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Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia

  1. Alison Leaf
  2. Mark Von Zastrow  Is a corresponding author
  1. University of California, San Francisco, United States
Research Article
Cite this article as: eLife 2015;4:e06996 doi: 10.7554/eLife.06996
8 figures and 1 table

Figures

D1Rs specifically localize to primary cilia.

(AC) Representative epifluorescence microscopy images of Flag-D1R (panel A), Flag-SSTR3 (panel B), and Flag-DOR (panel C) localization on the surface of inner medullary collecting duct (IMCD3) cells. Insets show a cropped region of the plasma membrane containing the cilium, with Flag immunoreactivity marking receptor (top) and acetylated tubulin (AcTub) immunoreactivity marking the cilium (middle). Merged view is at bottom with Flag in green and AcTub in red. Flag-D1R and Flag-SSTR3 localize robustly to cilia, while Flag-DOR is detectable in the extra-ciliary plasma membrane but not on cilia. (D) Quantification of ciliary localization by determining the fraction of receptor (Flag)-positive cilia, judged by the presence of Flag immunoreactivity visible by epifluorescence microscopy, and expressed as a percentage of total cilia counted in the transfected cell population. (E) Scheme for quantification of ciliary localization by determining enrichment of receptor (Flag) signal in an ROI containing the cilium, when compared to an adjacent region of the extra-ciliary plasma membrane. Representative ROIs are shown for a Flag-D1R-transfected cell. (F) Fold-enrichment calculated as a ratio of background-subtracted Flag signal present in the ciliary ROI divided by background-subtracted Flag signal present in the adjacent extra-ciliary plasma membrane ROI (cilia/PM). Error bars represent SEM from n = 3 independent experiments, with 10–15 cilia analyzed for each receptor in each experiment. (***) p < 0.001. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.003
Figure 2 with 7 supplements
D1Rs are mobile in the ciliary membrane and delivered from the extra-ciliary plasma membrane.

(A) Schematic for local labeling of D1-type dopamine receptor (D1R) in a cilium using PA-GFP. IMCD3 cells expressing Flag-D1-PAGFP were labeled with anti-Flag antibody conjugated to Alexa555 to visualize the overall surface receptor pool. A point-focused 405-nm laser spot was used to locally photoactivate receptors on the mid-portion of the cilium. Non-fluorescent PA-GFP is depicted in gray, fluorescent state in green. (B) Live cell confocal images of a representative cilium showing the frame immediately before the photoactivation pulse (left column), and frames acquired 1 s (middle column) and 10 s (right column) after local photoactivation. The Flag-Alexa555 signal labeling the entire surface receptor pool (top row) was present throughout the cilium at all time points. PA-GFP fluorescence representing the photoactivated pool was non-uniformly distributed at 1 s and uniformly distributed along the cilium within 10 s. (C) Line scan analysis of PA-GFP fluorescence along the cilium from the example in panel B. (D) Integrated PA-GFP fluorescence signal in the cilium as a function of time after the 405-nm laser pulse. The PA-GFP fluorescence at time = 0 was set at 100%. Points represent the mean fraction of PA-GFP fluorescence present in the cilium over an 80-s imaging interval. Error bars represent SD from analysis of n = 6 cilia. There was no detectable loss of ciliary PA-GFP signal quantified over an 80-s interval. (E) Confocal images of a representative cilium acquired immediately after (0 min) and 10 min after photoactivation, showing that the locally photoactivated receptor pool was largely retained in the cilium even after this longer interval. (F) Assessing new D1R delivery to the cilium by saturation photoactivation and the sequential ‘image-photoactivate-image’ scheme described in the ‘Materials and methods’. Bars represent mean fractional increase in ciliary PA-GFP fluorescence elicited by the subsequent test pulse. Error bars represent SD for n = 7 cilia. (G) Schematic for modifying the saturation photoactivation method to assess source of newly delivered D1Rs, based on the ratio of integrated PA-GFP/Alexa555 fluorescence (PA/555) measured in the cilium as a function of time. The initial condition is depicted on the left (‘0 min’) with the fluorescence ratio (PA/555) arbitrarily set to 1. If new receptors enter the cilium from an internal membrane pool during the 30-min incubation period (depicted in center, ‘30 min’), they contribute neither Alexa555 nor PA-GFP signal, so the fluorescence ratio is unchanged from the initial condition (PA/555 = 1). After the subsequent 405-nm test pulse (depicted at right, ‘30 min + PA’), the PA-GFP signal increases without any change in Alexa555 signal, elevating the fluorescence ratio above the initial condition (PA/555 > 1). If new receptors enter the cilium from the extra-ciliary plasma membrane pool, they contribute Alexa555 but not PA-GFP signal during the 30-min incubation, reducing the fluorescence ratio from the initial condition (PA/555 < 1). The subsequent 405-nm pulse restores the fluorescence ratio to the initial value (PA/555 = 1). (H) Experimental results from the strategy depicted in panel G. Bars represent the mean ratio of integrated PA-GFP/Alexa555 fluorescence measured in the cilium. Error bars represent SD from n = 6 cilia. (***) p < 0.001. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.004
Figure 2—figure supplement 1
Whole-cell images corresponding to the images shown in Figure 2B.

