Figure 4.

FRAP analysis reveals that the R1747Q missense mutation affects the mobility of ciliary molecules. Cells were cotransfected with indicated mCherry- or EGFP-tagged ciliary proteins and vector or mCherry/EGFP-Cep290 constructs and starved for 24 hrs prior to FRAP experiments. Representative images show changes in fluorescence intensity prior (pre; t = −3.146 s), at (bleach; t = 0.000) and after (post; t = 115.191 s) photobleaching of (A) Smo-EGFP, (B) 5HT6-EGFP, (C) SSTR3-EGFP, (D) IA-EGFP, and (E) Arl13b-mCherry in primary cilium. Scale bar = 2 μm. (A’–E’) Fluorescence recovery of indicated bleached proteins are plotted as recovery curves in each transfected condition. Data was corrected for background fluorescence and for FRAP-unrelated bleaching effects using an unbleached ROI and thereafter normalised to the fluorescence intensity immediately before bleaching. (A”–E”) Recovered fraction after bleaching was obtained from the plateau of the recovery curves. n ≥ 18 for Smo-EGFP, n ≥ 32 for 5HT6-EGFP, n ≥ 29 for SSTR3-EGFP, n ≥ 23 for IA-EGFP, and n ≥ 33 for Arl13B-mCherry. n = cells per category, three independent experiments. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vector, one-way ANOVA post hoc Holm-Sidak’s multiple comparisons test.