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. 2021 Jun 17;24(7):102739. doi: 10.1016/j.isci.2021.102739

Figure 7.

Figure 7

MRTF is necessary for the correct localization of CEP290 at the ciliary base

(A and B) To assess the localization of CEP290 in relation to PC-related structures, serum-starved RPE cells were fixed and co-stained with antibodies against CEP290 and either the BB marker γ-tubulin (A) or the PC marker (shaft) Ac-Tubulin (B). Nuclei were visualized by Dapi, and cells were viewed by confocal microscopy. Bar 10 μm.

(C) Co-staining of CEP290 and MRTF in a monolayer of RPE cells. The merged image (last panel) shows strong colocalization between distinct MRTF-positive patches and CEP290. Bar: 20 μm. (C’) Magnified view of the two areas demarcated by the dashed rectangles in C. Note that MRTF- and CEP290-containing clusters strongly co-localize but do not completely overlap.

(D and E) MRTF and CEP290 are in a complex. (D) RPE cells were serum-deprived, followed by lysis and immunoprecipitation of endogenous MRTF. Whole cell lysates (input) and precipitates were probed with anti-MRTF and anti-CEP290 antibodies. (E) RPE cells were transfected with Myc-tagged WT (full-length) MRTF or the Myc-tagged N- or C-terminal half of the MRTF protein. Immunoprecipitation was performed with anti-Myc antibody. Note that the full-length and the N-terminal MRTF exhibited strong association with CEP290, while the C-terminal segment showed marginal if any binding.

(F) To determine the impact of MRTF on CEP290 localization, RPE cells were transfected with siNR or siMRTF and allowed to ciliate for 48 hr under serum-free conditions. Cells were then co-stained for CEP290 and acetylated tubulin to determine the localization of CEP290 vis-à-vis the PC. In cells transfected with siNR CEP290 unequivocally localized to the base of the PC. In cells transfected with siMRTF CEP290 exhibited varied localization, in three different subsets; CEP290 resided 1) at the base of PC or 2) at the tip of the PC or 3) at both locations. Representative images for these categories are shown. Bar 10 μm. The insets depict further enlarged images of the corresponding cilium tips to document ectopic CEP290 staining.

(G) Quantification of CEP290 localization, as percentage of cells in each category (localization at the base, tip, or both). Only those cells were counted where the localization could be unambiguously determined (mean ± SD, n = 3 independent experiments, ≈100 cells/experiment/condition); ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001, determined by ANOVA.