Flag immunoreactivity is shown in red and PA-GFP in green. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.005
Figure 2—figure supplement 2
Whole-cell images corresponding to the images shown in Figure 2E.

Channels are shown individually in gray scale. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.006
Figure 2—figure supplement 3
New D1R delivery to cilia increases over time.

The sequential ‘image-photoactivate-image’ scheme was applied as described in Figure 2F except that the time interval between the initial 405-nm pulse series and the subsequent assessment of PA-GFP fluorescence increment was varied from 2 min to 50 min. Each square represents an individual determination. The line indicates a least squares best fit.

https://doi.org/10.7554/eLife.06996.007
Figure 2—figure supplement 4
Control experiment for the ciliary delivery assay described in Figure 2F.

The scheme used in Figure 2F was applied to fixed cells. Error bars represent SD for n = 6 cilia. There was no significant PA-GFP fluorescence increment at either time point.

https://doi.org/10.7554/eLife.06996.008
Figure 2—figure supplement 5
Images of cilia from ciliary delivery assay.

Representative images of a cilium from the ‘image-photoactivate-image’ scheme described in Figure 2F,G showing the PA-GFP signal and the Flag signal separately in gray scale. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.009
Figure 2—figure supplement 6
Bleaching control for the ciliary delivery assay.

Cilia were photoactivated and consecutive PA-GFP and Alexa555 images were acquired. The integrated fluorescence intensity in the cilium was normalized to that measured in the initial image after photoactivation. Error bars represent SD from analysis of n = 3 cilia. There was no detectable loss of ciliary PA-GFP signal or ciliary Alexa555 (Flag) signal after 28 consecutive images.

https://doi.org/10.7554/eLife.06996.010
Figure 2—figure supplement 7
Bleed-through control for the ciliary delivery assay.

Representative images of cilia expressing Flag-D1-GFP in the absence and presence of M1-555, which recognizes Flag, showing negligible bleed-through of the GFP signal into the 555 channel. The merged image displays the GFP channel in green and 555 (Flag-receptor) in red. Insets show a cropped region of the plasma membrane containing the cilium. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.011
Figure 3 with 5 supplements
The D1R cytoplasmic tail is necessary and sufficient for ciliary receptor targeting.

