Primary Cilia

1 Dopamine receptors reveal an essential role of IFT-B, 2 KIF17, and Rab23 in delivering specific receptors to 3 primary cilia. 4 5 6 7 1,2 1,3,4 8 Alison Leaf and Mark von Zastrow 9 1 2 3 10 Program in Cell Biology, Department of Biochemistry and Biophysics, Department of 4 11 Psychiatry, and Department of Cellular and Molecular Pharmacology, University of 12 California, San Francisco, CA 94158 13 14 15 16 17 18 Running Title: Ciliary targeting of dopamine receptors 19 20 21 22 Address correspondence to: Mark von Zastrow, MC 2140, 600 16th Street, 23 San Francisco, CA 94158-2140. Fax: 415-514-0169 E-mail: [email protected] 24 25 26 Keywords: Cilia, sorting, G protein-coupled receptor, Rab23, signaling, KIF17, 27 intraflagellar transport 28 29 30 31 32

33 Abstract 34 35 Appropriate physiological signaling by primary cilia depends on the specific targeting of 36 particular receptors to the ciliary membrane, but how this occurs remains poorly 37 understood. Here we show that D1-type dopaminergic receptors are delivered to cilia 38 from the extra-ciliary plasma membrane by a mechanism requiring the receptor 39 cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. 40 This targeting mechanism critically depends on Rab23, a small GTP-binding protein that 41 has important effects on physiological signaling from cilia but was not known previously 42 to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine 43 receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non- 44 ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the 45 ciliary membrane. Dopamine receptors thus reveal a previously unrecognized 46 mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this 47 process.

48 Introduction 49 50 Primary cilia are microtubule-based protrusions of the plasma membrane that support a

51 wide range of specialized receptor-mediated signaling functions. Physiological signaling

52 from cilia critically depends on the selectivity of receptor targeting to the ciliary

53 membrane, and disturbances in this targeting are thought to underlie a variety of

54 pathological conditions (Hsiao et al., 2012). The remarkable specificity of ciliary

55 membrane targeting is clear among G protein-coupled receptors (GPCRs). Some

56 members of this large receptor family robustly accumulate in the ciliary membrane while

57 others, including closely related homologues, are found throughout the extra-ciliary

58 plasma membrane but are effectively excluded from cilia (Marley and von Zastrow,

59 2010; Schulz et al., 2000). Understanding how particular GPCRs localize to primary cilia

60 with such exquisite selectivity is a fundamental problem with broad physiological

61 significance (Emmer et al., 2010).

63 The ciliary membrane compartment is separated from the surrounding extra-ciliary

64 plasma membrane by a transition zone complex that impedes lateral exchange of

65 membrane proteins (Chih et al., 2011; Gilula and Satir, 1972; Hu et al., 2010; Williams

66 et al., 2011). This can explain how GPCRs are retained in cilia once delivered, but not

67 how they are delivered in the first place. Two basic routes of ciliary membrane delivery

68 have been described: First, receptors can originate from an intracellular source, through

69 fusion of post-Golgi transport vesicles with the ciliary membrane in or near the transition

70 zone. A number of membrane proteins are targeted to cilia by this route, and molecular

71 machineries supporting it have been identified (Deretic and Papermaster, 1991; Geng et

72 al., 2006; Mazelova et al., 2009). Second, receptors can originate from the extra-ciliary

73 plasma membrane. This route, first described in a seminal study of flagellar agglutinins

74 in Chalmydomonas (Hunnicutt et al., 1990), contributes to ciliary targeting of the atypical

75 seven-transmembrane protein Smoothened (Smo; Milenkovic et al., 2009) in

76 mammalian cells. Is the lateral delivery route relevant to ciliary localization of

77 conventional GPCRs?

79 Molecular mechanisms that underlie specific ciliary delivery pathways also remain

80 incompletely understood. A number of proteins are already known to play a role,

81 including the BBSome (Jin et al., 2010; Nachury et al., 2007; Berbari et al., 2008b),

82 Tulp3 (Mukhopadhyay et al., 2010, 2013), Arf4 (Deretic et al., 2005), ASAP1 (Wang et

83 al., 2012) and IFT-B and IFT-A (Mukhopadhyay et al., 2010; Crouse et al., 2014; Keady

84 et al., 2011; Kuzhandaivel et al., 2014; Keady et al., 2012). Are there additional

85 machineries not yet identified that function in targeting specific GPCRs to cilia?

87 We addressed these questions through study of the D1-type dopamine receptor (D1R),

88 a conventional GPCR that robustly localizes to cilia in diverse cell types (Domire et al.,

89 2011; Marley and von Zastrow, 2010). Here we show that D1Rs are delivered to the

90 cilium from the extra-ciliary plasma membrane. Further, we show that ciliary D1R

91 cytoplasmic tail is both necessary and sufficient to direct receptor targeting to the ciliary

92 membrane, and this requires a distinct set of cellular proteins including the anterograde

93 intraflagellar transport (IFT-B) complex and ciliary kinesin, KIF17. Moreover, we identify

94 an essential role of the small GTP-binding protein, Rab23, in the ciliary targeting

95 mechanism. Rab23 is not only necessary for D1R access to cilia, it is also sufficient to

96 drive strong ciliary localization of a non-ciliary GPCR. D1Rs thus reveal a discrete route

97 and mechanism of ciliary GPCR targeting, in which Rab23 plays an unprecedented and

98 essential role.

100 Results 101

102 D1Rs are robustly targeted to the primary cilium

103 The D1R is a cilia-localized GPCR whose mechanism of targeting to the cilium is poorly

104 understood (Marley and von Zastrow, 2010; Domire et al., 2011; Zhang et al., 2013). We

105 investigated this question using recombinant receptors expressed in inner medullary

106 collecting duct (IMCD3) cells. Using an N-terminal Flag tag on the D1R to label the

107 overall surface pool, D1Rs were visualized throughout the plasma membrane and highly

108 enriched in cilia marked by acetylated tubulin (AcTub) (Figure 1A), like the cilia-localized

109 somatostatin-3 receptor (SSTR3) (Figure 1B; Berbari et al., 2008a; Händel et al., 1999;

110 Schulz et al., 2000). In contrast, the delta opioid peptide receptor (DOP-R or DOR)

111 localized throughout the extra-ciliary plasma membrane, but was not detectable on cilia

112 (Figure 1C).

113

114 We first quantified ciliary localization by counting the number of receptor-expressing

115 cells with visible receptor immunoreactivity on the cilium. This normative metric verified

116 ubiquitous D1R localization to cilia, similar to SSTR3, and high specificity of ciliary

117 localization relative to DOR (Figure 1D). Second, because cilia scored as receptor-

118 positive varied in degree of apparent receptor concentration, we determined average

119 fold-enrichment of receptors on the cilium relative to the extra-ciliary plasma membrane

120 (Figure 1E). This graded metric further verified robust ciliary localization of the D1R and

121 SSTR3 (but not DOR), and indicated that the D1R is enriched on cilia even more

122 strongly than the SSTR3 (Figure 1F).

123 D1Rs are mobile in the ciliary membrane and accumulated by continuous delivery

124 from the extra-ciliary plasma membrane pool

125 In principle, D1Rs could be concentrated on cilia relative to the extra-ciliary plasma

126 membrane by immobilization or by a diffusion barrier at the base of the cilium (Francis

127 et al., 2011; Hu et al., 2010; Huang et al., 2007). To distinguish these possibilities, we

128 fused the D1R to photoactivatable GFP (Flag-D1-PAGFP) and investigated mobility by

129 live cell imaging coupled to local photoactivation (Figure 2A). We verified appropriate

130 ciliary localization of engineered receptors by anti-Flag Alexa555 surface labeling

131 (Figure 2B, top row). Locally photoactivated D1Rs were distributed non-uniformly on

132 cilia immediately after the 405 nm illumination pulse and then equilibrated throughout

133 the cilium within seconds (Figure 2B,C; whole cell images shown in Figure 2—figure

134 supplement 1). However, there was not visible spread of labeled receptors outside of

135 the cilium on this time scale. Consistent with this, total PA-GFP fluorescence intensity

136 integrated over the ciliary length remained unchanged throughout an 80 sec interval

137 after local photoactivation (Figure 2D). Further, a robust fluorescence signal

138 representing photoactivated D1Rs was still visible when the same cilia were re-imaged

139 minutes thereafter (Figure 2E; whole cell images shown in Figure 2—figure supplement

140 2). Together, these observations indicate that ciliary D1Rs are laterally mobile in the

141 ciliary membrane compartment, but restricted from freely diffusing into the extra-ciliary

142 plasma membrane.

143

144 Achieving net ciliary concentration of receptors that are laterally mobile requires the

145 ability of new receptors to enter the cilium. We sought to measure this delivery by taking

146 advantage of the irreversible nature of PA-GFP photoactivation (Lippincott-Schwartz et

147 al., 2003). Multiple 405 nm light pulses were delivered in rapid succession to

148 photoactivate the majority of D1Rs present in the cilium. We then assessed the effect of

149 administering a subsequent 405 nm pulse at a later time. We reasoned that, because

150 D1Rs delivered from outside the cilium would not have been previously photoactivated,

151 their arrival in the cilium would result in an increment in the ciliary PA-GFP signal elicited

152 by a subsequent 405 nm pulse. This was indeed the case: When a subsequent 405 nm

153 pulse was administered shortly (~ 30 sec) after the initial photoactivation series, little

154 (~15%) increase of ciliary PA-GFP fluorescence was observed, consistent with a small

155 residual fraction of D1Rs escaping photoactivation in the initial pulse series (Figure 2F,

156 left bar). However, when the subsequent 405 nm pulse was administered 30 minutes

157 after the initial series, the increment of ciliary PA-GFP fluorescence was markedly

158 increased (Figure 2F, right bar). Additionally, the degree of the PA-GFP fluorescence

159 increment increased with time (Figure 2—figure supplement 3), and we verified that the

160 PA-GFP fluorescence increment was negligible when measured in fixed cells (Figure

161 2—figure supplement 4). These results directly verify active delivery of D1Rs to the

162 ciliary membrane compartment and provide a rough estimate of the rate of this delivery,

163 on the order of ~1% of the total ciliary D1R pool per min.

