The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

Abstract

Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.

Figure 1.

Rpgrip1l localizes at the ciliary TZ in G₀ phase and at centrosomes during mitosis. (A–C) Immunofluorescence on MEFs (isolated from E12.5 embryos). (A and B) Plots indicate fluorescent intensities of each channel along the cilium from base (left) to tip (right). (A) The ciliary axoneme is marked by acetylated α-tubulin (α-Tub) and the BB by γ-tubulin. (B) The ciliary axoneme is marked by acetylated α-tubulin (α-Tub), and the TZ is marked by Cep290. (C) Centrosomes are marked by γ-tubulin, and cell nuclei are marked by DAPI. Insets illustrate higher magnifications of boxed regions. (D) Immunofluorescence on E12.5 murine limbs. The ciliary axoneme is marked by acetylated α-tubulin and centrosomes by Pcnt2. Bars: (A and B) 1 µm; (C) 10 µm; (D, overview) 5 µm; (D, magnification) 1 µm.

Figure 2.

Rpgrip1l deficiency affects ciliary length. (A and B) MEFs were isolated from E12.5 WT and Rpgrip1l^−/− embryos. (A) Comparison of WT and Rpgrip1l^−/− ciliary length. Embryonic organs were separated from E12.5 embryos. At least 20 cilia per embryo were analyzed. n values (n refers to the number of embryos) are as follows: MEFs: WT, n = 4; Rpgrip1l^−/−, n = 5; limbs: WT, n = 4; Rpgrip1l^−/−, n = 5; hearts: WT, n = 4; Rpgrip1l^−/−, n = 5; lungs: WT, n = 4; Rpgrip1l^−/−, n = 4; livers: WT, n = 4; Rpgrip1l^−/−, n = 4. Error bars show standard error of the mean. (B) Elongation of E12.5 Rpgrip1l^−/− limb cilia are confirmed in comparison to fluorescence based cilia length measurement (10 cilia; SEM: 2.88 ± 0.14 µm; immunofluorescence: 2.44 ± 0.265 µm). Magnifications (termed as 1 and 2) show two examples of Rpgrip1l^−/− limb cilia. Bars: (overview) 10 µm; (magnification) 1 µm. (C) Absolute values of the quantified ciliary length of WT (n = 4 embryos) and Rpgrip1l^−/− (n = 5 embryos) limbs at E12.5 based on immunofluorescence. Rpgrip1l-negative cilia (2.44 ± 0.265 µm [standard error of the mean]) are significantly longer than WT cilia (1.25 µm ± 0.05 µm [standard error of the mean]). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Rpgrip1l deficiency leads to elongation of limb cilia and an increase of the Gli3-190/Gli3-83 ratio but does not alter overall cellular proteasomal activity. (A and B) Western blot analysis of MEFs and E12.5 limbs (WT: n = 3 embryos; Rpgrip1l^−/−: n = 3 embryos, respectively). Gapdh (A) and Actin (B) serve as loading controls. Evaluation of the Gli3-190/Gli3-83 ratio depicts a significant increase in the Rpgrip1l^−/− situation of 3.6-fold (MEFs, A) and 8.1-fold (limbs, B). (C) Proteasomal activity was measured in total cell lysates of MEFs isolated from E12.5 WT (n = 8) and Rpgrip1l^−/− embryos (n = 4). No alteration is detectable in Rpgrip1l^−/− MEFs in comparison to WT MEFs either without ATP (activity of the 20S proteasomal subunit) or after ATP addition (activity of the 20S proteasomal subunit and 26S proteasome together). There is also no alteration in the activity of the 26S proteasome alone (ATP-w/o ATP), demonstrating that the activity of the 19S proteasomal subunit is unchanged in the absence of Rpgrip1l. Error bars show standard error of the mean. *, P < 0.05; **, P < 0.01.

Figure 4.