(A) Schematic representation of D1R C-tail mutations used in the present analysis. (B) Representative images of cells expressing Flag-tagged wild-type D1R (D1R) or a receptor construct truncated at residue 415 (D1-415T), showing robust ciliary localization of both. The merged image at bottom displays Flag-receptor in green and AcTub in red. (C) Representative image of a receptor construct truncated at residue 382 (D1-382T), showing near complete loss of receptor localization to cilia marked by AcTub. (D) Representative images of a D1R construct with internal deletion of residues 381–395 (D1Δ381-395), showing the range of phenotypes observed, from a complete loss of receptor localization in cilia to a pronounced reduction of ciliary receptor localization. (E) Quantification of the fraction of receptor (Flag)-positive cilia for Flag-tagged wild-type (D1R) or mutant (D1Δ381-395) receptor. The analysis is described in Figure 1D. (F) Quantification of the average fold-enrichment of wild-type (D1R) or mutant (D1Δ381-395) receptors on cilia. The analysis is described in Figure 1E,F. (G) Schematic representation of chimeric mutant receptors containing portions of the D1R cytoplasmic tail (in red) fused to the delta opioid peptide receptor (DOR) cytoplasmic tail (in green). (H) Representative images of cilia in cells expressing Flag-DOR, Flag-DOR-D1(338–446), or Flag-DOR-D1(368–446) showing that the D1R C-tail is sufficient to drive ciliary targeting of chimeric receptors. (I) Fraction of receptor (Flag)-positive cilia. (J) Average fold-enrichment of receptor (Flag) signal on cilia. Error bars represent SEM from n = 3 experiments with 10–20 cilia analyzed per experiment. (***) p < 0.001. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.012
Figure 3—figure supplement 1
Whole-cell images corresponding to images shown in Figure 3B–D.

The merged image displays Flag-receptor immunoreactivity in green and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.013
Figure 3—figure supplement 2
Overall surface expression of D1R C-tail mutant.

Surface-accessible Flag immunoreactivity was quantified for the cilia-defective mutant D1R (D1∆381–395) relative to wild-type D1R by fluorescence flow cytometry, as described in the ‘Materials and methods’. The ciliary targeting defective mutant receptor was expressed in the overall plasma membrane at a level indistinguishable from the cilia-localized wild-type D1R, supporting the hypothesis that the C-tail region mutated is required specifically for targeting surface receptors to cilia, but not for targeting receptors to the extra-ciliary surface.

https://doi.org/10.7554/eLife.06996.014
Figure 3—figure supplement 3
Whole-cell images corresponding to images shown in Figure 3H.

The merged image displays Flag-receptor immunoreactivity in green and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.015
Figure 3—figure supplement 4
Overall surface expression of DOR-derived constructs.

Surface-accessible Flag immunoreactivity for the indicated DOR-derived chimeric mutant constructs expressed relative to wild-type DOR. The chimeric mutant receptors were expressed at similar levels, further supporting the specific function of the D1R-derived sequence in targeting receptors to the ciliary plasma membrane compartment. (*) p < 0.05.

https://doi.org/10.7554/eLife.06996.016
Figure 3—figure supplement 5
The 15 residue sequence required for full ciliary targeting of D1R is not sufficient to confer ciliary localization on DOR.

Representative image of a cilium in cells expressing the chimeric mutant receptor Flag-DOR-D1(379–400), which includes the D1R residues 381–395, showing that this sequence is not sufficient to confer ciliary targeting of receptors. The merged image displays Flag-receptor immunoreactivity in green and AcTub in red. Insets show a cropped region of the plasma membrane containing the cilium. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.017
Figure 4 with 3 supplements
IFT-B complex proteins are necessary for D1R localization to cilia.

(A) Representative images of cilia on cells stably transfected with Flag-D1R and transiently transfected with a non-silencing duplex (control) or siRNA targeting IFT57 (IFT57-1 and IFT57-4) or IFT172 (IFT172-3 and IFT172-4). In the merged image, Flag-D1R immunoreactivity is shown in green and AcTub in red. Duplexes targeting IFT57 and IFT172 caused a visually obvious reduction in ciliary D1R localization. The right column of images shows the rescue condition in which cells transfected with siRNA against IFT57 were additionally transfected with IFT57 that is not targetable by the IFT57 siRNA (HA-IFT57-NTM). Scale bar, 5 μm. (B) Effect of siRNAs on the fraction of cells in the population possessing a visible cilium marked by AcTub. Error bars represent SEM from 150 cells counted in n = 3 independent experiments. (C) Fraction of D1R (Flag)-positive cilia. Error bars represent SEM for n = 3 experiments with 50 cells counted in each experiment. (D) Average fold-enrichment of D1R (Flag) signal on cilia. Error bars represent SEM for n = 3 experiments with 15–40 cilia analyzed per experiment. (E) Association of IFT57 with the D1R but not DOR demonstrated by co-immunoprecipitation. Cells were transfected with the constructs indicated above each lane. Cell extracts were blotted for HA and Flag; HA-IFT57 resolved as a sharp band at its expected apparent molecular mass and Flag-tagged receptors resolved as heterogeneous species consistent with complex glycosylation as shown previously. Specific co-immunoprecipitation is indicated by HA-IFT57 detected in the Flag-D1R pull-down but not in Flag-DOR pull-down. Molecular mass markers (in kDa) are shown on right side of blots. The results in panel E are representative of n = 3 independent experiments. (F) Increased association of IFT57 with D1Δ381-395 relative to D1R demonstrated by co-IP. Cells were transfected with the constructs indicated above each lane. Cell extracts were blotted for HA and Flag. More HA-IFT57 was detected in the Flag-D1Δ381-395 pull-down than the Flag-D1R pull-down. Molecular mass markers (in kDa) are shown on right side of blots. The results in panel E are representative of n = 3 independent experiments. (G) Immunoblots from multiple experiments were scanned in the linear range, as described in ‘Materials and methods’, to estimate the amount of IFT57 co-IPed with the indicated receptors. Expressed as a fold increase over control where control is D1R. Error bars represent SD from n = 3 experiments. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