164

165 D1Rs delivered to the cilium could originate from an internal vesicular pool or from the

166 extra-ciliary plasma membrane (Deretic and Papermaster, 1991; Milenkovic et al., 2009;

167 Wang et al., 2009). To distinguish these possibilities, we further elaborated the

168 sequential photoactivation technique by taking advantage of dual labeling of surface

169 D1Rs using anti-Flag conjugated to Alexa555, whose fluorescence was not affected by

170 the 405 nm pulses (Figure 2G). If D1R delivery originates from an internal membrane

171 pool we expected 1) unchanged PA-GFP / Alexa555 ratio over the 30 min incubation

172 after initial photoactivation because receptors delivered during this interval would not

173 contribute fluorescence in either channel, and 2) increased PA-GFP / Alexa555 ratio

174 above the initial value after the subsequent photoactivation pulse because newly

175 delivered receptors would contribute PA-GFP but not Alexa555 signal. On the other

176 hand, if ciliary D1R delivery originates from a plasma membrane pool we expected 1)

177 decreased PA-GFP / Alexa555 ratio during the 30 min incubation after initial

178 photoactivation because newly delivered D1Rs would be labeled with Alexa555 but lack

179 PA-GFP signal, and 2) return to the initial value after the subsequent 405 nm pulse

180 because newly delivered D1Rs would then contribute both Alexa555 and PA-GFP

181 signal. We observed precisely the latter result: PA-GFP / Alexa555 decreased by

182 approximately 50% during the 30 min incubation after initial photoactivation, and

183 returned to a value close to the initial ratio after the subsequent photoactivation pulse

184 (Figure 2H; example images from separate fluorescence channels shown in Figure 2—

185 figure supplement 5). These observations indicate that D1Rs are delivered to the ciliary

186 membrane compartment primarily from the extra-ciliary plasma membrane pool.

187 Supporting the validity of this fluorescence ratio determination, bleaching of both

188 fluorophores was negligible after sequential image acquisitions exceeding the number

189 required for this experiment (Figure 2—figure supplement 6), and there was negligible

190 bleed-through from the 488 channel into the 555 channel (Figure 2—figure supplement

191 7).

192

193 Ciliary targeting of D1Rs is directed by the receptor’s cytoplasmic tail

194 To begin to explore the biochemical mechanism of D1R ciliary targeting, we searched

195 for structural determinants within the receptor that are required for ciliary localization.

196 Previous studies of other cilia-localized receptors have identified targeting determinants

197 located either in a cytoplasmic loop (Berbari et al., 2008a) or the cytoplasmic tail (C-tail;

198 Corbit et al., 2005; Deretic et al., 1998; Geng et al., 2006; Jenkins et al., 2006).

199 Progressive truncation of the D1R C-tail (Figure 3A) strongly reduced ciliary localization

200 of receptors. Truncating the distal end had no effect (e.g. D1-415T; Figure 3B) but

201 removing a larger portion strongly reduced ciliary receptor localization (D1-382T; Figure

202 3C). A 15-residue sequence within this region markedly reduced D1R ciliary localization

203 when selectively deleted (D1∆381-395; Figure 3D; whole cell images for Figure 3B-D

204 are shown in Figure 3—figure supplement 1). This effect was verified by both metrics of

205 ciliary receptor targeting (Figure 3E,F), and the deletion did not disrupt overall surface

206 expression of receptors (Figure 3—figure supplement 2).

207

208 We next asked if the D1R C-tail is sufficient to confer ciliary localization on a non-ciliary

209 GPCR. Fusion of the entire D1R C-tail to DOR (Figure 3G) conferred robust ciliary

210 localization. However, fusion of a shorter fragment of the D1R C-tail was not sufficient to

211 confer ciliary localization (Figure 3H-J; whole cell images for Figure 3H shown in Figure

212 3—figure supplement 3), even though it contained the necessary 381-395 sequence,

213 and chimeras exhibited similar levels of overall surface expression (Figure 3 -figure

214 supplement 4). Additionally, we found that fusion of a smaller region of the D1R C-tail

215 that also contains residues 381-395 (Flag-DOR-D1(379-400) was not sufficient to confer

216 ciliary localization on DOR. 23 out of 23 cells expressing the chimeric receptor had no

217 visible enrichment of receptor in the cilium (Figure 3—figure supplement 5). Together,

218 these results indicate that the structural information required for D1R ciliary targeting is

219 contained in the receptor's C-tail and requires residues 381-395 for full activity.

220 However, the targeting determinant is clearly not restricted to this 15-residue sequence,

221 and it likely represents a more extended structure including residues in the proximal C-

222 tail.

223

224 Ciliary targeting of D1Rs is promoted by IFT-B complex proteins and KIF17

225 We next pursued a candidate-based RNA interference screen to search for trans-acting

226 proteins required for ciliary D1R targeting (Table I). Duplexes were transfected

227 individually into IMCD3 cells stably expressing the Flag-D1R, using a clone with

228 particularly strong ciliary receptor accumulation, and ciliary D1R localization was scored

229 visually. No effect was found for siRNAs targeting several proteins implicated in ciliary

230 localization other GPCRs, including TULP3 and BBSome components important for

231 ciliary localization of SSTR3, MCHR1, and Gpr161 (Table I; Berbari et al., 2008b; Jin et

232 al., 2010; Mukhopadhyay et al., 2010, 2013). Instead, two components of the

233 intraflagellar transport (IFT) B complex, IFT57 and IFT172, were identified (Figure 4A;

234 whole cell images shown in Figure 4—figure supplement 1) and knockdown was verified

235 by qRT-PCR (Figure 4 -figure supplement 2). IFT-B knockdown was complicated by

236 reduced ciliogenesis (Figure 4B), but in the ciliated cells remaining in the transfected

237 cell population, reduced D1R targeting was clearly evident. This was verified by both

238 quantitative metrics of ciliary D1R localization, and this was specific to ciliary targeting

239 because knockdown produced little or no effect on the overall surface expression of

240 receptors (Figure 4C,D; Figure 4 -figure supplement 3). As IFT57 tolerates an N-

241 terminal epitope tag while IFT172 does not (Follit et al., 2009), we tested rescue of the

242 IFT57 knockdown effect using an N-terminally HA-tagged IFT57 construct engineered

243 with silent mutations in the sequence targeted by IFT57 siRNA (HA-IFT57-NTM).

244 Verifying a specific requirement for IFT57 in ciliary D1R targeting, HA-IFT57-NTM

245 restored normal ciliary D1R localization in 30 out of 30 cells analyzed (Figure 4A,

246 rescue; whole cell images verifying HA-IFT57-NTM expression in Figure 4—figure

247 supplement 1).

248

249 Co-immunoprecipitation analysis detected physical association of HA-IFT57 with Flag-

250 D1R, and this was specific because HA-IFT57 did not detectably co-IP with Flag-DOR

251 even when the latter was expressed at higher levels than Flag-D1R (Figure 4E). To ask

252 if IFT57 interaction with D1R is directly congruent with ciliary targeting activity, we

253 carried out co-immunoprecipitation analysis comparing HA-IFT57 pull-down with wild

254 type Flag-D1R and FlagD1Δ381-395. Deletion of resides 381-395 of the D1R C-tail, a

255 mutation that profoundly impairs ciliary targeting, did not reduce IFT57 co-IP. To the

256 contrary, the co-IP signal was significantly enhanced (Figure 4F-G). This suggests that

257 the D1R C-tail, while both necessary and sufficient for ciliary receptor targeting, likely

258 functions in a more complex manner than can be explained by a single interaction

259 surface with IFT-B.

260

261 IFT-B is known to associate with KIF3A (kinesin-II) and KIF17, plus end-directed

262 kinesins that mediate anterograde cargo movement toward or within cilia (reviewed in

263 Pedersen & Rosenbaum, 2008). KIF3A is required for delivery of axonemal components

264 and overall ciliogenesis, while KIF17 is not essential for ciliogenesis and is proposed to

265 have more specialized cargo transport functions (Jenkins et al., 2006; Zhao et al.,

266 2012). We verified the presence of KIF17 - IFT57 complexes in IMCD3 cells, as

267 reported previously by others (Howard et al., 2013; Insinna et al., 2008), by co-

268 immunoprecipitation (Figure 5A). Thus, we hypothesized that KIF17 may function as a

269 specific motor supporting D1R delivery to cilia. To test this, we introduced a point

270 mutation in the KIF17 motor domain at a conserved residue in the switch II region

271 (KIF17-G234A) that is essential for kinesin motor activity (Figure 5—figure supplement

272 1; Rice et al., 1999). D1Rs were visible on cilia in essentially all cells expressing the

273 motor-defective HA-KIF17-G234A, but the degree of ciliary receptor enrichment was

274 greatly reduced (Figure 5B). In contrast, expression of wild-type KIF17 did not visibly

275 affect D1R ciliary localization (Figure 5B; whole cell images verifying HA-KIF17-G234A

276 and HA-KIF17 expression shown in Figure 5—figure supplement 2). Verifying this,

277 KIF17-G234A, but not KIF17, selectively reduced the fold-enrichment metric while

278 having little effect on the fraction of receptor-positive cilia (Figure 5C,D). As an

279 independent assessment of the role of KIF17 on D1R ciliary localization, we examined

280 the effect of expressing a different dominant negative KIF17 construct (HA-KIF17-DN)

281 containing only the cargo-binding domain (Jenkins et al., 2006). Expression of HA-

282 KIF17-DN also significantly reduced D1R ciliary enrichment (Figure 5—figure

283 supplement 3). Further, this effect was specific to D1Rs because KIF17-G234A did not

284 reduce ciliary enrichment of SSTR3 (Figure 5E,F; whole cell images verifying HA-

285 KIF17-G234A expression in Figure 5—figure supplement 4). Expression of HA-KIF17-

286 G234A did not significantly affect overall surface expression of either D1R or SSTR3

287 (Figure 5—figure supplement 5)

288

289 A discrete and essential function of Rab23 in the ciliary targeting mechanism

290 Our siRNA screen also identified Rab23 as a candidate whose knockdown caused a

291 pronounced reduction of D1R localization to cilia (Figure 6A; whole cell images in Figure

292 6—figure supplement 1) without affecting ciliogenesis (Figure 6B). Knockdown was

293 verified by qRT-PCR (Figure 6 -figure supplement 2). Rab23 knockdown strongly

294 reduced both quantitative metrics of ciliary D1R targeting, without affecting overall

295 surface expression of receptors (Figure 6C,D; Figure 6 -figure supplement 3). Rab23

296 knockdown also blocked ciliary targeting activity of the D1R C-tail assessed through

297 fusion to the normally cilia-excluded DOR (Figure 6E,F). This was unexpected because

298 Rab23 was not known previously to be required for ciliary targeting of any signaling

299 receptor or other membrane cargo.

