Figure 1.
Key ciliary signaling proteins are expressed in RCTE and MO6-G3 cells.
Renal cortical tubular epithelial (RCTE) cells and tooth derived odontoblasts (MO6-G3) cells were lysed and probed with antibodies directed against indicated proteins. PC1 using NM002 pAb (A, B), PC2 using Santa Cruz pAb (C), OFD1 using Santa Cruz pAb [3] (D), EGFR using Santa Cruz pAb (E), ErbB2 using US Biological pAb (F), flotillin-1 using BD Transduction mAb (H), and flotillin-2 using BD Transduction mAb (I) were found to be expressed in both cell types. Actin mAb from Millipore (G) was used as a loading control for PC1, PC2, EGFR, ErbB2, and OFD1; α/β-tubulin pAb from Cell Signaling (J) was used as a loading control for flotillin-1 and flotillin-2. Actin or α/β-tubulin was used to normalize results for quantification. Bar graph showing densitometric quantification of PC1 (a, b), PC2 (c), OFD1 (d), EGFR (e), ErbB2 (f), flotillin-1 (g), flotillin-2 (h). Bar graph represents the mean ± SD of four independent experiments. (*) p = 0.01 to 0.05, (***) p<0.001.
Figure 2.
Transiently transfected GFP-OFD1 and flotillin-2-GFP localize to primary cilia of RCTE and MO6-G3 cells.
Polarized RCTE and MO6-G3 cells were transiently transfected with either GFP-OFD1 or flotillin-2-GFP constructs. Upon 16 hours post-transfection, cells were fixed and acetylated α-tubulin (Sigma Aldrich mAb) was labeled to identify cilia. GFP-OFD1 localized to primary cilia in RCTE cells (A) and MO6-G3 cells (C). GFP-Flotillin-2 localized to primary cilia of RCTE (B) and MO6-G3 (D) cells. Images captured using a Zeiss LSM510 confocal microscope (63× objective). Images are representative of at least 3 independent experiments. Arrows denote cilia. Scale bar 10 µm.
Figure 3.
Endogenous OFD1, PC1, PC2, EGFR and flotillin-1 localize to primary cilia of RCTE and MO6-G3 cells.
Polarized RCTE and MO6-G3 cells were fixed and stained using the indicated antibodies. OFD1 from Novus Biologicals (A, F), PC1 using pAb NM002 (B, G), PC2 using AbCam pAb (C, H), EGFR using GeneTex pAb (D, I) and flotillin-1 using AbCam pAb (E, J) were found localized to 100% of primary cilia analyzed for both cell types. Acetylated α-tubulin (Sigma-Aldrich mAb) labeling identifies cilia. Zeiss LSM510 confocal microscope images (63× objective). Arrows denote the protein of interest within a cilium. Representative results from at least 5 independent experiments. Secondary antibody only controls were negative (not shown). Scale bar 10 µm.
Figure 4.
PC1, PC2, EGFR, OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3 cells.
RCTE and MO6-G3 cells were grown 5 days post-confluency to allow for polarization and ciliogenesis. PC1 was immunoprecipitated using NM002 pAb and precipitated proteins were separated by SDS-PAGE and probed for PC1 using NM005 pAb (A, B), PC2 using AbCam pAb (C), EGFR using GeneTex pAb (D), OFD1 using Novus Biologicals pAb (E) and flotillin-2 using Cell Signaling Rabbit mAb (F). Bar graph showing a densitometric quantification of PC1 co-immunoprecipitation for PC1 (a, b), PC2 (c), EGFR (d), OFD1 (e), and flotillin-2 (f). Normal rabbit IgG was used as a negative control. In a reciprocal experiment EGFR was immunoprecipitated using Santa Cruz pAb from RCTE and MO6-G3 cell lysates. Immunoprecipitated proteins were separated by SDS-PAGE and probed for EGFR using GeneTex pAb (G), PC1 using NM002 pAb (H), PC2 using Santa Cruz pAb (I), OFD1 using Novus Biologicals pAb (J) and flotillin-2 using Cell Signaling Rabbit mAb (K). Bar graph showing a densitometric quantification of EGFR co-immunoprecipitation probed for EGFR (g), PC1 (h), PC2 (i), OFD1 (j), and flotillin-2 (k). Normal rabbit IgG was used as a negative control. Lysate lane inputs were 5% of immunoprecipitations. 25 µg of total protein was loaded into each well. Arrows indicate the quantified band of interest in each immunoblot panel. Bar represents the mean ± SD of at least three independent experiments. (*) p = 0.01 to 0.05, (**) p = 0.001 to 0.01, (***) p<0.001.