https://doi.org/10.7554/eLife.06996.019
Figure 4—figure supplement 1
Whole-cell images corresponding to the images shown in Figure 4A.

The merged image displays Flag-D1R immunoreactivity in green and AcTub in red. Expression of HA-IFT57-NTM is verified by HA immunoreactivity. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.020
Figure 4—figure supplement 2
Verification of IFT-B knockdown.

Cells stably expressing Flag-D1R were transfected with either control siRNA or siRNA targeting intraflagellar transport (IFT)-B components, IFT57 and IFT172, and RNA levels were measured via qRT-PCR. Error bars represent SEM for n = 3 experiments. (**) p < 0.01; (***) p < 0.001.

https://doi.org/10.7554/eLife.06996.021
Figure 4—figure supplement 3
IFT-B knockdown has little effect on overall surface receptor expression.

Surface-accessible Flag immunoreactivity representing Flag-D1Rs present in the plasma membrane was quantified by flow cytometry. Results are normalized to control siRNA.

https://doi.org/10.7554/eLife.06996.022
Figure 5 with 5 supplements
KIF17 motor activity is required for full D1R enrichment in cilia.

(A) Association of IFT57 with KIF17 indicated by co-immunoprecipitation. Cells were transfected with expression constructs indicated at top of the panel, and extracts were blotted for HA to detect IFT57 and Flag to detect KIF17. HA-IFT57 resolved as expected and described in Figure 4E. KIF17 resolved as two species with the top band corresponding to the expected molecular mass of the full-length protein. Specific co-immunoprecipitation is indicated by HA-IFT57 detected in the Flag pull-down from cells expressing Flag-KIF17 pull-down (arrow) but not from cells in which Flag-KIF17 was not expressed. Molecular mass markers (in kDa) shown on right. (B) Representative images of cilia on cells co-transfected with Flag-D1R and control empty vector (+pcDNA), a plasmid encoding HA-tagged KIF17 (+KIF17), or a plasmid encoding an HA-tagged KIF17 construct harboring a point mutation in a conserved residue that disrupts kinesin motor activity (+KIF17-G234A). Robust ciliary localization of Flag-D1R was observed in cells expressing control plasmid or the wild-type KIF17 construct, but markedly reduced ciliary enrichment of D1Rs was observed in cells expressing motor-defective mutant KIF17. (C) Quantification of the effect of disrupting KIF17 motor activity on the fraction of D1R (Flag)-positive cilia. (D) Quantification of the effect of disrupting KIF17 motor activity on average fold-enrichment of D1R (Flag) on cilia. Disrupting KIF17 motor activity strongly reduced the degree of D1R enrichment on the ciliary membrane without blocking D1R access to cilia. (E) Representative images of cilia on cells co-transfected with Flag-SSTR3 and with control empty vector (+pcDNA) or a plasmid encoding motor domain-mutant KIF17 (+KIF17-G234A). Disrupting KIF17 motor activity did not detectably affect Flag-SSTR3 localization to cilia. (F) Quantification of the effect of disrupting KIF17 motor activity on average fold-enrichment of somatostatin-3 receptor (SSTR3) (Flag) on cilia. Disrupting KIF17 motor activity did not detectably affect ciliary enrichment of SSTR3. Error bars represent SEM from n = 3 independent experiments with 10–20 cilia analyzed in each experiment. (***) p < 0.001. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.023
Figure 5—figure supplement 1
Switch II mutation in KIF17.