300

301 To ask if Rab23 affects additional receptor cargoes, we carried out the same experiment

302 investigating ciliary localization of SSTR3. Duplexes were transfected individually into

303 IMCD3 cells stably expressing SSTR3-GFP. Rab23 knockdown significantly reduced

304 ciliary targeting of SSTR3, as assessed by epifluorescence microscopy and both

305 quantitative metrics (Figure 6G-I; whole cell images shown in Figure 6—figure

306 supplement 4).

307

308 Rab23 was not only necessary for ciliary localization of wild type D1Rs and the ciliary

309 targeting activity of the D1R C-tail, but it was also sufficient to rescue ciliary targeting of

310 targeting-defective D1Rs when fused to the C-tail. Fusing wild type Rab23 to the C-tail

311 of the ciliary localization-defective D1Δ381-395 mutant receptor ((D1Δ381-395-Rab23,

312 Figure 7A) rescued robust ciliary targeting (Figure 7B; whole cell images shown in

313 Figure 7—figure supplement 1). This effect was dependent on the nucleotide state of

314 Rab23 because a GTP binding-defective mutant allele (D1Δ381-395-Rab23-S23N)

315 failed to produce detectable ciliary receptor localization, while fusion of an activated

316 Rab23 allele (D1Δ381-395-Rab23-Q68L) drove ciliary localization even more robustly

317 than the D1R C-tail itself (Figure 7B – D; overall surface expression is shown in Figure 7

318 -figure supplement 2). Rab23 was detectable but not highly concentrated in cilia (Figure

319 7E). Therefore we do not think Rab23 fusion confers ciliary localization on receptors by

320 simple tethering.

321

322 To ask if Rab23 fusion is fully sufficient to promote ciliary targeting of receptors, we

323 carried out similar experiments fusing Rab23-Q68L to DOR, which is normally

324 undetectable on cilia. Remarkably, activated Rab23 drove robust ciliary localization of

325 DOR (DOR-Rab23-Q68L, Figure 7 F-I; whole cell images are shown in Figure 7—figure

326 supplement 3). While fusion of Rab23-Q68L to the mutant D1R increased overall

327 receptor surface expression (Figure 7 -figure supplement 2), the opposite was observed

328 for fusion to DOR (Figure 7 -figure supplement 4). This indicates that the ciliary targeting

329 activity of Rab23 is separable from its effects on overall surface receptor expression.

330 Moreover, this ciliary targeting activity was specific for Rab23 because fusion of an

331 activated allele of the closely related Rab paralogue, Rab11 (DOR-Rab11-Q70L), or of

332 Rab8 (DOR-Rab8-Q67L) that is known to localize to cilia, failed to drive detectable

333 ciliary targeting (Figure 7 F,G). The ability of activated Rab23 to confer ciliary

334 localization on a non-ciliary GPCR was not limited to DOR. Fusion of activated Rab23 to

335 the C-tails of the mu-opioid receptor (MOR) and beta-2-adrenergic receptor (B2AR),

336 which are normally excluded from cilia, conferred robust ciliary localization on both

337 receptors (Figure 7—figure supplement 5). Together, these results support the

338 hypothesis that Rab23 plays a key role in determining the specificity of ciliary receptor

339 targeting.

340

341 We next sought to investigate if the identified protein components required for D1R

342 ciliary targeting function in an integrated pathway. As noted above, disrupting KIF17

343 motor activity strongly reduced ciliary enrichment of the wild type D1R. In contrast,

344 ciliary enrichment driven by direct fusion of the activated Rab23 to the D1R (D1Δ381-

345 395-Rab23-Q68L) was unaffected by this manipulation (Figure 8A–C; whole cell images

346 verifying HA-KIF17-G234A expression are shown in Figure 8—figure supplement 1).

347 Additionally, full ciliary enrichment of D1Δ381-395-Rab23-Q68L remained in the

348 presence of IFT172 knockdown (Figure 8D), suggesting that fusion to activated Rab23

349 can also override the IFT-B requirement. We also noted that Rab23 knockdown did not

350 prevent or reduce D1R association with IFT-B, as estimated by co-immunoprecipitation

351 of IFT57 (Figure 8E). To the contrary, Rab23 knockdown tended to increase the IFT57-

352 D1R co-IP signal (Figure 8F). In contrast to its clear effect on the ciliary concentration of

353 D1Rs, disrupting KIF17 motor activity did not prevent Rab23 localization to cilia (Figure

354 8—figure supplement 2). Together, these results suggest that IFT-B/KIF17 and Rab23

355 are all required for efficient targeting of D1Rs to cilia and may indeed function in an

356 integrated pathway.

357

358 Discussion

359

360 The physiological signaling functions of primary cilia critically depend on the specificity

361 with which particular receptors are targeted to the ciliary membrane (Corbit et al., 2005;

362 Garcia-Gonzalo and Reiter, 2012). We identified a discrete mechanism of ciliary

363 receptor targeting through study of the D1R.

364

365 Past work has focused primarily on receptor delivery from a post-Golgi membrane

366 source. In contrast, we found that D1Rs are delivered to cilia from the extra-ciliary

367 plasma membrane. We also found that ciliary D1R delivery is directed by the receptor

368 C-tail. However, we were unable to find any sequence in the D1R C-tail conforming to a

369 previously defined ciliary targeting motif (Deretic et al., 1998; Geng et al., 2006; Jenkins

370 et al., 2006; Berbari et al., 2008a). Also, our mutational studies suggest that the

371 structural determinant required for ciliary D1R targeting is a relatively extended

372 structure. We identified a distinct set of trans-acting proteins important for ciliary

373 targeting of D1Rs. We were unable to detect a requirement for TULP3 or BBSome

374 components, although these are essential for ciliary localization of SSTR3 and MCHR1

375 (Berbari et al., 2008b; Jin et al., 2010; Mukhopadhyay et al., 2010). We also did not

376 detect a requirement for Arf4, ASAP1 or Rab11, which are essential for rhodopsin

377 delivery to the rod outer segment (Deretic et al., 2005; Wang et al., 2012). Thus D1Rs

378 add to a growing appreciation that there exist receptor-specific differences in

379 mechanisms of ciliary membrane targeting.

380

381 Nevertheless, some similarities are evident. The present results are consistent with IFT-

382 B functioning in anterograde cargo delivery to the cilium (Garcia-Gonzalo and Reiter,

383 2012; Silverman and Leroux, 2009). Several IFT-B components (IFT20, IFT57 and

384 IFT140) have been implicated in rhodopsin delivery to the rod outer segment (Crouse et

385 al., 2014; Keady et al., 2011; Krock and Perkins, 2008), and IFT172 was recently

386 implicated in Smo localization to primary cilia (Kuzhandaivel et al., 2014). The present

387 finding that IFT57 and IFT172 are required for ciliary D1R targeting provides further

388 evidence that IFT-B contributes to ciliary delivery of select membrane cargoes.

389

390 Our findings are also consistent with KIF17 functioning as an ancillary motor supporting

391 ciliary cargo delivery. KIF17 is necessary for ciliary localization of an olfactory CNG

392 channel (Jenkins et al., 2006) but, to our knowledge, KIF17 has not been shown

393 previously to function in localizing a GPCR to cilia. Our results identify a specific role of

394 KIF17, and of KIF17 motor activity, in promoting ciliary concentration of the D1R but not

395 the SSTR3. Further supporting the proposed role of KIF17 as an anterograde transport

396 motor for D1Rs, we found that D1Rs specifically co-IP IFT57 from intact cells (Figure

397 4E) and verified IFT57 association with KIF17 (Figure 5A; Howard et al., 2013; Insinna

398 et al., 2008). An interesting related observation is that deletion of residues 381-395 in

399 the D1R tail, a manipulation that inhibits D1R ciliary targeting, did not disrupt the D1R-

400 IFT57 interaction, but rather, increased it. This supports the idea that ciliary targeting of

401 D1Rs is likely a complex process.

402

403 A remarkable finding in the present study is that Rab23 is an essential component of the

404 D1R ciliary targeting mechanism. To our knowledge, this represents the first evidence

405 that Rab23 is required for ciliary localization of any signaling receptor or membrane

406 cargo. Previous studies of other cilia-localized membrane proteins, such as polycystin-2

407 and Smo, have not observed such a requirement (Hoffmeister et al., 2011; Boehlke et

408 al., 2010; Eggenschwiler et al., 2006). Thus we think it likely that Rab23’s function in

409 ciliary membrane targeting is specific to a subset of cilia-localized cargoes. We note that

410 ciliary localization of SSTR3 is also sensitive to Rab23 knockdown, even though, in

411 contrast to D1R, its ciliary targeting is insensitive to manipulation of KIF17 motor activity.

412 This provides further support for the existence of receptor-specific differences in the

413 ciliary targeting mechanism.

414

415 Altogether, the present findings support the conclusion that D1Rs are targeted to the

416 cilium from the extra-ciliary plasma membrane through a complex mechanism involving

417 IFT-B, KIF17, and Rab23. Our results support the hypothesis that these components

418 function together in an integrated ciliary delivery pathway and suggest that they have

419 distinguishable functional effects in the delivery pathway. First, disrupting KIF17 motor

420 activity strongly reduced ciliary D1R enrichment without affecting the fraction of receptor

421 positive cilia. This is potentially consistent with KIF17 motor activity promoting cililary

422 D1R concentration, but not being essential for ciliary D1R access. Second, direct fusion

423 of activated Rab23 to the D1R C-tail can promote ciliary targeting in a manner that is

424 apparently insensitive to KIF17 motor activity. Third, Rab23 knockdown did not disrupt

425 the D1R / IFT-B interaction as estimated by co-immunoprecipitation. These results also

426 support the idea that the lateral delivery mechanism is complex, but additional studies

427 are required to further delineate the precise functions of each of these identified

428 components.