Rpgrip1l deficiency causes impaired proteasomal activity at primary cilia. (A–F and I–L) MEFs were isolated from E12.5 WT and Rpgrip1l^−/− embryos. (A and B) Western blot analysis of WT and Rpgrip1l^−/− MEF lysates (n = 4 embryos, respectively). (C) Western blot analysis of WT and Rpgrip1l^−/− MEF lysates (n = 3 embryos, respectively). (A–C) Actin serves as a loading control. (A) Phospho-(S33/37/T41)-β-Catenin is significantly increased in serum-starved Rpgrip1l^−/− MEF lysates (82% of all cells had cilia; in serum-starved Rpgrip1l^+/+ MEF lysates, 88.67% of all cells possessed cilia) but not in non–serum-starved Rpgrip1l^−/− MEF lysates (4% of all cells displayed cilia; in non–serum-starved Rpgrip1l^+/+ MEF lysates, 6.67% of all cells carried cilia; C). (B) Non–phospho-(S33/37/T41)-β-Catenin is unaltered in serum-starved Rpgrip1l^−/− MEF lysates. Black lines indicate that intervening lanes have been spliced out. (D–F, I, and L) Immunofluorescence on MEFs of E12.5 WT and Rpgrip1l^−/− embryos (both genotypes: p-β-Catenin: n = 5; p-β-Catenin (3D-SIM, n = 3; Ubiquitin, n = 4; Gli3-190, n = 6; ZsProSensor-1, n = 3; n refers to the number of embryos, respectively). Per embryo, 15 cilia were quantified for p-β-Catenin, 10 cilia were quantified for p-β-Catenin (3D-SIM) and for Ubiquitin, and 20 cilia were quantified for Gli3-190. (G and H) Immunofluorescence on limbs of E12.5 WT and Rpgrip1l^−/− embryos (n = 3 embryos, respectively). Per embryo, 20 cilia were quantified for p-β-Catenin and Ubiquitin. All quantified proteins are shown in red (D–J), the ciliary axoneme is marked by acetylated α-tubulin (green; D–J), and the BB is marked by γ-tubulin (blue; D–F, H, and I) or by Pcnt2 (blue; G). (J and K) Immunofluorescence on MEFs of WT embryos (n = 4). 25 cilia per embryo were used for phospho-(S33/37/T41)-β-Catenin and cilia length quantification. (L) Proteasome activity assay on WT and Rpgrip1l^−/− MEFs. Cilia are marked by acetylated α-tubulin (α-Tub), and centrosomes/basal bodies are marked by γ-tubulin. Colored squares mark cilia with basal bodies (yellow squares) as well as centrosomes (red squares), which are presented magnified. The green ZsProSensor-1 protein signal is exclusively detected at the ciliary base in Rpgrip1l^−/− MEFs. Error bars show standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 1 µm.

Figure 5.

Components of the S19 and S20 proteasomal subunits localize at primary cilia. (A–E) Immunofluorescence on MEFs of E12.5 WT and Rpgrip1l^−/− embryos. Higher resolution was achieved by using SIM. The ciliary axoneme is marked by acetylated α-tubulin (blue). The TZ is marked by Tctn2 (A) or Rpgrip1l (B and C). (D and E) The ciliary axoneme is marked by acetylated α-tubulin (green) and the BB by γ-tubulin (blue). Psma5 is shown in red. (D) Z-stack of the single optical sections from E with the appropriate plot of the depicted representative image. Bars: (A–C) 0.5 µm; (D and E) 1 µm.

Figure 6.

Rpgrip1l deficiency results in an accumulation of proteasomal subunit components at the base of cilia. (A–D) Immunofluorescence on MEFs of E12.5 WT and Rpgrip1l^−/− embryos (both genotypes: Psmd2, n = 7 embryos; Psmd3, n = 3 embryos; Psmd4, n = 3 embryos; Psma5, n = 5 embryos). At least 10 cilia per embryo were used for Psmd2, Psmd3, and Psmd4 quantification, respectively, and 20 cilia per embryo were used for Psma5 quantification. The ciliary axoneme is marked by acetylated α-tubulin (green). The BB is marked by Pcnt (green; white arrowheads; A) or γ-tubulin (blue; B–D). Error bars show standard error of the mean. *, P < 0.05; ***, P < 0.001. Bars: (A, B, and D) 1 µm; (C) 0.5 µm.

Figure 7.

Rpgrip1l interacts with the 19S proteasomal subunit component Psmd2. (A) Coimmunoprecipitation experiments in NIH3T3 cells. Myc-tagged Psmd2 full-length protein and FLAG-tagged RID were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. Myc-Psmd2 coimmunoprecipitated with FLAG-RID (lane 3). Ctrl., control; IP, immunoprecipitation; WB, Western blot. (B) Myc-tagged Psmd2 full-length protein and FLAG/Strep/HA-tagged RID domain of Rpgrip1l were transiently overexpressed in HEK293T cells and tested for interaction by tandem affinity purification (TAP) tag experiments from total cell lysates. FLAG/Strep/HA-tagged RID was immunopurified using anti-FLAG beads, eluted with FLAG-protein, and recaptured on anti-Strep beads. The final eluation was performed by using biotin. After this sequential purification, we identified Myc-Psmd2 (lane 2) but not the unrelated protein Gatad1 (lane 6) in addition to purified FLAG/Strep/HA-RID. (C–E) Immunofluorescence on MEFs isolated from WT or Rpgrip1l^−/− E12.5 embryos (n = 3 embryos, respectively). (C) Centrosomes are marked by γ-tubulin as well as acetylated α-tubulin (α-Tub), and cell nuclei were marked by DAPI. Insets illustrate higher magnifications. (D) The ciliary axoneme and the BB are marked by acetylated α-tubulin and by γ-tubulin, respectively. The plot shows fluorescence intensities of the depicted representative image. (E) In situ proximity ligation assay (in situ PLA) on MEFs. Cell nuclei are marked by DAPI, and the ciliary axoneme are marked by transiently transfected Ift88-EYFP. Additional accumulation of Ift88-EYFP at the ciliary base is highlighted by yellow brackets. White arrowheads point to cilia. Bars: (C, all images; and D, overview) 10 µm; (D and E, magnifications) 1 µm.