Amino acid sequence alignment of the switch II regions of Kinesin-1 and KIF17 showing identical sequences. Switch II residues are shown in magenta with a box around the G residue mutated in the motor domain mutant.

https://doi.org/10.7554/eLife.06996.024
Figure 5—figure supplement 2
Whole-cell images corresponding to the images shown in Figure 5B.

HA-KIF17-G234A and HA-KIF17 expression is verified by HA immunoreactivity detected throughout the cytoplasm and accumulated in the nucleus. The HA signal detected in cilia is likely contributed in large part by cross reactivity of the secondary anti-rat antibody with the mouse primary antibody recognizing AcTub. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.025
Figure 5—figure supplement 3
Independent verification of KIF17 requirement for D1R ciliary enrichment.

Cells were co-transfected with Flag-D1R and an empty vector (+pcDNA) or a plasmid encoding an HA-tagged version of a previously reported dominant negative KIF17 (+KIF17-DN). Quantification of the effect of KIF17-DN on average fold-enrichment of D1R (Flag) on cilia. Expression of KIF17-DN strongly reduced the degree of D1R ciliary enrichment. Error bars represent SEM from n = 4 independent experiments with 10–20 cilia analyzed per experiment. (***) p < 0.001.

https://doi.org/10.7554/eLife.06996.026
Figure 5—figure supplement 4
Whole-cell images corresponding to the images shown in Figure 5E.

HA-KIF17-G234A expression is verified by HA immunoreactivity. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.027
Figure 5—figure supplement 5
The KIF17 motor domain mutation has little effect on overall surface expression of receptors.

Effects of KIF17 transfection on surface-accessible Flag immunoreactivity from Flag-tagged D1R (D1R) or Flag-tagged SSTR3 (SSTR3) were quantified by fluorescence flow cytometry and normalized to the mock-transfected (pcDNA) condition. Bars represent mean normalized surface expression. (*) p < 0.05; (**) p < 0.01.

https://doi.org/10.7554/eLife.06996.028
Figure 6 with 4 supplements
Rab23 is necessary for D1R localization to cilia.

(A) Representative images of cilia on cells stably transfected with Flag-D1R and transiently transfected with a non-silencing duplex (control) or siRNA targeting Rab23 (Rab23-4 and Rab23-2). Rab23 knockdown strongly reduced Flag-D1R localization to cilia. (B) Quantification of the siRNA effect on the fraction of cells in the population possessing a visible cilium marked by AcTub. Error bars represent SEM from 150 cells counted in n = 3 experiments. (C) Quantification of the Rab23 knockdown effect on the fraction of D1R (Flag)-positive cilia. Error bars represent SEM from n = 3 experiments with 50 cells counted per experiment. (D) Quantification of the Rab23 knockdown effect on average fold-enrichment of D1R (Flag) signal on cilia. Error bars represent SEM from n = 3 independent experiments with 20–30 cilia analyzed per experiment. (E) Schematic representation of wild-type D1R and the cilia-targeted DOR-D1(338–446) chimeric mutant receptor (duplicated from Figure 3G). (F) Quantification of the Rab23 knockdown effect on average fold-enrichment of Flag-D1R (D1R) and the Flag-tagged chimeric mutant receptor (DOR-D1(338–446)) on cilia of transiently transfected cells. Error bars represent SEM from n = 3 independent experiments with 10–20 cilia analyzed per experiment. (G) Representative images of cilia on cells stably transfected with SSTR3-GFP and transiently transfected with a non-silencing duplex (control) or siRNA targeting Rab23 (Rab23-4 and Rab23-2). Rab23 knockdown strongly reduced SSTR3-GFP localization to cilia. (H) Quantification of the Rab23 knockdown effect on the fraction of SSTR3-GFP (GFP) positive cilia. Error bars represent SEM from n = 3 experiments with 50 cells counted per experiment. (I) Quantification of the Rab23 knockdown effect on average fold-enrichment of SSTR3 (GFP) signal on cilia. Error bars represent SEM from n = 3 experiments with 10–20 cilia analyzed in each experiment. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.029
Figure 6—figure supplement 1
Whole-cell images for corresponding images shown in Figure 6A.