429

430 The present results raise a number of interesting questions for future study. First, given

431 that receptor accumulation in the ciliary membrane is dependent on Rab23 nucleotide

432 state, an important next question is how this nucleotide state is controlled. A second

433 question, which may be related to the first, is how selective cargo engagement with the

434 ciliary delivery mechanism is determined. The present data strongly suggest a

435 functional interaction between Rab23 and the D1R ciliary targeting determinant, but we

436 have been unable, so far, to establish direct physical interaction between the D1R and

437 Rab23. It is conceivable that IFT-B links D1Rs and Rab23, or that unidentified additional

438 protein(s) explain the functional interaction observed. A third question is to ascertain

439 precisely how Rab23 directs receptor delivery to the ciliary membrane. Based on

440 precedent of other Rab protein functions, we speculate that there is a specific effector of

441 Rab23 that operates at or near the ciliary diffusion barrier. A fourth question is what

442 broader physiological significance the discrete, Rab23-dependent ciliary targeting

443 mechanism has. We note that a mutation in Rab23 produces excessive Hedgehog

444 signaling in vivo (Eggenschwiler et al., 2001). One possibility is that this reflects a

445 disruption of ciliary signaling normally mediated by a Rab23-dependent receptor. Thus,

446 further investigation of the receptor-specific ciliary targeting mechanism identified here

447 may provide fundamental insight into the role of primary cilia as physiological signaling

448 devices, and toward understanding pathologies associated with ciliary defects.

449 Materials and Methods

450

451 Cell culture and transfection

452 IMCD3 cells (ATCC) were grown in DMEM/Ham’s F-12 Medium supplemented with 10%

453 fetal bovine serum (UCSF Cell Culture Facility, San Francisco, California, USA).

454 Flag-D1R, Flag-DOR, Flag-MOR, and Flag-B2AR constructs were described

455 previously (Vickery and von Zastrow, 1999; Yu et al., 2010; Tanowitz and Von Zastrow,

456 2003). KIF17 and KIF17-DN cDNA (Jenkins et al., 2006) was a gift from Kristen Verhey

457 (University of Michigan, Ann Arbor). 2XHA-KIF17 was created using PCR and ligation

458 into pIRES. HA-KIF17-DN was created using PCR and ligation into pIRES. 2XHA-

459 KIF17-G234A was generated using site-directed mutagenesis (Phusion Site-Directed

460 Mutagenesis Kit, Thermo Scientific). SSTR3-GFP IMCD3 stable cells were a gift from

461 Maxence Nachury (Stanford University). IFT57 cDNA was a gift from Wallace Marshall

462 (UCSF, San Francisco). HA-IFT57 was created using PCR and ligation into pIRES. HA-

463 IFT57-NTM was generated using site directed mutagenesis (Phusion Site-Directed

464 Mutagenesis Kit, Thermo Scientific) of HA-IFT57 to change the site targeted by IFT57-4

465 siRNA using primers 5’-

466 GTCACCCCAGAGTCTGCGATAGGGTTCTACTAAACACGTGGGCTTCC-3’ and 5’-

467 AGCTGCATGCATGTCCCTGGTCATGTTGC-3’.

468 Flag-tagged D1-415T, D1-382T, and D1Δ381-395 were created by site-directed

469 mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific). Receptor

470 chimeras, DOR-D1(338-446), DOR-D1(368-446), and DOR-D1(379-400) were

471 generated using PCR and homology-directed ligation (In-Fusion HD Cloning kit,

472 Clontech). D1 oligos were fused to DOR residue 340. Flag-D1-PAGFP was generated

473 using PCR and ligation into p-PAGFP-N1. Flag-SSTR3 was created using PCR and

474 ligation into pIRES. Rab8a and Arl13b-YFP cDNA were gifts from Jeremy Reiter (UCSF,

475 San Francisco). Rab23, Rab23-S23N, Rab23-Q68L, and Rab11 cDNA were gifts from

476 Keith Mostov (UCSF, San Francisco). Flag-Rab23-Q68L was created using PCR and

477 ligation into pIRES. Receptor chimeras, Flag-D1Δ381-395-Rab23, Flag-D1Δ381-395-

478 Rab23-S23N, Flag-D1Δ381-395-Rab23-Q68L, Flag-DOR-Rab23-Q68L, and Flag-DOR-

479 Rab11-Q70L, Flag-DOR-Rab8-Q67L, Flag-MOR-Rab23-Q68L and Flag-B2AR-Rab23-

480 Q68L, were generated using PCR and homology-directed ligation into pIRES (In-Fusion

481 HD Cloning kit, Clontech) Rab23 constructs were fused to the C-terminal end of

482 D1Δ381-395 with a 2 residue linker. Rab23-Q68L, Rab11-Q70L, and Rab8-Q67L were

483 fused to the C-terminal end of DOR with an 8 residue linker.

484 Transfection of constructs was performed using Lipofectamine 2000 and RNAi-

485 max (Invitrogen) for cDNA or siRNA, respectively, in accordance with manufacturer’s

486 instructions. Stably transfected cell clones expressing Flag-D1R were generated by

487 selecting for neomycin resistance with 500 ug / ml G418 (Geneticin, Invitrogen). Target

488 sequences for knockdown mIFT57 (1: 5’-CAGCAATTGGCTTCTATTAAA-3’, 2: 5’-

489 TACAATGAATATAGTATTTAA-3’), mIFT172 (1: 5’-AAGGAGCATTTACAAGAACAA-3’, 2:

490 5’-CCCACAGAATTTCAACATCTA-3’), mRab23 (1: 5’-AAGATTGGTGTCTTTAATGCA-

491 3’, 2: 5’-TAGCCACTAAATGCATGGTAA-3’), and control (1027281, Qiagen). Duplex

492 RNA (30 pm, Qiagen) was transfected into 40% confluent cells in a 6-well dish 72 h

493 before experimentation.

494

495 Antibodies

496 Antibodies used were rabbit anti-Flag (Sigma), mouse anti-Flag M1 (Sigma), rat anti-HA

497 (Roche Applied Science), and mouse anti-acetylated tubulin (Sigma).

498

499 Coimmunoprecipitation

500 Cells expressing indicated constructs were grown to confluency in 10 cm dishes. 48

501 hours after transfection, cells were lysed in 0.2% Triton X-100, 200 mM NaCl, 50 mM

502 Tris pH 7.4, and 1 mM CaCl2 supplemented with a standard protease inhibitor mixture

503 (Roche Applied Science), and cleared by centrifugation (12000 x g for 10 min). Samples

504 were pre-cleared by incubation with mouse IgG agarose (Sigma) at 4 degrees C for 30

505 minutes. Samples were incubated with anti-Flag M2 affinity gel IgG (Sigma) at 4

506 degrees C for 1 h, washed with lysis buffer 5 times and incubated with SDS sample

507 buffer (Invitrogen) supplemented with DTT to elute proteins. Western immunoblot

508 analysis was performed using rat anti-HA-peroxidase (Roche) or rabbit anti-Flag

509 (Sigma). Immunoprecipitation signals were quantified by scanning densitometry of films

510 exposed in the linear range. Linearity was verified by generating a standard curve using

511 a dilution series of the indicated sample.

512

513 Fixed cell microscopy

514 Cells were transfected with the indicated construct(s) and then plated on glass

515 coverslips 24 hours later. Cells were grown to confluency to induce ciliation over 24

516 hours and then fixed. Surface Flag-tagged receptors were labeled by addition of rabbit

517 anti-Flag antibody (1:500; Sigma) to the media for 20 minutes at 37 degrees C. Cells

518 were then washed with PBS 2X and fixed by incubation in 4% formaldehyde (Fisher

519 Scientific) diluted in PBS for 15 minutes at room temperature. Cells were permeabilized

520 and blocked in 0.1% Triton X-100 and 3% milk diluted in PBS. Primary labeling of

521 acetylated tubulin and HA was performed with mouse anti-acetylated tubulin (1:1000;

522 Sigma) or rat anti-HA (1:1000; Roche Applied Science) respectively, for 1 hour.

523 Secondary labeling was performed using donkey anti-rabbit Alexa Fluor 488 (1:1000;

524 Invitrogen), goat anti-mouse Alexa Fluor 594 (1:1000; Invitrogen) and goat anti-rat Alexa

525 Fluor 647 (1:1000; Invitrogen). Specimens were mounted using ProLong Gold antifade

526 reagent (Life Technologies). Fixed cells were imaged by epifluorescence microscopy

527 using a Nikon inverted microscope, 60X NA 1.4 objective (Nikon), mercury arc lamp

528 illumination and standard dichroic filter sets (Chroma).

529

530 Live cell microscopy

531 Cells were imaged at 37 degrees C in Dulbecco’s Modified Eagle Medium, D-MEM

532 Glucose (DME H-21), w/o Phenol Red supplemented with 30 mM Hepes. Surface Flag-

533 D1-PAGFP receptors were labeled by addition of mouse anti-Flag M1 antibody (1:500;

534 Sigma) conjugated to Alexa Fluor 555. Cells were imaged on a spinning disk confocal

535 microscope (Nikon TE-2000 with Yokogawa confocal scanner unit CSU22) using a 100X

536 NA 1.45 objective. To photoactivate Flag-D1-PAGFP specifically in the cilium, 405 nm

537 laser illumination was directed through a second light path via a single mode fiber and

538 focused in the image plane. Photoactivation was achieved by delivering brief (100 ms)

539 pulses of 405 nm illumination.

540

541 Microscope Image Acquisition

542 For epifluorescence microscopy of fixed cells, images were acquired using a cooled

543 CCD camera (Princeton Instruments MicroMax) with illumination and exposure times

544 adjusted to remain in the linear range of the camera. For spinning disc confocal

545 microscopy of live cells, images were collected at 37 degrees C using an electron

546 multiplying CCD camera (Andor iXon 897) operated in the linear range. Images were

547 processed at full bit depth for all analysis, and rendered for display by converting to 8 bit

548 format using ImageJ software (http://imagej.nih.gov/ij/) and a linear lookup table.

549 To measure the receptor fluorescence in the cilium, an ROI was manually created by

550 outlining the cilium in the image. To correct for background fluorescence, the ROI was

551 moved to a region outside the cell to measure representative fluorescence. This value

552 was subtracted from the ciliary fluorescence. To measure the D1-PAGFP diffusion in

553 cilium, the total PAGFP fluorescence was measured and normalized to the Flag-555

554 label to account for accumulation of receptor or focal plane (orientation) of the cilium.

