RP2 localizes to primary cilia. (A) RP2 localizes to cilia in MDCK cells. MDCK cells were grown 7 days post-confluence, fixed, permeabilized and immuno-stained using antibodies against RP2 (green) and polyglutamylated tubulin (red). (B) RP2-EGFP localizes to primary cilia. MDCK cells stably expressing EGFP-tagged RP2 (green) were grown for 7 days post-calcium switch to allow cilia formation. Cilia were visualized using an antibody against the cilia marker acetylated tubulin (red). (C) Mutation of the N-terminal myristoylation motif prevents cilia localization of RP2-EGFP and mistargets it to nuclei. MDCK cells stably expressing RP2-EGFP G2A (green) were grown for 7 days post-confluence on trans-well filters, fixed, permeabilized and immuno-stained with an anti-acetylated tubulin antibody (red). (D) RP2-EGFP C3S fails to target to cilia. MDCK cells stably expressing the palmitoylation mutant C3S (green) were treated as above. All scale bars (white) represent 10 µm.

RP2 regulates polycystin 2 trafficking. (A) Ablation of RP2 results in increased mobility of cilia-localized polycystin 2. Representative FRAP confocal images from live control (Luc shRNA) or RP2 knockdown (RP2 shRNA) MDCK cells stably expressing EmGFP-tagged polycystin 2. Images show fluorescent EGFP signal pre-bleach, immediately after the bleach (Bleach) and after recovery (Post-bleach). (B) Average recovery after photobleach for cilia-localized polycystin2-EGFP in control (Luc shRNA, blue) and RP2 knockdown (RP2 shRNA, red). Data shown are mean ± SE, n = 5 cells. The solid line through the data is a single-exponential fit to the average data. (C) Average recovery after photobleaching for cilia-localized Crb3-EGFP in control (Luc shRNA, blue) and RP2 knockdown (RP2 shRNA, red). Data shown are mean ± SE, n = 5 cells. The solid line through the data is a single-exponential fit to the average data. (D) Ablation of RP2 results in reduced secretion of polycystin 2. Conditioned media from control (Luc shRNA) and RP2 knockdown (RP2 shRNA) cells were subjected to sequential centrifugation, and input and the 100 000g pellet (100K Pellet) were analyzed by western blot. (E) Knockdown of IFT88 results in shortened or no cilia in MDCK cells. MDCK cells were retrovirally infected with either a luciferase-specific shRNA construct (Luc) or one of two separate canine-specific IFT88 shRNA constructs (IFT88 shRNA no. 1 and IFT88 shRNA no. 2) and stable pools selected. All scale bars represent 10 µm. (F) Ablation of IFT88 results in reduced secretion of polycystin 2. Conditioned media from control (Luc shRNA) and IFT88 knockdown (IFT88 shRNA no. 1 and shRNA no. 2) cells were subjected to sequential centrifugation, and input and the 100 000g pellet (100K Pellet) were analyzed by western blot.

RP2 knockdown in zebrafish results in multiple developmental defects. (A) Zebrafish embryos were injected with one of two (AUG1 0.5 ng, AUG2 2 ng) translation-blocking morpholinos or a 5 bp mismatch morpholino (2 ng). (B) RP2 morphants have no detectable RP2 protein. Zebrafish embryos were injected with 0.5 ng of AUG1 morpholino and whole fish lysate was prepared 24 h post-injection. Lysates were subjected to SDS–PAGE and blotted with anti-RP2 antibody and alpha-tubulin as a loading control. (C) RP2 morphants display hydrocephalus (arrowhead) and pericardiac effusion (arrow). (D) Methylene blue-stained plastic-embedded section of RP2 morphants displaying prominent hydrocephalus. (E) RP2 morphants exhibit pronephric cysts. RP2 mismatch morphants (left panel) and RP2 AUG1 morphants were fixed at 60 hpf and plastic-embedded sections stained with methylene blue. Cysts are marked with asterisks. (F) RP2 morphants show left–right symmetry defects as shown by mislocalization of the lateral mesoderm marker southpaw. RP2 morphants were fixed at the 23 somite stage and in situ hybridization performed with an antisense southpaw probe. (G) RP2 morphants exhibit heart looping defects. Mismatch or RP2 AUG1 morpholinos were injected into embryos from the CMLC:EGFP zebrafish strain and heart looping was observed by fluorescence microscopy.

