Figure 6. Targeted disruption of the BBSome-TOM1L2 interaction blocks the regulated exit of GPCRs from cilia.

A. IMCD3-[pEF1α^Δ-^APSSTR3; pEF1α-BirA•ER] were transfected with plasmids expressing the BBSome interacting region of TOM1L2 (BIR) fused to the ciliary targeting signal of NPHP3 (CTS) and GFP or indicated variants. Ciliary ^APSSTR3 was pulse-labeled with Alexa647-labeled monovalent streptavidin (mSA647) for 5 to 10 min, and cells were then treated with or without sst for 2 h, before fixation and staining for acetylated tubulin (acTub; magenta) and DNA (cyan). The CTS fusions were visualized through the intrinsic fluorescence of GFP (yellow) and ^APSSTR3 was visualized via mSA647 (white). Scale bars, 5 μm (main panel), 1 μm (inset). B. The fluorescence intensities of ciliary ^APSSTR3 are represented as violin plots. Asterisks indicate statistical significance value calculated by one-way ANOVA followed by Tukey’s post hoc test. **, p ≤ 0.01; *, p ≤ 0.05. n = 38–64 cilia. C. RPE1-hTERT cells transfected with the indicated constructs were treated with SAG or vehicle (DMSO) for 2 h and then fixed and stained for acetylated tubulin (magenta), GPR161 (white), and DNA (cyan). The CTS fusions were visualized through the intrinsic fluorescence of GFP (yellow). Scale bars, 5 μm (main panel), 1 μm (inset). D. The fluorescence intensities of ciliary GPR161 are represented as violin plots. Asterisks indicate ANOVA significance value. ****, p ≤ 0.0001. E. IMCD3-[pCrys-SMO^FLAG] cells transfected with the indicated constructs were fixed and stained for acetylated tubulin (magenta), FLAG (SMO, white), and DNA (cyan). The CTS fusions were visualized through the intrinsic fluorescence of GFP (yellow). F. The fluorescence intensities of ciliary SMO are represented as violin plots. Asterisks indicate ANOVA significance value. ****, p ≤ 0.0001. n = 42–64 cilia.