555

556 To estimate lateral mobility of receptors in the cilium, the 405 nm laser spot was

557 positioned so that it illuminated the center of the cilium but not the ends. A single

558 photoactivation pulse was delivered with continuous confocal imaging at 0.5 Hz to

559 monitor changes in the distribution of photoactivated Flag-D1-PAGFP in the cilium over

560 time. To estimate new receptor delivery to the cilium, three 405 nm pulses were

561 delivered over 10 sec, determined empirically to photoactivate the majority of Flag-D1-

562 PAGFP present in the cilium. We then acquired a subsequent GFP image, delivered

563 another (single) 405 nm pulse, and acquired the GFP image again. This sequential

564 ”image-photoactivate-image” sequence was applied either immediately (approximately

565 30 sec) after the initial 405 nm pulse series or 30 min after. New delivery was estimated

566 by the increment of integrated PA-GFP fluorescence intensity measured after the

567 subsequent 405 nm pulse minus before, normalized to the integrated fluorescence

568 intensity measured after. The anti-Flag Alex555 channel (unaffected by 405 nm pulses)

569 was used to optimize focus on the cilium and to verify that the specimen did not move

570 significantly between the ‘before’ and ‘after’ images. To assess the source of Flag-D1-

571 PAGFP delivery to the cilium we used a similar strategy as described above but

572 quantified in the photoactivation series both PA-GFP and Alexa555 channels and

573 determined their ratio. New receptor delivery from internal relative to plasma membrane

574 sources was distinguished by changes in the PA-GFP / Alexa555 ratio, based on

575 selective labeling with Alexa555 of only the plasma membrane pool, as discussed in

576 text.

577

578 Image analysis and statistical analysis

579 For line scan analysis, a straight line was drawn on the cilium and the PlotProfile tool in

580 ImageJ was used to determine the fluorescence intensity along the line. For integrated

581 fluorescence determinations, the rectangular ROI tool was used. Results are displayed

582 as mean of results from each experiment involving imaging of multiple specimens and

583 cilia (specified in the figure legends). Error bars represent standard error of the mean

584 based on at least n= 3 independent experiments done on different days unless noted

585 otherwise. The statistical significance between conditions was analyzed using Student’s

586 unpaired t-test, calculated using Prism 6.0 software (GraphPad Software, Inc) and

587 applying the Hochberg correction for multiple comparisons. The threshold for

588 significance was p < 0.05 with exact p value ranges indicated in the figure legends.

589

590 Fluorescence Flow Cytometry

591 Surface-accessible Flag immunoreactivity was quantified by fluorescence flow

592 cytometry as described previously (Tsao and Von Zastrow, 2000). Briefly, cells were

593 dissociated from culture dishes, labeled in suspension 4˚C with anti-Flag M1 conjugated

594 to Alexa 647 and analyzed using a FACS-Calibur instrument (Becton Dickenson). In

595 each experiment, mean fluorescence intensity was determined from 10,000 cells, and

596 averaged over triplicate determinations for each construct. For all conditions shown,

597 experiments were performed in triplicate, on separate days and from separate

598 transfections. Error bars represent SEM across the experimental days. For

599 determination of mutant receptor surface expression, transiently transfected cells were

600 analyzed 48 hours after transfection. For evaluation of the effects of siRNA knockdown,

601 stably transfected cells were analyzed 72 hours after transfection with the indicated

602 siRNA duplex.

603

604 Acknowledgments

605 We thank Aaron Marley for assistance at an early stage of this project and for

606 generating the Flag-D1R stable cell line used in this study. We thank Dave Bryant,

607 Wallace Marshall, Jeremy Reiter and Kristen Verhey for valuable advice and generously

608 providing reagents, and Henry Bourne, Roshanak Irannejad, Erik Jonsson, and Braden

609 Lobingier for useful discussion. This work was supported by grants from the US

610 National Institutes of Health (DA 010154, 010711 and 012864). A.E.L. is a recipient of a

611 predoctoral fellowship from the US National Science Foundation.

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749 Figure Legends 750 751 Figure 1. D1Rs specifically localize to primary cilia. (A - C) Representative 752 epifluorescence microscopy images of Flag-D1R (panel A), Flag-SSTR3 (panel B), and 753 Flag-DOR (panel C) localization on the surface of IMCD3 cells. Insets show a cropped 754 region of the plasma membrane containing the cilium, with Flag immunoreactivity 755 marking receptor (top) and acetylated tubulin (AcTub) immunoreactivity marking the 756 cilium (middle). Merged view is at bottom with Flag in green and AcTub in red. Flag-D1R 757 and Flag-SSTR3 localize robustly to cilia while Flag-DOR is detectable in the extra- 758 ciliary plasma membrane but not on cilia. (D) Quantification of ciliary localization by 759 determining the fraction of receptor (Flag) -positive cilia, judged by the presence of Flag 760 immunoreactivity visible by epifluorescence microscopy, and expressed as a 761 percentage of total cilia counted in the transfected cell population. (E) Scheme for 762 quantification of ciliary localization by determining enrichment of receptor (Flag) signal 763 in an ROI containing the cilium, when compared to an adjacent region of the extra- 764 ciliary plasma membrane. Representative ROIs are shown for a Flag-D1R -transfected 765 cell. (F) Fold-enrichment calculated as a ratio of background-subtracted Flag signal 766 present in the ciliary ROI divided by background-subtracted Flag signal present in the 767 adjacent extra-ciliary plasma membrane ROI (cilia / PM). Error bars represent SEM 768 from n = 3 independent experiments, with 10-15 cilia analyzed for each receptor in each 769 experiment. (**) p<0.01; (***) p < 0.001. Scale bars, 5 um. 770 771 Figure 2. D1Rs are mobile in the ciliary membrane and delivered from the extra- 772 ciliary plasma membrane. (A) Schematic for local labeling of D1R in a cilium using PA- 773 GFP. IMCD3 cells expressing Flag-D1-PAGFP were labeled with anti-Flag antibody 774 conjugated to Alexa555 to visualize the overall surface receptor pool. A point-focused 775 405 nm laser spot was used to locally photoactivate receptors on the mid-portion of the 776 cilium. Non-fluorescent PA-GFP is depicted in grey, fluorescent state in green. (B) Live 777 cell confocal images of a representative cilium showing the frame immediately before 778 the photoactivation pulse (left column), and frames acquired 1 sec (middle column) and 779 10 sec (right column) after local photoactivation. The Flag-Alexa555 signal labeling the 32

780 entire surface receptor pool (top row) was present throughout the cilium at all time 781 points. PA-GFP fluorescence representing the photoactivated pool was non-uniformly 782 distributed at 1 sec and uniformly distributed along the cilium within 10 sec. (C) Line 783 scan analysis of PA-GFP fluorescence along the cilium from the example in panel B. (D) 784 Integrated PA-GFP fluorescence signal in the cilium as a function of time after the 405 785 nm laser pulse. The PA-GFP fluorescence at time=0 was set at 100%. Points represent 786 the mean fraction of PA-GFP fluorescence present in the cilium over an 80 sec imaging 787 interval. Error bars represent SD from analysis of n = 6 cilia. There was no detectable 788 loss of ciliary PA-GFP signal quantified over an 80 sec interval. (E) Confocal images of 789 a representative cilium acquired immediately after (0 min) and 10 min after 790 photoactivation, showing that the locally photoactivated receptor pool was largely 791 retained in the cilium even after this longer interval. (F) Assessing new D1R delivery to 792 the cilium by saturation photoactivation and the sequential “image-photoactivate-image” 793 scheme described in the Materials and methods. Bars represent mean fractional 794 increase in ciliary PA-GFP fluorescence elicited by the subsequent test pulse. Error bars 795 represent SD for n= 7 cilia. (G) Schematic for modifying the saturation photoactivation 796 method to assess source of newly delivered D1Rs, based on the ratio of integrated PA- 797 GFP / Alexa555 fluorescence (PA/555) measured in the cilium as a function of time. The 798 initial condition is depicted on the left (‘0 min’) with the fluorescence ratio (PA/555) 799 arbitrarily set to 1. If new receptors enter the cilium from an internal membrane pool 800 during the 30 min incubation period (depicted in center, ‘30 min’), they contribute neither 801 Alexa555 nor PA-GFP signal, so the fluorescence ratio is unchanged from the initial 802 condition (PA/555 = 1). After the subsequent 405 nm test pulse (depicted at right, ‘30 803 min +PA’), the PA-GFP signal increases without any change in Alexa555 signal, 804 elevating the fluorescence ratio above the initial condition (PA/555 > 1). If new receptors 805 enter the cilium from the extra-ciliary plasma membrane pool, they contribute Alexa555 806 but not PA-GFP signal during the 30 min incubation, reducing the fluorescence ratio 807 from the initial condition (PA/555 < 1). The subsequent 405 nm pulse restores the 808 fluorescence ratio to the initial value (PA/555 = 1). (H) Experimental results from the 809 strategy depicted in panel G. Bars represent the mean ratio of integrated PA-GFP /

810 Alexa555 fluorescence measured in the cilium. Error bars represent SD from n = 6 cilia. 811 (***) p < 0.001. Scale bars, 5 um. 812 813 Figure 2—figure supplement 1. Whole cell images corresponding to the images 814 shown in Figure 2B. Flag immunoreactivity is shown in red and PA-GFP in green. 815 Dashed blue line indicates outline of an individual cell. Scale bar, 5 um. 816 817 Figure 2—figure supplement 2. Whole cell images corresponding to the images 818 shown in Figure 2E. Channels are shown individually in grey scale. Dashed blue line 819 indicates outline of an individual cell. Scale bar, 5 um. 820 821 Figure 2—figure supplement 3. New D1R delivery to cilia increases over time. The 822 sequential “image-photoactivate-image” scheme was applied as described in Figure 2F 823 except that the time interval between the initial 405nm pulse series and the subsequent 824 assessment of PA-GFP fluorescence increment was varied from 2 min to 50 min. Each 825 square represents an individual determination. The line indicates a least squares best 826 fit. 827 828 Figure 2—figure supplement 4. Control experiment for the ciliary delivery assay 829 described in Figure 2F. The scheme used in Figure 2F was applied to fixed cells. Error 830 bars represent SD for n= 6 cilia. There was no significant PA-GFP fluorescence 831 increment at either time point. 832 833 Figure 2—figure supplement 5. Images of cilia from ciliary delivery assay. 834 Representative images of a cilium from the “image-photoactivate-image’ scheme 835 described in Figure 2F-G showing the PA-GFP signal and the Flag signal separately in 836 grey scale. Scale bar, 5 um. 837 838 Figure 2—figure supplement 6. Bleaching control for the ciliary delivery assay. Cilia 839 were photoactivated and consecutive PA-GFP and Alexa555 images were acquired.

