Fig. 2.
Importin-binding motifs and domains increase the ciliary localization of membrane reporters. (A,B) SV40-cNLS increased the ciliary localization of CD8a chimeras tagged with 1–3 GFP molecules. Images showing representative ciliated RPE1 cells co-expressing Arl13b–mCherry and CD8a chimeras. The cilium of interest is indicated by an arrow. The three inserts in each image show the cilium in GFP (monochromatic, left), mCherry (monochromatic, middle) and merge (color, right) channel. The bar graph shows CPIR values of CD8a chimeras. (C,D) IBB and the PY-NLS signal of hnRNP-M increased the ciliary localization of CD8a–GFP or CD8a–GFP×3. The organization of images and bar graph is similar to those described for A,B. (E) Representative two-dimensional time-lapse images of cilia expressing various fluorescence chimeras during whole cilium FRAP analysis. Live ciliated RPE1 cells expressing the indicated fluorescence chimeras were imaged by using a spinning disk confocal microscope. The whole cilium was photobleached at 0 s. Time is indicated at the upper left of each image. The half lives and immobile fractions of FRAP are plotted in F and G, respectively. Both CD8a-f-CTS–GFP and CFF–GFP contain the CTS of fibrocystin. Note that the half life value of CD8a–GFP in Fig. 1H (control) is duplicated here for comparison. The number of cells, n, is labeled in each bar graph. Error bars are s.e.m. The mean value is indicated at the top of each column. P-values (t-test) of selected pairs are denoted.