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. 2018 May 7;217(5):1847–1868. doi: 10.1083/jcb.201709041

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© 2018 Ye et al.

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Figure 5. — PKA antagonizes the recruitment of the BBSome to the tip of cilia by Kif7. (A–C) Corecruitment of BBS5 and Kif7 to the tip of cilia in live IMCD3-[pEF1α-^NG3BBS5; pCMV-Kif7^mScarlet] cells. (A) Bottom: Representative images showing SAG- and PKI-induced accumulation of ^NG3BBS5 and Kif7^mScarlet at ciliary tip. Cells were treated with SAG, PKI, or vehicle for 40 min before imaging. White boxes mark the ciliary tip. b, base; t, tip. Top: Individual channel crops of the tip area. (B) Correlation between the fluorescence signal of ^NG3BBS5 and Kif7^mScarlet at the ciliary tip. Fluorescence signals of ^NG3BBS5 and Kif7^mScarlet were measured at ciliary tip of live cells after 40 min treatment with vehicle, SAG, or PKI. Micrographs of ^NG3BBS5 and Kif7^mScarlet at the ciliary tip for three representative data points are shown. Linear regressions (dotted lines) highlight the positive correlation between ciliary tip levels of ^NG3BBS5 and Kif7^mScarlet in the presence of SAG or PKI. The Pearson correlation coefficient (r) is shown. Student’s t test of the Pearson’s correlation coefficient (r) returned a nonsignificant p-value under control conditions (P > 0.7) but a significant value after SAG or PKI treatment (P < 10⁻⁵). n = 40–49 cilia. (C) In SAG-treated cells where a second spot of Kif7 is occasionally found along cilia, a second spot of BBS5 was observed at the same location as Kif7. The yellow arrows mark the location of ^NG3BBS5 foci that accumulated at the ectopic tip. Bars, 2 µm. B, base; T, tip. (D and E) Kif7 is necessary for the redistribution of BBS5 to the tip of cilia. (D) Line scans of ^NG3BBS5 fluorescence intensities along cilia of live cells. Cells were transfected with siRNAs for 72 h and treated with sst for 40 min before live imaging of ^NG3BBS5 fluorescence. The line marks the mean intensity along length-normalized cilia. The shaded areas show the 95% CI (not depicted for the vehicle control). n = 20–27 cilia. (E) The total number of BBS5 molecules at the tip was calculated as in Fig. 3 C. n = 20–28 cilia from three independent experiments. (F) Kif7 interacts with BBSome, and PKA antagonizes this interaction. HEK293 cells cotransfected with Kif7^GFP and ^MycBBS1 were treated with the cAMP phosphodiesterase inhibitor IBMX or vehicle for 30 min before lysis. Complexes were immunoprecipitated (IP) with anti-GFP antibodies. Lysates and eluates were immunoblotted (IB) for Myc. The capture efficiency of ^MycBBS1 by Kif7^GFP was decreased 29 ± 3% upon treatment with IBMX. Molecular weights (MWs; kD) are indicated on the right. n = 3 independent experiments. (G) Kif7 is necessary for SSTR3 exit from cilia. IMCD3-[^APSSTR3^NG] cells were treated with siRNA targeting Kif7 or luciferase, pulse-labeled with mSA647, and imaged every 10 min after addition of sst. The resulting loss in mSA647 fluorescence was plotted and linearly fitted to determine the rate of SSTR3 retrieval. Asterisks indicate Mann-Whitney test significance values. *, P < 0.05; ***, P < 0.0005. Error bars represent SD. n = 13 cilia.