RP2 is enriched in distinct regions of the kidney. (A) RP2 staining in mouse kidney. PFA-perfused frozen mouse kidney sections were permeabilized and stained with anti-RP2 antibodies (red) and DAPI (blue) to mark nuclei. (B) RP2 does not localize to distal convoluted tubules. Mouse kidney sections were stained as above with the addition of peanut lectin agglutinin (PNA-FITC) to label distal convoluted tubules. (C) RP2 localizes to Tamm Horsfall protein (THF) positive (green) segments of the nephron. Mouse kidney sections were stained as above with the addition of anti-THF antibody. (D) RP2 localizes to primary cilia in mouse kidney tubules. Mouse kidney sections were stained as above with the addition of anti-acetylated tubulin to label primary cilia (red).

RP2 forms a complex with polycystin 2. (A) Knockdown of RP2 by retroviral shRNA. MDCK cells were retrovirally infected with either a luciferase-specific shRNA construct (Luc) or one of two separate canine-specific RP2 shRNA constructs (RP2 shRNA no. 1 and RP2 shRNA no. 2) and stable pools selected. Cell lysates were subjected to SDS–PAGE and western blotting with the indicated antibodies. (B) Ablation of RP2 results in swelling of the cilia tip as shown by SEM. Control or RP2 shRNA were grown 7 days post-confluence on trans-well filters, fixed and prepared for SEM imaging. (C) Exogenous RP2 co-immunoprecipitates endogenous polycystin 2. Cell lysates (Input) from MDCK cells stably expressing either empty vector (pQCXIP) or RP2-FLAG wild-type (WT) were subjected to anti-FLAG immunoprecipitation in the presence or absence of 5 mm calcium chloride or EGTA. Immunoprecipitates were washed and subjected to SDS–PAGE and western blotting with the antibodies indicated. (D) Quantification of the effect of calcium and EGTA on the co-immunoprecipitation of RP2 and polycystin 2. The western blots in (C) were subjected to densitometric analysis. (E) Endogenous RP2 forms a complex with endogenous polycystin 2 in retina. Lysate (Input) from bovine retina was subjected to anti-RP2 immunoprecipitation. Immunoprecipitates were washed and subjected to SDS–PAGE and western blotting with the antibodies indicated. (F) The membrane association of RP2 is required for the interaction with polycystin 2. HEK293 cells were transiently transfected with polycystin 2-V5 and either wild-type (WT) RP2-FLAG or the membrane targeting mutant (G2A) RP2-FLAG. Lysates were subjected to immunoprecipitation as described above. (G) Ablation of RP2 results in accumulation of endogenous polycystin 2 at the distal region of cilia. Control (Luc shRNA) or RP2 shRNA were grown 7 days post-confluence on trans-well filters, fixed and stained with antibodies against polycystin 2 (green) and glutamylated tubulin (red). All scale bars (white) represent 10 µm.

RP2 expression in zebrafish. (A) Gene structure of the Zebrafish rp2. Protein coding region shown in grey. Shown are the primer sets used for RT–PCR. (B) RT–PCR analysis of rp2 transcript at different time points post-fertilization (hpf). NTC, no template control. (C) rp2 is expressed in the olfactory placode (arrowheads). In situ hybridization was performed on 24 hpf embryo with an rp2 antisense probe. (D) rp2 is expressed in the pronephric duct at 24 hpf. In situ hybridization was performed on a 24 hpf embryo with an rp2 antisense probe. Arrowheads show signal along the pronephric duct and asterisk shows cloaca. (E) rp2 is expressed in the photoreceptor layer (arrowheads) of the retina at 72 hpf. In situ hybridization was performed on a 72 hpf embryo with an rp2 antisense probe. (F) Zebrafish RP2 localizes to cilia in MDCK cells. MDCKII cells stably expressing zebrafish RP2-EGFP were grown for 7 days post-calcium switch to allow cilia formation. Cilia were visualized using an antibody against the cilia marker acetylated tubulin (red).

Combined knockdown of RP2 and PKD2 increases incorrect heart looping. (A) RP2 and PKD2 morpholinos were injected into CMLC:GFP zebrafish embryos at the indicated dosages. Heart looping was analyzed by fluorescence microscopy 48 hpf. (B) RP2 morphants have normal Kupffer's vesicle development. Zebrafish embryos were injected with either mismatch or RP2 morpholinos. Embryos were fixed at the 10–12 somite stage and stained for acetylated tubulin (red).