The merged images display Flag-D1R immunoreactivity in green and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.030
Figure 6—figure supplement 2
Verification of Rab23 knockdown.

Results of qRT-PCR analysis. Error bars represent SEM for n = 3 experiments. (**) p < 0.01.

https://doi.org/10.7554/eLife.06996.031
Figure 6—figure supplement 3
Rab23 knockdown has little effect on overall surface receptor expression.

Results of flow cytometric analysis. Results are normalized to control siRNA. Bars represent mean normalized surface expression.

https://doi.org/10.7554/eLife.06996.032
Figure 6—figure supplement 4
Whole-cell images for corresponding images shown in Figure 6G.

The merged images display SSTR3-GFP in green and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.033
Figure 7 with 5 supplements
Rab23 is sufficient to drive ciliary localization of a non-ciliary GPCR.

(A) Schematic representation of the D1R-derived constructs examined. D1R-derived sequence is depicted in red and Rab23 sequence in blue. Flag-tagged wild-type D1R was compared to the ciliary targeting-impaired mutant D1R (D1Δ381-395), and to the ciliary targeting-impaired mutant D1R fused to wild-type Rab23 (D1Δ381-395-Rab23), inactive mutant Rab23 (D1Δ381-395-Rab23-S23N) or activated mutant Rab23 (D1Δ381-395-Rab23-Q68L). (B) Representative images of cilia on cells transiently expressing Flag-tagged versions of the indicated receptor constructs. Fusion of either wild-type or activated Rab23 to the cilia targeting-defective D1R visibly enhanced ciliary localization of receptors. (C) Quantification of the fraction of receptor (Flag)-positive cilia. (D) Average fold-enrichment of receptor (Flag) on cilia. (E) Representative live-cell images of GFP-tagged Rab23 or Rab23-Q68L localization relative to cilia marked by Arl13b-mRuby after transient co-transfection. (F) Schematic representation of the DOR-Rab fusions. DOR-derived sequence is depicted in green, Rab23 in blue, Rab11 in violet, and Rab8 in orange. (G) Representative images of cilia on cells transiently expressing Flag-tagged versions of the indicated receptor constructs. Wild-type DOR was not detected on cilia. Fusion of activated (Q68L) Rab23 produced strong ciliary localization, while fusion of activated (Q70L) Rab11 or activated (Q67L) Rab8 failed to do so. (H) Fraction of receptor (Flag)-positive cilia. (I) Average fold-enrichment of receptor (Flag) signal on cilia. Error bars represent SEM from n = 3 independent experiments with 10–20 cilia analyzed in each experiment. (**) p < 0.01; (***) p < 0.001. Scale bars, 5 μm. GPCR, G protein-coupled receptor.

https://doi.org/10.7554/eLife.06996.034
Figure 7—figure supplement 1
Whole-cell images corresponding to images shown in Figure 7B.

The merged images display Flag-receptor immunoreactivity in green and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.035
Figure 7—figure supplement 2
Overall surface expression of D1R-Rab23 fusion constructs.

Results of flow cytometric analysis. Bars represent mean normalized to Flag-D1R surface expression. (**) p < 0.01.

https://doi.org/10.7554/eLife.06996.036
Figure 7—figure supplement 3
Whole-cell images corresponding to images shown in Figure 7G.

The merged images display Flag-receptor immunoreactivity in green and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.037
Figure 7—figure supplement 4
Overall surface expression of DOR-Rab23 fusion.

Results of flow cytometric analysis. Bars represent mean normalized to Flag-DOR surface expression. (**) p < 0.01.

https://doi.org/10.7554/eLife.06996.038
Figure 7—figure supplement 5
Rab23 is sufficient to drive ciliary localization of several non-ciliary GPCRs.