840 The integrated fluorescence intensity in the cilium was normalized to that measured in 841 the initial image after photoactivation. Error bars represent SD from analysis of n = 3 842 cilia. There was no detectable loss of ciliary PA-GFP signal or ciliary Alexa555 (Flag) 843 signal after 28 consecutive images. 844 845 Figure 2—figure supplement 7. Bleed-through control for the ciliary delivery assay. 846 Representative images of cilia expressing Flag-D1-GFP in the absence and presence of 847 M1-555, which recognizes Flag, showing negligible bleed-through of the GFP signal into 848 the 555 channel. The merged image displays the GFP channel in green and 555 (Flag- 849 receptor) in red. Insets show a cropped region of the plasma membrane containing the 850 cilium. Dashed blue line indicates outline of an individual cell. Scale bar, 5 um. 851 852 Figure 3. The D1R cytoplasmic tail is necessary and sufficient for ciliary receptor 853 targeting. (A) Schematic representation of D1R C-tail mutations used in the present 854 analysis. (B) Representative images of cells expressing Flag-tagged wild type D1R 855 (D1R) or a receptor construct truncated at residue 415 (D1-415T), showing robust ciliary 856 localization of both. The merged image at bottom displays Flag-receptor in green and 857 AcTub in red. (C) Representative image of a receptor construct truncated at residue 382 858 (D1-382T), showing near complete loss of receptor localization to cilia marked by 859 AcTub. (D) Representative images of a D1R construct with internal deletion of residues 860 381 - 395 (D1Δ381-395), showing the range of phenotypes observed, from a complete 861 loss of receptor localization in cilia to a pronounced reduction of ciliary receptor 862 localization. (E) Quantification of the fraction of receptor (Flag) -positive cilia for Flag- 863 tagged wild type (D1R) or mutant (D1Δ381-395) receptor. The analysis is described in 864 Figure 1D. (F) Quantification of the average fold-enrichment of wild type (D1R) or 865 mutant (D1Δ381-395) receptors on cilia. The analysis is described in Figure 1E,F. (G) 866 Schematic representation of chimeric mutant receptors containing portions of the D1R 867 cytoplasmic tail (in red) fused to the DOR cytoplasmic tail (in green). (H) Representative 868 images of cilia in cells expressing Flag-DOR, Flag-DOR-D1(338-446), or Flag-DOR- 869 D1(368-446) showing that the D1R C-tail is sufficient to drive ciliary targeting of chimeric

870 receptors. (I) Fraction of receptor (Flag) -positive cilia. (J) Average fold-enrichment of 871 receptor (Flag) signal on cilia. Error bars represent SEM from n = 3 experiments with 872 10-20 cilia analyzed per experiment. (***) p < 0.001. Scale bars, 5 um. 873 874 Figure 3—figure supplement 1. Whole cell images corresponding to images shown 875 in Figure 3B-D. The merged image displays Flag-receptor immunoreactivity in green 876 and AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bars, 5 877 μm. 878 879 Figure 3—figure supplement 2. Overall surface expression of D1R C-tail mutant. 880 Surface-accessible Flag immunoreactivity was quantified for the cilia-defective mutant 881 D1R (D1∆381-395) relative to wild type D1 dopamine receptor (D1R) by fluorescence 882 flow cytometry, as described in the Materials and methods. The ciliary targeting 883 defective mutant receptor was expressed in the overall plasma membrane at a level 884 indistinguishable from the cilia-localized wild type D1R, supporting the hypothesis that 885 the C-tail region mutated is required specifically for targeting surface receptors to cilia, 886 but not for targeting receptors to the extra-ciliary surface. 887 888 Figure 3—figure supplement 3. Whole cell images corresponding to images shown 889 in Figure 3H. The merged image displays Flag-receptor immunoreactivity in green and 890 AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm. 891 892 Figure 3—figure supplement 4. Overall surface expression of DOR-derived 893 constructs. Surface-accessible Flag immunoreactivity for the indicated DOR-derived 894 chimeric mutant constructs expressed relative to wild type DOR. The chimeric mutant 895 receptors were expressed at similar levels, further supporting the specific function of the 896 D1R-derived sequence in targeting receptors to the ciliary plasma membrane 897 compartment. (*) p <0.05. 898

899 Figure 3—figure supplement 5. The 15 residue sequence required for full ciliary 900 targeting of D1R is not sufficient to confer ciliary localization on DOR. 901 Representative image of a cilium in cells expressing the chimeric mutant receptor Flag- 902 DOR-D1(379-400), which includes the D1R residues 381-395, showing that this 903 sequence is not sufficient to confer ciliary targeting of receptors. The merged image 904 displays Flag-receptor immunoreactivity in green and AcTub in red. Insets show a 905 cropped region of the plasma membrane containing the cilium. Dashed blue line 906 indicates outline of an individual cell. Scale bar, 5 μm. 907 908 Figure 4. IFT-B complex proteins are necessary for D1R localization to cilia. (A) 909 Representative images of cilia on cells stably transfected with Flag-D1R and transiently 910 transfected with a non-silencing duplex (control) or siRNA targeting IFT57 (IFT57-1 and 911 IFT57-4) or IFT172 (IFT172-3 and IFT172-4). In the merged image, Flag-D1R 912 immunoreactivity is shown in green and AcTub in red. Duplexes targeting IFT57 and 913 IFT172 caused a visually obvious reduction in ciliary D1R localization. The right column 914 of images shows the rescue condition, in which cells transfected with siRNA against 915 IFT57 were additionally transfected with IFT57 that is not targetable by the IFT57 siRNA 916 (HA-IFT57-NTM). Scale bar, 5 μm. (B) Effect of siRNAs on the fraction of cells in the 917 population possessing a visible cilium marked by AcTub. Error bars represent SEM from 918 150 cells counted in n = 3 independent experiments. (C) Fraction of D1R (Flag) -positive 919 cilia. Error bars represent SEM for n = 3 experiments with 50 cells counted in each 920 experiment. (D) Average fold-enrichment of D1R (Flag) signal on cilia. Error bars 921 represent SEM for n =3 experiments with 15-40 cilia analyzed per experiment. (E) 922 Association of IFT57 with the D1R but not DOR demonstrated by co- 923 immunoprecipitation. Cells were transfected with the constructs indicated above each 924 lane. Cell extracts were blotted for HA and Flag; HA-IFT57 resolved as a sharp band at 925 its expected apparent molecular mass and Flag-tagged receptors resolved as 926 heterogeneous species consistent with complex glycosylation as shown previously. 927 Specific co-immunoprecipitation is indicated by HA-IFT57 detected in the Flag-D1R pull- 928 down but not in Flag-DOR pull-down. Molecular mass markers (in kDa) are shown on

929 right side of blots. The results in panel E are representative of n = 3 independent 930 experiments. (F) Increased association of IFT57 with D1Δ381-395 relative to D1R 931 demonstrated by co-IP. Cells were transfected with the constructs indicated above each 932 lane. Cell extracts were blotted for HA and Flag. More HA-IFT57 was detected in the 933 Flag-D1Δ381-395 pull-down than the Flag-D1R pull-down. Molecular mass markers (in 934 kDa) are shown on right side of blots. The results in panel E are representative of n = 3 935 independent experiments. (G) Immunoblots from multiple experiments were scanned in 936 the linear range, as described in Materials and methods, to estimate the amount of 937 IFT57 co-IPed with the indicated receptors. Expressed as a fold increase over control 938 where control is D1R. Error bars represent SD from n = 3 experiments. (*) p <0.05; (**) 939 p<0.01; (***) p < 0.001. 940 941 Figure 4—figure supplement 1. Whole cell images corresponding to the images 942 shown in Figure 4A. The merged image displays Flag-D1R immunoreactivity in green 943 and AcTub in red. Expression of HA-IFT57-NTM is verified by HA immunoreactivity. 944 Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. 945 946 Figure 4—figure supplement 2. Verification of IFT-B knockdown. Cells stably 947 expressing Flag-D1R were transfected with either control siRNA or siRNA targeting IFT- 948 B components, IFT57 and IFT172, and RNA levels were measured via qRT-PCR. Error 949 bars represent SEM for n =3 experiments. (**) p<0.01; (***) p < 0.001. 950 951 Figure 4—figure supplement 3. IFT-B knockdown has little effect on overall surface 952 receptor expression. Surface-accessible Flag immunoreactivity representing Flag- 953 D1Rs present in the plasma membrane was quantified by flow cytometry. Results are 954 normalized to control siRNA. 955 956 Figure 5. KIF17 motor activity is required for full D1R enrichment in cilia. (A) 957 Association of IFT57 with KIF17 indicated by co-immunoprecipitation. Cells were 958 transfected with expression constructs indicated at top of the panel and extracts were

959 blotted for HA to detect IFT57 and Flag to detect KIF17. HA-IFT57 resolved as expected 960 and described in Figure 4E. KIF17 resolved as two species with the top band 961 corresponding to the expected molecular mass of the full-length protein. Specific co- 962 immunoprecipitation is indicated by HA-IFT57 detected in the Flag pull-down from cells 963 expressing Flag-KIF17 pull-down (arrow) but not from cells in which Flag-KIF17 was not 964 expressed. Molecular mass markers (in kDa) shown on right. (B) Representative 965 images of cilia on cells co-transfected with Flag-D1R and control empty vector 966 (+pcDNA), a plasmid encoding HA-tagged KIF17 (+KIF17), or a plasmid encoding an 967 HA-tagged KIF17 construct harboring a point mutation in a conserved residue that 968 disrupts kinesin motor activity (+KIF17-G234A). Robust ciliary localization of Flag-D1R 969 was observed in cells expressing control plasmid or the wild type KIF17 construct, but 970 markedly reduced ciliary enrichment of D1Rs was observed in cells expressing motor- 971 defective mutant KIF17. (C) Quantification of the effect of disrupting KIF17 motor activity 972 on the fraction of D1R (Flag) -positive cilia. (D) Quantification of the effect of disrupting 973 KIF17 motor activity on average fold-enrichment of D1R (Flag) on cilia. Disrupting KIF17 974 motor activity strongly reduced the degree of D1R enrichment on the ciliary membrane 975 without blocking D1R access to cilia. (E) Representative images of cilia on cells co- 976 transfected with Flag-SSTR3 and with control empty vector (+pcDNA) or a plasmid 977 encoding motor domain-mutant KIF17 (+KIF17-G234A). Disrupting KIF17 motor activity 978 did not detectably affect Flag-SSTR3 localization to cilia. (F) Quantification of the effect 979 of disrupting KIF17 motor activity on average fold-enrichment of SSTR3 (Flag) on cilia. 980 Disrupting KIF17 motor activity did not detectably affect ciliary enrichment of SSTR3. 981 Error bars represent SEM from n = 3 independent experiments with 10-20 cilia analyzed 982 in each experiment. (***) p < 0.001. Scale bars, 5 μm. 983 984 Figure 5—figure supplement 1. Switch II mutation in KIF17. Amino acid sequence 985 alignment of the switch II regions of Kinesin-1 and KIF17 showing identical sequences. 986 Switch II residues are shown in magenta with a box around the G residue mutated in 987 the motor domain mutant. 988

989 Figure 5—figure supplement 2. Whole cell images corresponding to the images 990 shown in Figure 5B. HA-KIF17-G234A and HA-KIF17 expression is verified by HA 991 immunoreactivity detected throughout the cytoplasm and accumulated in the nucleus. 992 The HA signal detected in cilia is likely contributed in large part by cross reactivity of the 993 secondary anti-rat antibody with the mouse primary antibody recognizing AcTub. 994 Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. 995 996 Figure 5—figure supplement 3. Independent verification of KIF17 requirement for 997 D1R ciliary enrichment. Cells were co-transfected with Flag-D1R and an empty vector 998 (+pcDNA) or a plasmid encoding an HA-tagged version of a previously reported 999 dominant negative KIF17 (+KIF17-DN). Quantification of the effect of KIF17-DN on 1000 average fold-enrichment of D1R (Flag) on cilia. Expression of KIF17-DN strongly 1001 reduced the degree of D1R ciliary enrichment. Error bars represent SEM from n = 4 1002 independent experiments with 10-20 cilia analyzed per experiment. (***) p < 0.001. 1003 1004 Figure 5—figure supplement 4. Whole cell images corresponding to the images 1005 shown in Figure 5E. HA-KIF17-G234A expression is verified by HA immunoreactivity. 1006 Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. 1007 1008 Figure 5—figure supplement 5. The KIF17 motor domain mutation has little effect 1009 on overall surface expression of receptors. Effects of KIF17 transfection on surface- 1010 accessible Flag immunoreactivity from Flag-tagged D1R (D1R) or Flag-tagged SSTR3 1011 (SSTR3) was quantified by fluorescence flow cytometry and normalized to the mock- 1012 transfected (pcDNA) condition. Bars represent mean normalized surface expression. (*) 1013 p <0.05; (**) p<0.01. 1014 1015 Figure 6. Rab23 is necessary for D1R localization to cilia. (A) Representative 1016 images of cilia on cells stably transfected with Flag-D1R and transiently transfected with 1017 a non-silencing duplex (control) or siRNA targeting Rab23 (Rab23-4 and Rab23-2). 1018 Rab23 knockdown strongly reduced Flag-D1R localization to cilia. (B) Quantification of

1019 the siRNA effect on the fraction of cells in the population possessing a visible cilium 1020 marked by AcTub. Error bars represent SEM from 150 cells counted in n = 3 1021 experiments. (C) Quantification of the Rab23 knockdown effect on the fraction of D1R 1022 (Flag) -positive cilia. Error bars represent SEM from n =3 experiments with 50 cells 1023 counted per experiment. (D) Quantification of the Rab23 knockdown effect on average 1024 fold-enrichment of D1R (Flag) signal on cilia. Error bars represent SEM from n = 3 1025 independent experiments with 20-30 cilia analyzed per experiment. (E) Schematic 1026 representation of wild type D1R and the cilia-targeted DOR-D1(338-446) chimeric 1027 mutant receptor (duplicated from Figure 3G). (F) Quantification of the Rab23 1028 knockdown effect on average fold-enrichment of Flag-D1R (D1R) and the Flag-tagged 1029 chimeric mutant receptor (DOR-D1(338-446)) on cilia of transiently transfected cells. 1030 Error bars represent SEM from n = 3 independent experiments with 10-20 cilia analyzed 1031 per experiment. (G) Representative images of cilia on cells stably transfected with 1032 SSTR3-GFP and transiently transfected with a non-silencing duplex (control) or siRNA 1033 targeting Rab23 (Rab23-4 and Rab23-2). Rab23 knockdown strongly reduced SSTR3- 1034 GFP localization to cilia. (H) Quantification of the Rab23 knockdown effect on the 1035 fraction of SSTR3-GFP (GFP) positive cilia. Error bars represent SEM from n =3 1036 experiments with 50 cells counted per experiment. (I) Quantification of the Rab23 1037 knockdown effect on average fold-enrichment of SSTR3 (GFP) signal on cilia. Error 1038 bars represent SEM from n =3 experiments with 10-20 cilia analyzed in each 1039 experiment. (*) p <0.05; (**) p<0.01; (***) p < 0.001. Scale bars, 5 μm. 1040 1041 Figure 6—figure supplement 1. Whole cell images for corresponding images shown 1042 in Figure 6A. The merged images display Flag-D1R immunoreactivity in green and 1043 AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm. 1044 1045 Figure 6—figure supplement 2. Verification of Rab23 knockdown. Results of qRT- 1046 PCR analysis. Error bars represent SEM for n =3 experiments. (**) p<0.01. 1047

1048 Figure 6—figure supplement 3. Rab23 knockdown has little effect on overall surface 1049 receptor expression. Results of flow cytometric analysis. Results are normalized to 1050 control siRNA. Bars represent mean normalized surface expression. 1051 1052 Figure 6—figure supplement 4. Whole cell images for corresponding images shown 1053 in Figure 6G. The merged images display SSTR3-GFP in green and AcTub in red. 1054 Dashed blue line indicates outline of an individual cell. Scale bar, 5 μm. 1055 1056 Figure 7. Rab23 is sufficient to drive ciliary localization of a non-ciliary GPCR. (A) 1057 Schematic representation of the D1R-derived constructs examined. D1R-derived 1058 sequence is depicted in red and Rab23 sequence in blue. Flag-tagged wild type D1R 1059 was compared to the ciliary targeting-impaired mutant D1R (D1Δ381-395), and to the 1060 ciliary targeting-impaired mutant D1R fused to wild type Rab23 (D1Δ381-395-Rab23), 1061 inactive mutant Rab23 (D1Δ381-395-Rab23-S23N) or activated mutant Rab23 1062 (D1Δ381-395-Rab23-Q68L). (B) Representative images of cilia on cells transiently 1063 expressing Flag-tagged versions of the indicated receptor constructs. Fusion of either 1064 wild type or activated Rab23 to the cilia targeting-defective D1R visibly enhanced ciliary 1065 localization of receptors. (C) Quantification of the fraction of receptor (Flag) -positive 1066 cilia. (D) Average fold-enrichment of receptor (Flag) on cilia. (E) Representative live-cell 1067 images of GFP-tagged Rab23 or Rab23-Q68L localization relative to cilia marked by 1068 Arl13b-mRuby after transient co-transfection. (F) Schematic representation of the DOR- 1069 Rab fusions. DOR-derived sequence is depicted in green, Rab23 in blue, Rab11 in 1070 violet, and Rab8 in orange. (G) Representative images of cilia on cells transiently 1071 expressing Flag-tagged versions of the indicated receptor constructs. Wild type DOR 1072 was not detected on cilia. Fusion of activated (Q68L) Rab23 produced strong ciliary 1073 localization while fusion of activated (Q70L) Rab11 or activated (Q67L) Rab8 failed to 1074 do so. (H) Fraction of receptor (Flag) -positive cilia. (I) Average fold-enrichment of 1075 receptor (Flag) signal on cilia. Error bars represent SEM from n = 3 independent 1076 experiments with 10-20 cilia analyzed in each experiment. (**) p<0.01; (***) p < 0.001. 1077 Scale bars, 5 μm.

1078 1079 Figure 7—figure supplement 1. Whole cell images corresponding to images shown 1080 in Figure 7B. The merged images display Flag-receptor immunoreactivity in green and 1081 AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. 1082 1083 Figure 7—figure supplement 2. Overall surface expression of D1R-Rab23 fusion 1084 constructs. Results of flow cytometric analysis. Bars represent mean normalized to 1085 Flag-D1R surface expression. (**) p<0.01. 1086 1087 Figure 7—figure supplement 3. Whole cell images corresponding to images shown 1088 in Figure 7G. The merged images display Flag-receptor immunoreactivity in green and 1089 AcTub in red. Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. 1090 1091 Figure 7—figure supplement 4. Overall surface expression of DOR-Rab23 fusion. 1092 Results of flow cytometric analysis. Bars represent mean normalized to Flag-DOR 1093 surface expression. (**) p<0.01. 1094 1095 Figure 7—figure supplement 5. Rab23 is sufficient to drive ciliary localization of 1096 several non-ciliary GPCRs. Both MOR and B2AR were fused to activated mutant 1097 Rab23 (MOR-Rab23-Q68L, B2AR-Rab23-Q68L). Representative images of cells 1098 transiently expressing Flag-tagged versions of the indicated constructs are shown. Wild 1099 type MOR and B2AR were not detected on cilia, while fusion of MOR and B2AR to 1100 activated (Q68L) Rab23 produced strong chimeric receptor ciliary localization. The 1101 merged images display Flag-receptor immunoreactivity in green and AcTub in red. 1102 Insets show a cropped region of the plasma membrane containing the cilium. Dashed 1103 blue line indicates outline of an individual cell. Scale bars, 5 μm. 1104 1105 Figure 8. Evidence IFT-B, KIF17, and Rab23 function in an integrated ciliary 1106 delivery pathway. (A) Representative images showing the effect of motor domain- 1107 mutant KIF17 (+KIF17-G234A) on Flag-tagged wild type D1R localization to the cilium

