XB-IMG-155175
Xenbase Image ID: 155175
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Figure 5. Cp110 coiled-coil domains are required for cilia inhibition and centriolar functions.(A) Cp110 coiled-coil domains are required for MCC cilia inhibition and formation of supernumerary centrioles. Controls and embryos injected with full-length gfp-cp110, gfp-cp110ΔCentral or gfp-cp110ΔCCD1+2 (all green) were analyzed for ciliation by immunofluorescent staining (Ac.-α-tub., red). Upper panels: red fluorescence channel; merge channel insets show magnifications outlined by dashed boxes. Lower panels: green/red merge; insets show magnifications of non-MCCs outlined by dashed boxes. Related to Figure 5—figure supplement 1A–B. (B) Cp110 coiled-coil domains are required for basal body aggregation. Embryos injected with centrin4-cfp (basal bodies, blue), clamp-rfp (rootlets, red), and either full-length gfp-cp110 or gfp-cp110ΔCentral or gfp-cp110ΔCCD1+2 (green). Upper panels: red/blue merge; insets show magnifications of basal bodies outlined by dashed boxes. Lower panels: green/blue merge; insets show magnifications of basal bodies outlined by dashed boxes. n embryos/MCCs: control (7/21), gfp-cp110 (7/21), gfp-cp110ΔCentral (7/21), gfp-cp110ΔCCD1+2 (7/21). Related to Figure 5—figure supplement 1D. (C) GFP-Cp110 constructs show different localization patterns at basal bodies (bb) and rootlets (r). Upper panels: Individual basal bodies from MCCs shown in (B) (solid boxes). Lower panels: heat maps of GFP-Cp110 intensity. Color code shown right. Related to Figure 5—figure supplement 1D. (D) Cp110 constructs generated in this study. Different colors indicate predicted functional domains. Green, CDK phosphorylation sites; blue, coiled-coil domains; yellow, Cyclin binding domain (RXL); pink, KEN domain (proteasomal degradation); brown, CaM-binding domains; red asterisk indicates the position of miR-34/449 target site in the cp110 mRNA. See also:DOI:
http://dx.doi.org/10.7554/eLife.17557.019Figure 5—figure supplement 1. Cp110 coiled-coil domains are required for cilia inhibition and centriolar functions.(A) Related to Figure 5A. Embryos injected with gfp-cp110ΔN or gfp-cp110ΔC (all green) were analyzed for ciliation by immunofluorescent staining against Acetylated-α-tubulin (cilia, Ac.-α-tub., red). Upper panels: red fluorescence channel only. Lower panels: green/red merge channels. (B) Related to (A) and Figure 5A. Quantification of MCC cilia phenotype in controls and after overexpression of full-length gfp-cp110 and deletion constructs. ***p<0.001 from χ²-test. (C) Immunoblot of GFP-Cp110 (α-GFP antibody) constructs to monitor expression levels. Please note that GFP-Cp110ΔCCD1+2 levels are not reduced as compared to cilia inhibiting constructs. (D) Related to Figure 5B. Embryos were injected with centrin4-cfp (basal bodies, blue), clamp-rfp (rootlets, red), and either gfp-cp110ΔN or gfp-cp110ΔC (all green). Upper panels: red/blue merge channels; insets show magnifications of basal bodies outlined by dashed boxes. Lower panels: green/blue merge channels; insets show magnifications of basal bodies outlined by dashed boxes. n embryos/MCCs: gfp-cp110ΔN (3/9), gfp-cp110ΔC (3/9). (E) Left panels: Individual basal bodies from MCCs shown in (D) as red/green/blue merge channels. Right panels: heat maps showing GFP-Cp110 intensity levels. Basal body (bb), rootlet (r). Color code shown on the right. (F) gfp-cp110 overexpression induces apical Actin defects in MCCs. Embryos were injected with centrin4-cfp (basal bodies, white) and gfp-cp110 constructs (not shown) and stained for Actin (red). Upper panels: apical view on MCCs. Lower panels: lateral projections show localization of basal bodies along the apical-basal axis on MCCs. n embryos/MCCs: control (9/27), gfp-cp110 (7/12), gfp-cp110ΔCentral (6/18), gfp-cp110ΔCCD1+2 (6/18), gfp-cp110ΔN (3/9), gfp-cp110ΔC (3/9).DOI:
http://dx.doi.org/10.7554/eLife.17557.020 Image published in: Walentek P et al. (2016) © 2016, Walentek et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |