Axoneme polyglutamylation regulated by Joubert syndrome protein ARL13B controls ciliary targeting of signaling molecules

Abstract

Tubulin polyglutamylation is a predominant axonemal post-translational modification. However, if and how axoneme polyglutamylation is essential for primary cilia and contribute to ciliopathies are unknown. Here, we report that Joubert syndrome protein ARL13B controls axoneme polyglutamylation, which is marginally required for cilia stability but essential for cilia signaling. ARL13B interacts with RAB11 effector FIP5 to promote cilia import of glutamylase TTLL5 and TTLL6. Hypoglutamylation caused by a deficient ARL13B-RAB11-FIP5 trafficking pathway shows no effect on ciliogenesis, but promotes cilia disassembly and, importantly, impairs cilia signaling by disrupting the proper anchoring of sensory receptors and trafficking of signaling molecules. Remarkably, depletion of deglutamylase CCP5, the predominant cilia deglutamylase, effectively restores hypoglutamylation-induced cilia defects. Our study reveals a paradigm that tubulin polyglutamylation is a major contributor for cilia signaling and suggests a potential therapeutic strategy by targeting polyglutamylation machinery to promote ciliary targeting of signaling machineries and correct signaling defects in ciliopathies.

Fig. 1

ARL13B associates with FIP5 and regulates the latter’s enrichment at the ciliary base in a temporal-spatial manner. a Indicated plasmids were transfected in HEK-293T cells for 48 h and the interaction between exogenous FIP5 and ARL13B proteins was detected by immunoprecipitation. b Direct interaction between FIP5 and ARL13B was detected by GST pull-down assay. GST-fused WT ∆19, N domain, C domain and Proline-rich region of ARL13B and His-FIP5 proteins were used. c In hTERT-RPE-1 cells, endogenous ARL13B strongly associates with FIP5 during the early stage of ciliogenesis but not in non-ciliated cells or the cells with mature cilia. d FIP5-positive vesicles specifically translocate to the ciliary base during ciliogenesis. Endogenous FIP5 (upper), acetylated tubulin (Ac-Tub) and FBF1 were immunostained by corresponding antibodies and FIP5-mcherry (lower) was shown by direct fluorescence. Ac-Tub was used as a ciliary marker and FBF1 was used as the transition fiber marker. Scar bar: 10 μm. e ARL13B-EGFP RCTE stable cells were serum starved for 2 h. Structure illumination microscopic study shows that FIP5-positive vesicles locate immediately adjacent to ARL13B signal at the ciliary base in a newly synthesized cilium. Endogenous FIP5 was immunostained by antibody and ARL13B-EGFP was shown by direct fluorescence. Association of FIP5-positive vesicles and ARL13B were indicated by white triangles. Scar bars: 500 nm. f Depletion of ARL13B abolished the recruitment of FIP5-positive vesicles at the ciliary base in hTERT-RPE-1 cells. Quantification of FIP5 total intensity at mother centriole (non-ciliated cells) or cilia base (30 μm² area were measured). Scar bar: 10 μm. Center values represent mean. Error bars represent s.d. N values: cilia number accessed from at least six fields. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01

Fig. 2

ARL13B and FIP5 regulate axoneme polyglutamylation by controlling the ciliary import of glutamylase TTLL5 and TTLL6. a hTERT-RPE-1 cells were treated with indicated siRNAs for 48 h and serum starved for 24 h. Axoneme polyglutamylation was immunostained by antibody GT335. ARL13B was used as a ciliary marker. (right) Quantitation of the (GT335 staining)/(Cilia length) ratio (siNC: n = 43, siFIP5#1: n = 39, siFIP5#2: n = 41 cilia). b CCP5 ciliary localization was not affected by FIP5 depletion. hTERT-RPE-1 stable cells expressing CCP5-EYFP were treated with indicated siRNAs for 48 h, staved for 24 h. c TTLL5 and TTLL6 were detected inside cilia and on vesicle-like organelles at the ciliary base. hTERT-RPE-1 stable cells expressing TTLL5-EYFP and TTLL6-EYFP were starved for 24 h. Quantification of the percentage of different TTLL5/6 localization patterns in transfected RPE cells (n = 50 cilia). d SIM images show that FIP5-positive vesicles (Red) associate with TTLL5- or TTLL6-positive vesicles (Green) at the ciliary base of hTERT-RPE-1 stable cells expressing TTLL5-EYFP and TTLL6-EYFP. Enlarged image of the box area in left panel. e, f Depletion of FIP5 abolished the enrichment of TTLL5- (e) or TTLL6-positvie (f) vesicles at the ciliary base. hTERT-RPE-1 stable cells expressing TTLL5-EYFP and TTLL6-EYFP were treated with the indicated siRNAs for 48 h and serum staved for 24 h. Quantified of TTLL5/6-EYFP total intensity at cilia base (30 μm² area were measured). g Depletion of TTLL5 or TTLL6 impaired axoneme polyglutamylation. Quantified data shown in right panels (siNC: n = 39, siTTLL5: n = 35, siTTLL6: n = 36, siTTLL5 + 6: n = 38 cilia). h The residual polyglutamylation locates above transition fibers, presumably at the transition zone. hTERT-RPE-1 cells were treated with indicated siRNAs for 48 h and serum starved for 24 h. FBF1 was used as a marker of transition fibers. ARL13B, Ac-Tub, FIP5, FBF1 and glutamylation were immunostained by antibodies. TTLL5/6-EYFP and CCP5-EYFP was shown by direct fluorescence. Scale bars in a, b, d, g and h are 2 μm. Scale bars in c, e and f are 5 μm. Center values represent mean. Error bars represent s.d. N values: cilia number accessed from at least six fields. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01

