Figure 3.
Figure 3. A Gαi–PKA axis promotes BBSome tip accumulation. (A) Near-endogenous expression of NG3BBS5. IMCD3 and IMCD3-[pEF1α-NG3BBS5] cells were subjected to immunoprecipitation (IP) with an anti-BBS5 antibody, and lysates and eluates were immunoblotted (IB) for BBS5. Molecular weights (MWs; kD) are indicated on the left. Measurement of band intensities with Image Lab (Bio-Rad Laboratories) indicates that the molar ratios between NG3BBS5 and endogenous BBS5 is 1.27. (B) IMCD3-[pEF1αΔ-APSSTR3, pEF1α-NG3BBS5] were treated with sst or vehicle for 40 min. Top: Representative images of cilia from live cells. NG3BBS5 is in fire scale, and white crosses mark the location of the basal body (see Fig. S2 J). Bottom: Line scans of NG3BBS5 fluorescence intensities along cilia of live cells. The line marks the mean intensity along length-normalized cilia. The shaded area shows the 95% CI. n = 20–29 cilia. (C) NG3BBS5 tip fluorescence intensities were quantified in live cells after 40 min of incubation with sst, SAG, Rp-cAMPs, or PKI. Cells were preincubated with PTX for 16 h to fully inactivate Gαi. Representative images are shown in the bottom panels with tips marked by yellow arrows. The total number of BBS5 molecules at the tip was calculated using the NG3 calibrator and the measured ratio of NG3BBS5 to total BBS5. The whiskers represent 1.5× the interquartile range. Asterisks indicate Mann-Whitney test significance values. ***, P < 0.0005. n = 20–29 cilia from three independent experiments. (D) IMCD3 or IMCD3-[pEF1αΔ-APSSTR3NG] cells were treated with vehicle, sst, or SAG for 40 min before fixation. The bar graph shows the percentage of BBS9-positive tips detected by immunofluorescence staining of endogenous BBS9. Error bars represent SD. n = 52–99 cilia. (E) The pathways downstream of Smoothened (SMO) and SSTR3 and the site of action of the pharmacological perturbations are shown. (F) Representative immunofluorescence images of ciliary BBS9 (red) in IMCD3-[pEF1α-NPHP3GFP-PKI] and IMCD3-[pEF1α-NPHP3GFP-PKIdead] cells (Mick et al., 2015). Optical sections were deconvolved, and X/Z projections are shown. Arrows mark tips of cilia. Percentages of BBS9-positive ciliary tips are plotted on the right. n = 29–50 cilia. Bars: (B) 1 µm; (C and F) 2 µm.

A Gαi–PKA axis promotes BBSome tip accumulation. (A) Near-endogenous expression of NG3BBS5. IMCD3 and IMCD3-[pEF1α-NG3BBS5] cells were subjected to immunoprecipitation (IP) with an anti-BBS5 antibody, and lysates and eluates were immunoblotted (IB) for BBS5. Molecular weights (MWs; kD) are indicated on the left. Measurement of band intensities with Image Lab (Bio-Rad Laboratories) indicates that the molar ratios between NG3BBS5 and endogenous BBS5 is 1.27. (B) IMCD3-[pEF1αΔ-APSSTR3, pEF1α-NG3BBS5] were treated with sst or vehicle for 40 min. Top: Representative images of cilia from live cells. NG3BBS5 is in fire scale, and white crosses mark the location of the basal body (see Fig. S2 J). Bottom: Line scans of NG3BBS5 fluorescence intensities along cilia of live cells. The line marks the mean intensity along length-normalized cilia. The shaded area shows the 95% CI. n = 20–29 cilia. (C) NG3BBS5 tip fluorescence intensities were quantified in live cells after 40 min of incubation with sst, SAG, Rp-cAMPs, or PKI. Cells were preincubated with PTX for 16 h to fully inactivate Gαi. Representative images are shown in the bottom panels with tips marked by yellow arrows. The total number of BBS5 molecules at the tip was calculated using the NG3 calibrator and the measured ratio of NG3BBS5 to total BBS5. The whiskers represent 1.5× the interquartile range. Asterisks indicate Mann-Whitney test significance values. ***, P < 0.0005. n = 20–29 cilia from three independent experiments. (D) IMCD3 or IMCD3-[pEF1αΔ-APSSTR3NG] cells were treated with vehicle, sst, or SAG for 40 min before fixation. The bar graph shows the percentage of BBS9-positive tips detected by immunofluorescence staining of endogenous BBS9. Error bars represent SD. n = 52–99 cilia. (E) The pathways downstream of Smoothened (SMO) and SSTR3 and the site of action of the pharmacological perturbations are shown. (F) Representative immunofluorescence images of ciliary BBS9 (red) in IMCD3-[pEF1α-NPHP3GFP-PKI] and IMCD3-[pEF1α-NPHP3GFP-PKIdead] cells (Mick et al., 2015). Optical sections were deconvolved, and X/Z projections are shown. Arrows mark tips of cilia. Percentages of BBS9-positive ciliary tips are plotted on the right. n = 29–50 cilia. Bars: (B) 1 µm; (C and F) 2 µm.

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