1108 (from Figure 5B). (B) Representative images from an identical experiment examining 1109 localization of the Flag-tagged D1R fusion to activated Rab23 (D1Δ381-395-Rab23- 1110 Q68L). Scale bars, 5 μm. (C) Average fold-enrichment of D1R and D1Δ381-395-Rab23- 1111 Q68L (Flag) signal on cilia. Wild type D1R localization to cilia was strongly reduced by 1112 motor-defective KIF17, but direct Rab23 fusion effectively bypassed this requirement. 1113 (D) Effect of IFT172 knockdown on average fold-enrichment of D1R and D1Δ381-395- 1114 Rab23-Q68L (Flag) signal on cilia. Wild type D1R localization to cilia was strongly 1115 reduced by IFT172 knockdown, but direct Rab23 fusion effectively bypassed this 1116 requirement. Error bars represent SEM from n = 3 independent experiments with 10-20 1117 cilia analyzed in each experiment. (***) p < 0.001. (E) Co-immunoprecipitation analysis 1118 showing that Rab23 is not necessary for D1R association with IFT57. The analysis and 1119 presentation of data are described in Figure 4E. (F) Immunoblots from multiple 1120 experiments were quantified in the linear range to estimate the amount of IFT57 co- 1121 IPed. The result is expressed as a fold-increase over the control siRNA condition. Error 1122 bars represent SD from n =3 experiments. 1123 1124 Figure 8—figure supplement 1. Whole cell images corresponding to the images 1125 shown in Figure 8B. HA-KIF17-G234A expression is verified by HA immunoreactivity. 1126 Dashed blue line indicates outline of an individual cell. Scale bars, 5 μm. 1127 1128 Figure 8—figure supplement 2. Disruption of KIF17 motor activity does not affect 1129 Rab23 ciliary localization. Representative live-cell images of cells co-transfected with 1130 Flag-Rab23-Q68L, Arl13b-YFP as a cilia marker, and either empty vector (+pcDNA) or 1131 motor domain mutant KIF17 (+KIF17-G234A). In merged image, Flag-Rab23-Q68L 1132 (Flag) immunoreactivity is shown in red, Arl13b-YFP in green, and HA-KIF17-G234A in 1133 blue. Rab23-Q68L was observed in 8/18 cilia in cells expressing pcDNA. Similarly, 1134 Rab23-Q68L was clearly visible in 9/15 cilia in cells expressing KIF17-G234A. Scale 1135 bar, 5 μm. 1136

1137 Table I

Table I: siRNA knockdown screen

Gene Implication +/- effect on

D1 in cilia

TULP3 SSTR3, MCHR1 cilia localization -

Bbs4, Bbs2 SSTR3, MCHR1 cilia localization -

Arf4 Rhodopsin localization to rod outer -

segment

Asap1 Rhodopsin localization to rod outer -

segment

Kif7, Kif27 Hedgehog signaling -

Vps35 Receptor trafficking -

Rab15, Rab14, Rab8, Cilia associated Rabs -

Rab11

Rab4, Rab35 Trafficking of receptors -

Rab23 Hedgehog signaling +

Arl6 Cilia associated small GTPase -

IFT57 Intraflagellar transport +

IFT172 Intraflagellar transport +

Clathrin heavy chain Receptor endocytosis -

Pacs1 Olfactory CNG channel cilia localization -

Kif5c Apical trafficking of cargo -

Septin2 Cilia diffusion barrier -

1138

46 Figure 1

ABC D1R SSTR3 DOR

Flag- Flag- Flag- receptor receptor receptor

AcTub AcTub AcTub

merge merge merge

*** D1R *** D *** E F *** 10 100 M P /

80 a 8 i l i c

60 n 6 e m

40 h 4 c i r n e 20 2 d l o f receptor positive cilia (%) cilia positive receptor 0 0

D1R DOR SSTR3 D1R DOR SSTR3 Figure 2 photoactivation ABphotoactivate C cilia 1s 10s 1 sec post PA 555 555 1.0 10 sec post PA

Flag 0.8

405nm PA PA 0.6 PA-GFP 555 0.4 555 0.2 normalized PA-GFP normalized

merge intensity fluorescence PA PA 0.0 012345 cilia length (+m) DEF PA series PA PA 0 min 10 min post PA 125 time (min) 0 30

100 *** 60 ilium (%) PA-GFP 75 ilium 40 50

escence in c in escence 25 20 Integrated D1-PAGFP Flag fluor 0 0 1020304050607080 c D1R in % new 0 time after PA (sec) 030 time post PA series (min)

GH0 min 30 min 30 min + PA

ns 1.25 *** 1.00 Delivery from PA/555= 1 PA/555= 1 PA/555 > 1 internal pool 0.75

0.50

0.25 ? Alexa555 / PA-GFP 0.00 A 0 min 30 min

30 min + P time post PA series

Delivery from PA/555= 1 PA/555 < 1 PA/555= 1 PM pool ? Figure 3 A B C D EF *** *** D1R D1-415T D1Δ381-395 100 10 D1-382T D1R 338 446 M P /

80 a 8 i ilia (%) Flag- l D1-415T 415 i

338 c

receptor t

60 n 6 e

D1-382T 338 382 m 40 h 4 c i r

AcTub n

338 446 e D1¨381-395 20 2 d l o f receptor positive c positive receptor 0 0

merge D1R D1R 381-395 381-395 D1 D1 GH IJ DOR- DOR-

*** DOR D1(338-446) D1(368-446) *** 100 8

DOR P Flag- 80 / a i l receptor i 6 c

DOR- 338 446 60 t n

D1(338-446) e 4 m

40 h 368 c

446 i

DOR- AcTub r n 2 D1(368-446) e 20 d l o f receptor positive cilia (%) cilia positive receptor 0 0 merge DOR DOR

(338-446) -D1 R DOR-D1(338-446)DOR-D1(368-446) DO DOR-D1(368-446) Figure 4

AB siRNA treatment rescue

IFT57 siRNA 100 IFT57 IFT172 control +IFT57-NTM * 80 * Flag-D1R 60

AcTub % ciliated cells 20

0 merge IFT57-1IFT57-4 control IFT172-3IFT172-4 siRNA

CD EHA-IFT57 + + + * *** *** *** Flag-D1R – + – *** ** 100 *** Flag-DOR – – + 20 MW

M IP: Flag 80 P 55 /

a blot: HA i l

i 15 c

60 t

n lysate

e 55 10 blot: HA

40 m h c i r 70 20 n 5 e

d lysate l receptor positive cilia (%) cilia positive receptor

o blot: Flag

0 f 0 40

IFT57-1IFT57-4 control IFT172-3IFT172-4 IFT57-1IFT57-4 siRNA control IFT172-3IFT172-4 siRNA

FGHA-IFT57 + + + Flag-D1R – + – 4 * Flag-D1Δ381-395 – – + MW 3

IP: Flag 55 blot: HA 2

lysate 1 blot: HA 55 fold over control

lysate D1R blot: Flag 55 381-395 D1 Figure 5

AB+pcDNA +KIF17-G234A +KIF17 HA-IFT57 ++ Flag-KIF17 – + Flag-D1R MW IP: Flag blot: HA 55

lysate 55 AcTub blot: HA

Iysate 150 blot: Flag merge 70

CDD1R D1R 12 *** M P 100 / 10 a i l i c 80 8 t ilia (%) n e 6 60 m h c i

r 4 40 n e

d 2 l

20 o f 0 receptor positive c positive receptor 0

KIF17 KIF17 pcDNA pcDNA KIF17-G234A KIF17-G234A

SSTR3 12 EF

+pcDNA +KIF17-G234A M P / 10 a i l i

Flag-SSTR3 c 8 t n.s. n e 6 m h

AcTub c i

r 4 n e

d 2 l o f merge 0

pcDNA

KIF17-G234A Figure 6

siRNA treatment ADB C *** control Rab23 *** 15 *** 100 100 M P /

80 80 a i ilia (%) Flag-D1R l i c

10 t

60 60 n e m

40 40 h c i

AcTub r 5 n % ciliated cells % ciliated e

20 20 d l o f receptor positive c positive receptor 0 0 0 merge

controlRab23-4 Rab23-2 controlRab23-4 Rab23-2 controlRab23-4Rab23-2 siRNA siRNA siRNA

E F DOR-D1 D1R (338-446)

10 ***

M P /

a 8 i ** l 338 446 i

D1R c

n 6 e

338 446 m DOR-D1(338-446) h 4 c i r n e 2 d l o f 0

control Rab23 control Rab23 siRNA

G siRNA treatment H ** I * 8 *** control Rab23

M **

100 P / a i l

i 6

SSTR3-GFP 80 c

t n 60 e 4 m h c i

40 r AcTub n 2 e

20 l o f

receptor positive cilia (%) cilia positive receptor 0 0 merge control Rab23-4 Rab23-2 control Rab23-4 Rab23-2 siRNA siRNA Figure 7

ABD1ǻ381-395- D1ǻ381-395- D1ǻ381-395- D1R D1ǻ381-395 Rab23 Rab23-S23N Rab23-Q68L

D1R Flag- receptor D1Δ381-395

Rab23 D1Δ381-395 -Rab23 AcTub

D1Δ381-395 Rab23 -Rab23-S23N S ->N

D1Δ381-395 Rab23 merge -Rab23-Q68L Q -> L

CDE** *** 100 GFP-Rab23 Arl13b-mRuby merge 20 **

P *** 75 / a i l

i 15

c ***

t n

50 e 10 m GFP-Rab23-Q68L Arl13b-mRuby merge h c i 25 r n 5 e

d l o receptor positive cilia (%) cilia positive receptor 0 f 0

D1R D1R -395 381-395 381 D1 D1 381-395-Rab23 381-395-Rab23 D1 381-395-Rab23-S23N381-395-Rab23-Q68L D1 381-395-Rab23-S23N381-395-Rab23-Q68L D1 D1 D1 D1 F GHI DOR- DOR- DOR- *** DOR Rab23-Q68L Rab11-Q70L Rab8-Q67L *** 15 100

DOR M P

Flag- / a i

80 l receptor i c 10 t

DOR- Rab23 n Rab23-Q68L Q -> L 60 e m h c i

40 r 5 n e DOR- Rab11

AcTub d 20 l

Rab11-Q70L Q -> L o f 0 receptor positive cilia (%) cilia positive receptor 0

DOR DOR- Rab8 DOR Rab8-Q67L Q -> L merge

DOR-Rab23-Q68L DOR-Rab23-Q68L Figure 8

D1ǻ381-395- ABCD1R Rab23-Q68L

+pcDNA +KIF17-G234A +pcDNA +KIF17-G234A n.s.

20 M P

Flag- / a i

D1ǻ381-395- l Flag-D1R i 15

c ***

Rab23-Q68L t n e 10 m h c

AcTub i

AcTub r n 5 e

d l o f merge 0 merge

pcDNA pcDNA

KIF17-G234A KIF17-G234A

DED1ǻ381-395- F D1R Rab23-Q68L n.s. control Rab23 5 n.s. siRNA siRNA 15 HA-IFT57 – + + – + + 4 M *** P / Flag-D1R ++– + – + a *** MW i 3 l i c

10 t IP: Flag 55 n blot: HA 2 e m

h lysate fold over control c

i 1

r 5 blot: HA 55 n e

d 0 l lysate

o 50 f blot: Flag 0 Rab23 control siRNA control IFT172-3IFT172-4 control IFT172-3IFT172-4 siRNA