Fig. 3

RAB11 regulates the ciliary import of TTLL5 and TTLL6 in FIP5- and ARL13B-dependent manner. a Depletion of FIP5 or ARL13B impaired ciliary base enrichment for RAB11A-positive vesicles in hTERT-RPE-1 cells. Quantified of RAB11A total intensity at cilia base (30 μm² area were measured). Scale bar: 10 μm. b hTERT-RPE-1 stable cells expressing TTLL5-EYFP and TTLL6-EYFP were treated with 20 nM RAB11A + B siRNAs for 48 h and serum starved for 24 h, the impaired enrichment of TTLL5- or TTLL6-positive vesicles at the ciliary base was observed. Quantified of TTLL5/6-EYFP total intensity at cilia base (30 μm² area were measured). Ac-Tub was immunostained by antibody. Scale bar: 5 μm. c Depletion of ARL13B compromised the enrichment of TTLL5- and TTLL6-positive vesicles at the ciliary base as seen. Quantified of TTLL5/6-EYFP total intensity at cilia base (30 μm² area were measured). Ac-Tub was immunostained by antibodies and TTLL5/6-EYFP was shown by direct fluorescence. Scale bar: 5 μm. d, e Depletion of RAB11 impaired axoneme polyglutamylation (scale bar: 2 μm). Quantification of the length of GT335 signal (d, right panel) and the (GT335 staining)/(Cilia length) ratio (siNC: n = 57, siRAB11A + B: n = 55 cilia) (e) were shown. Center values represent mean. Error bars represent s.d. N value: cilia number accessed from at least six fields. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01

Fig. 4

A balanced enzymatic activity between CCP5 and TTLL5/TTLL6 is critical for maintaining a proper level of axoneme polyglutamylation. Axoneme polyglutamylation was detected by antibody GT335 or polyE. GT335 antibody detects all polyglutamate side chains independently of their length (≥1 glutamates) while polyE antibody is specific to long side chains (≥3 glutamates). a Depletion of CCP5 increased axoneme polyglutamylation and cilia length. Quantification in right panels. b–d Depletion of CCP5 corrected FIP5- (b, siNC: n = 54, siFIP5: n = 42, siFIP5 + siCCP5: n = 49 cilia), TTLL5- (c, n = 50 cilia for each group), or TTLL6- (d, n = 50 cilia for each group) knockdown-induced defective axoneme polyglutamylation as detected by GT335 antibody. Quantification in right panels. e Depletion of CCP5 could not restore long-chain axoneme glutamylation as detected by polyE antibody. Quantification in right panel (n = 50 cilia for each group). f Depletion of CCP5 restored axoneme polyglutamylation in ARL13B-deficient cells as detected by GT335 antibody. Quantification in right panel. Scale bars: 2 μm. Center values represent mean. Error bars represent s.d. N values: cilia number accessed from at least six fields. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01

Fig. 5

Axonemal hypoglutamylation promotes cilia disassembly. a, b hTERT-RPE-1 cells were serum starved for 24 h and then cultured in the medium containing 20% FBS for the indicated times. Relative cilia length (a) and resorption rate (b) were measured and quantified at the indicated times. c, d CCP5 knockdown restores relative cilia length (c) and resorption rate (d) in FIP5-depleted hTERT-RPE-1 cells. e, f CCP5 knockdown restores relative cilia length (e) and resorption rate (f) in TTLL5-depleted hTERT-RPE-1 cells. g, h CCP5 knockdown partially restored ciliogenesis defect in ARL13B knockdown cells by rescuing both cilia length (g) and ciliation ratio (h). Center values represent mean. Error bars represent s.d. N value: cilia number accessed from at least six fields. In all experiments, hTERT-RPE-1 cells were treated with indicated siRNAs for 48 h and serum starved for 24 h. Data in a–f and h are the statistical analysis of three independent experiments. Statistical significance was determined using unpaired Student’s t test. * p < 0.05; ** p < 0.01

Fig. 6

Axoneme hypoglutamylation compromises the ciliary localization of polycystins, which can be restored by concomitant depletion of deglutamylase CCP5. a–c hTERT-RPE-1 cells were treated with indicated siRNAs for 48 h and serum starved for 24 h. Ciliary localizations of endogenous Polycystin 2 (PC2) were examined by immunofluorescence staining. The (PC2 staining)/(cilia length) ratios were quantified in the right panel (n = 50 cilia for each group). d CCP5-depleted stable hTERT-RPE-1 cells were constructed by shRNA lentivirus system. Scramble shRNA sequence was used as negative control. Ciliary localization of PC2 was examined by immunofluorescence staining. The length, total intensity and relative intensity/μm² of ciliary PC2 were quantified in the right panels. e–g Depletion of CCP5 restored the ciliary localizations of PC2 in FIP5- (e), TTLL5- (f), or TTLL6- (g) depleted hTERT-RPE-1 cells. Cells were treated with indicated siRNAs and the (PC2 staining)/(cilia length) ratios were quantified in the right panels (n = 40 cilia for each group). h CCP5-depleted stable GANAB^−/− RCTE cells were constructed by shRNA lentivirus system. Ciliary localizations of PC2 were examined by immunofluorescence staining. Scale bars in a–g are 2 μm. Scale bar in h is 5 μm. Center values represent mean. Error bars represent s.d. N values: cilia number accessed from at least six fields. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01

Fig. 7

Defective Shh signaling induced by axoneme hypoglutamylation can be restored by concomitant CCP5 depletion. a hTERT-RPE-1 cells were treated with the indicated siRNAs for 48 h, serum starved, and treated with 500 nM SAG for 24 h. GLI3 ciliary localizations were examined by immunofluorescent staining. Percentages of the cilia with GLI3 at cilia tip were quantified. Scale bar: 2 μm. b Depletion of either FIP5 or TTLL6 abolished GLI1 expression upon hedgehog activation. hTERT-RPE-1 cells were treated with the indicated siRNAs for 48 h, serum starved, and treated with 500 nM SAG for 24 h. The relative mRNA levels of GLI1 were measured by real-time PCR. c Depletion of CCP5 suppressed GLI3 localization to cilia tip. Percentages of the cilia with GLI3 in cilia tip were quantified. d Depletion of CCP5 restored the ciliary tip localization of GLI3 in FIP5 or TTLL5-depleted hTERT-RPE-1 cells. Percentages of the cilia with GLI3 in cilia tip were quantified. e Depletion of CCP5 rescued GLI1 expression induced by SAG as seen by real-time PCR. Data shown in a–e are the statistical analysis of three independent experiments. f Schematic model of polyglutamylation regulation and function in the context of cilia. During the early stage of ciliogenesis, ARL13B recruits RAB11/FIP5-positive vesicles, and then RAB11/FIP5-positive vesicles tether TTLL5/6-containing vesicles to the proximity of the ciliary base, which allowing cilia import of TTLL5/6. Inside cilia, axoneme polyglutamylation was controlled by the balanced enzymatic activities between TTLLs (mainly TTLL5 and TTLL6) and ciliary deglutamylase CCPs (CCP5 and other CCPs that shorten the long glutamate chains). Polyglutamylated axoneme anchors sensory receptor polycystins to the ciliary surface via unknown adaptor mechanism, which could interact with both the glutamylated chain and the intercellular domain of Polycystins. Additionally, axoneme polyglutamylation is required for proper translocation of hedgehog signaling molecule GLI3 to the ciliary tip, likely through a different adaptor mechanism that can recognize both polyglutamylated chain and intraflagellar transport machinery. Center values represent mean. Error bars represent s.d. Statistical significance was determined using two-way ANOVA test. * p < 0.05; ** p < 0.01. n.s. not significant