Both mu-opioid receptor (MOR) and B2AR were fused to activated mutant Rab23 (MOR-Rab23-Q68L, B2AR-Rab23-Q68L). Representative images of cells transiently expressing Flag-tagged versions of the indicated constructs are shown. Wild-type MOR and B2AR were not detected on cilia, while fusion of MOR and B2AR to activated (Q68L) Rab23 produced strong chimeric receptor ciliary localization. The merged images display Flag-receptor immunoreactivity in green and AcTub in red. Insets show a cropped region of the plasma membrane containing the cilium. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. GPCR, G protein-coupled receptor.

https://doi.org/10.7554/eLife.06996.039
Figure 8 with 2 supplements
Evidence IFT-B, KIF17, and Rab23 function in an integrated ciliary delivery pathway.

(A) Representative images showing the effect of motor domain-mutant KIF17 (+KIF17-G234A) on Flag-tagged wild-type D1R localization to the cilium (from Figure 5B). (B) Representative images from an identical experiment examining localization of the Flag-tagged D1R fusion to activated Rab23 (D1Δ381-395-Rab23-Q68L). Scale bars, 5 μm. (C) Average fold-enrichment of D1R and D1Δ381-395-Rab23-Q68L (Flag) signal on cilia. Wild-type D1R localization to cilia was strongly reduced by motor-defective KIF17, but direct Rab23 fusion effectively bypassed this requirement. (D) Effect of IFT172 knockdown on average fold-enrichment of D1R and D1Δ381-395-Rab23-Q68L (Flag) signal on cilia. Wild-type D1R localization to cilia was strongly reduced by IFT172 knockdown, but direct Rab23 fusion effectively bypassed this requirement. Error bars represent SEM from n = 3 independent experiments with 10–20 cilia analyzed in each experiment. (***) p < 0.001. (E) Co-immunoprecipitation analysis showing that Rab23 is not necessary for D1R association with IFT57. The analysis and presentation of data are described in Figure 4E. (F) Immunoblots from multiple experiments were quantified in the linear range to estimate the amount of IFT57 co-IPed. The result is expressed as a fold-increase over the control siRNA condition. Error bars represent SD from n = 3 experiments.

https://doi.org/10.7554/eLife.06996.040
Figure 8—figure supplement 1
Whole-cell images corresponding to the images shown in Figure 8B.

HA-KIF17-G234A expression is verified by HA immunoreactivity. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm.

https://doi.org/10.7554/eLife.06996.041
Figure 8—figure supplement 2
Disruption of KIF17 motor activity does not affect Rab23 ciliary localization.

Representative live-cell images of cells co-transfected with Flag-Rab23-Q68L, Arl13b-YFP as a cilia marker, and either empty vector (+pcDNA) or motor domain mutant KIF17 (+KIF17-G234A). In merged image, Flag-Rab23-Q68L (Flag) immunoreactivity is shown in red, Arl13b-YFP in green, and HA-KIF17-G234A in blue. Rab23-Q68L was observed in 8/18 cilia in cells expressing pcDNA. Similarly, Rab23-Q68L was clearly visible in 9/15 cilia in cells expressing KIF17-G234A. Scale bar, 5 μm.

https://doi.org/10.7554/eLife.06996.042

Tables

Table 1

siRNA knockdown screen

https://doi.org/10.7554/eLife.06996.018
GeneImplication+/− effect on D1 in cilia
TULP3SSTR3, MCHR1 cilia localization
Bbs4, Bbs2SSTR3, MCHR1 cilia localization
Arf4Rhodopsin localization to rod outer segment
Asap1Rhodopsin localization to rod outer segment
Kif7, Kif27Hedgehog signaling
Vps35Receptor trafficking
Rab15, Rab14, Rab8, Rab11Cilia associated Rabs
Rab4, Rab35Trafficking of receptors
Rab23Hedgehog signaling+
Arl6Cilia associated small GTPase
IFT57Intraflagellar transport+
IFT172Intraflagellar transport+
Clathrin heavy chainReceptor endocytosis
Pacs1Olfactory CNG channel cilia localization
Kif5cApical trafficking of cargo
Septin2Cilia diffusion barrier

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