Figure 8.

Proteasomal activity is unaltered at Rpgrip1l^−/− centrosomes. (A–C) Immunofluorescence on MEFs isolated from E12.5 WT and Rpgrip1l^−/− embryos (WT: p-β-Catenin (treated with DMSO or MG132): n = 3 embryos; both genotypes: p-β-Catenin and Ubiquitin: n = 3 embryos, respectively). The ciliary axoneme is marked by acetylated α-tubulin (green) and the centrosomes (basal bodies in case of ciliary presence) by γ-tubulin (blue). All quantified proteins are shown in red. An axonemal-like green staining is not visible, demonstrating that the blue staining marks centrosomes. (A) After treatment of WT MEFs with the proteasome inhibitor MG132, the amount of phospho-(S33/37/T41)-β-Catenin is significantly increased at the centrosome. (B and C) The amounts of phospho-(S33/37/T41)-β-Catenin and Ubiquitin are unaltered at the centrosome of Rpgrip1l^−/− MEFs. (A–C) Per embryo, 20 cilia were used in the quantifications. Error bars show standard error of the mean. *, P < 0.05. Bars, 0.5 µm.

Figure 9.

Knockdown of the 19S proteasomal subunit components Psmd2, Psmd3, and Psmd4 reduce the activity of the ciliary proteasome. (A–G) Immunofluorescence on MEFs of E12.5 WT embryos (both genotypes: n = 3 embryos). (A) The ciliary axoneme is marked by acetylated α-tubulin (acet. α-Tub). Transfection of MEFs with siRNA against Psmd2 and quantification of the ciliary Psmd2 amount (A) and the ciliary Ubiquitin amount (B). (C) Measurement of cilia length after treatment with siRNA against Psmd2. (D and E) Transfection of MEFs with siRNA against Psmd3. (D) Quantification of Ubiquitin amount at cilia. The ciliary axoneme is marked by acetylated α-tubulin (acet. α-Tub)and the BB by γ-tubulin. (E) Measurement of cilia length. (F and G) Transfection of MEFs with siRNA against Psmd4. (F) Quantification of Ubiquitin amount at cilia. The ciliary axoneme is marked by acetylated α-tubulin (acet. α-Tub) and the BB by γ-tubulin. (G) Measurement of cilia length. Error bars show SEM. *, P < 0.05; **, P < 0.01. Bars: (A) 0.5 µm; (D and F) 1 µm.

Figure 10.

Transfection of the RID domain and SFN treatment increase proteasomal activity and rescue the decreased activity of the ciliary proteasome caused by Rpgrip1l deficiency. (A and B) Immunofluorescence on NIH3T3 cells. Per control and per transfected cells, 20 cilia were used for quantification. The ciliary axoneme is marked by acetylated α-tubulin (acet. α-Tub). (A) Yellow lines encircle the shape of cilia. (C–I) The most important significance comparisons are written in bold with larger asterisks as are larger not significant comparisons. (C and D) Immunofluorescence on MEFs of E12.5 WT and Rpgrip1l^−/− embryos (both genotypes, n = 3 embryos). At least 10 cilia per embryo were used for quantification. The ciliary axoneme is marked by acetylated α-tubulin (acet. α-Tub) and the BB by γ-tubulin. (E–G) Immunofluorescence on MEFs isolated from E12.5 WT and Rpgrip1l^−/− embryos (both genotypes: p-β-Catenin and Ubiquitin [treated with DMSO or SFN], n = 3 embryos, respectively). Per embryo, 20 cilia were used in the quantifications. The ciliary axoneme is marked by acetylated α-tubulin (green), and the BB is marked by γ-tubulin (blue). All quantified proteins are shown in red. (H and I) Whole embryo culture treatment of embryos isolated on E12.5 and incubated for 5 h in 20 µmol SFN (both genotypes: Ubiquitin [treated with DMSO or SFN], n = 3 embryos, respectively]). Per embryo, 20 cilia were used in the quantifications. The ciliary axoneme is marked by acetylated α-tubulin (green), and the BB is given by γ-tubulin (blue). Bars, 1 µm. Error bars show standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.