Figure 5.
PC1 and EGFR interact in the primary cilium of MO6-G3 and RCTE cells.
Polarized MO6-G3 (A) and RCTE (B) cells were grown on coverslips 5 days post-confluency. Cells were incubated with antibodies against PC1 (Santa Cruz mAb) and EGFR (GeneTex pAb), followed by Duolink PLA Probes to identify points of PC1-EGFR interaction. Cilia identified by acetylated α-tubulin (Sigma-Aldrich mAb). Identical experiments performed without PC1 antibody are negative for fluorescent signal (C). Top panel shows XY plane, bottom panel shows XZ plane. Images from confocal microscope (63× objective). Scale bar is 5 µm. Quantification of puncta in 3 representative images (11–29 cells/field) from 2 independent experiments (D). Quantification of cilia containing PLA puncta (E). Puncta in cilia were counted based on colocalization with acetylated α-tubulin. Statistical evaluation based on two-tailed t-test. p = 0.0194.
Figure 6.
Key ciliary signaling proteins are significantly reduced in primary cilia of PKD cells.
Human RCTE and PKD Q4004X cells (with expression of mutant PC1) were grown on coverslips 5 days post-confluency to permit ciliogenesis. Cells were fixed and stained using antibodies directed against indicated proteins: PC1 (NM002 pAb); PC2 (AbCam pAb); EGFR (GeneTex pAb); and flotillin-1 (AbCam pAb). PC1 (A), PC2 (C), EGFR (E) and flotillin-1(G) are present in primary cilia of RCTE cells. PC1 (B), PC2 (D), EGFR (F) and flotillin-1 (H) were lacking in primary cilia of PKD Q4004X cells. Acetylated α-tubulin (Sigma-Aldrich mAb) staining identifies cilia. Arrows denote a small residual pool of EGFR detectable at the ciliary base of PKD cells. Zeiss LSM510 confocal microscope images (63× objective). Representative results from at least 5 independent experiments. Comparative images are from a single experiment and taken under identical settings. Arrows denote cilia. Scale bar 10 µm. Quantification shows individual ciliary protein intensities normalized to the respective ciliary volume (I–L). Each protein was quantified in 30–100 cilia for each cell type. z-stack images were imported into SlideBook and a volume mask for each cilium was created based on acetylated α-tubulin staining. Staining intensities for each protein were quantified within the respective ciliary volume mask. Statistical evaluation based on two-tailed t-test. (*) p = 0.0105, (***) p<0.0001.
Figure 7.
OFD1 ciliary localization is altered in human primary cell lines.
Human primary renal proximal tubule epithelial cells (RPTEC) and PKD (44M06) cells were grown on glass coverslips 5 days post confluency to allow for ciliogenesis, fixed and stained using an antibody directed against OFD1 (Santa Cruz pAb). OFD1 localizes to primary cilia in RPTEC cells (A) but is diminished in primary cilia PKD 46M06 cells (B). Acetylated α-tubulin (Sigma-Aldrich mAb) staining identifies cilia. Zeiss LSM510 confocal microscope images (63× objective). Representative results from 3 independent experiments. Comparative images are from a single experiment and taken under identical settings. Arrows denote cilia. Scale bar 10 µm. Quantification shows OFD1 staining intensities normalized to ciliary volumes, performed as detailed in methods and Figure 6 (C). z-stack images were imported into SlideBook and a mask for the cilia was created based on acetylated α-tubulin staining. Statistical evaluation based on two-tailed t-test. (**) p = 0.0036.
Figure 8.
Expression of PC1, OFD1, EGFR, and Flotillin-1 is decreased in ciliary fractions of PKD cells compared to RCTE cells.
RCTE cells and PKD Q4004X cells were grown 6 days post-confluency to allow for cell polarization and ciliogenesis and treated with 1.5 mM Dibucaine-HCl for 5 minutes to reduce cell loss and induce shedding of primary cilia. Cilia were collected by fractionation and the ciliary fraction was probed. PC1 (MN032 pAb), OFD1 (Novus Biologicals pAb), EGFR (GeneTex pAb), and flotillin-1 (BD Transduction mAb) were expressed in the ciliary fraction of deciliated RCTE cells but not PKD cells. α/β-tubulin (Cell Signaling pAb) was used as a marker of primary cilia. The nuclear marker lamin B (Santa Cruz pAb) was not present in the ciliary fraction (A). Quantification of 3 independent samples (B). Mean values were found to be statistically different (p<0.0001) using 1-way ANOVA.