. cp110 expression in MCCs is regulated through the conserved MCC signaling/transcriptional cascade. Embryos were injected with Notch-icd to inhibit MCC induction (red) or with Notch-icd together with multicilin (mci) to stimulate MCC induction (green). RNA-sequencing (RNA-seq) was performed on extracts from mucociliary organoids at MCC specification stage (st. 16). Normalized counts are shown as bar graphs. The foxj1 expression analysis confirmed successful manipulation. (B) Related to Figure 6B,E–F. Expression of miRNA miR-34a is not activated by ciliary transcription factors. ChIP-seq and RNA-seq was performed as described in Figure 6B. miRNA location is indicated by red box.DOI: http://dx.doi.org/10.7554/eLife.17557.024|
|Figure 6—figure supplement 2. Model of the transcriptional/post-transcriptional regulatory module required to achieve optimal Cp110 levels in MCC ciliogenesis.A schematic model of ciliary transcription factors and miRNAs is shown. Activation is shown as arrow. Inhibition is shown as T-shaped arrow.DOI: http://dx.doi.org/10.7554/eLife.17557.025|
|Author response image 1. Left panel shows Centrin-CFP (blue) and GFP-Cp110 (green) in images where green channel brightness is low.In this image Cp110 localizes adjacent to the basal body. Right panel shows Centrin-CFP (blue) and GFP-Cp110 (green) in images where green channel brightness is high. In this image we can visualize the low-level localization of GFP-Cp110 to the rootlet tip, which was not visible in the left panel. Bottom panel shows Clamp-RFP staining of the ciliary rootlet (red). Top row, middle panel shows schematic localization of Cp110 relative to the basal body and the rootlet. Green = Cp110, blue = Centrin4, red = Clamp.DOI: http://dx.doi.org/10.7554/eLife.17557.026|
|Author response image 2. Alignment of the cp110 region, where the missing Adenine was identified in the FS-clone.BC167469, Xenopus tropicalis genome 9.0 sequence and the current Xenopus tropicalis cp110 reference sequence (XM_0129708) are shown.DOI: http://dx.doi.org/10.7554/eLife.17557.027|