Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome
Brooke L. Latour, … , Ronald Roepman, Dan Doherty
Brooke L. Latour, … , Ronald Roepman, Dan Doherty
Published May 26, 2020
Citation Information: J Clin Invest. 2020;130(8):4423-4439. https://doi.org/10.1172/JCI131656.
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Research Article Genetics

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome

Abstract

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif–containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.

Authors

Brooke L. Latour, Julie C. Van De Weghe, Tamara D.S. Rusterholz, Stef J.F. Letteboer, Arianna Gomez, Ranad Shaheen, Matthias Gesemann, Arezou Karamzade, Mostafa Asadollahi, Miguel Barroso-Gil, Manali Chitre, Megan E. Grout, Jeroen van Reeuwijk, Sylvia E.C. van Beersum, Caitlin V. Miller, Jennifer C. Dempsey, Heba Morsy, University of Washington Center for Mendelian Genomics, Michael J. Bamshad, Genomics England Research Consortium, Deborah A. Nickerson, Stephan C.F. Neuhauss, Karsten Boldt, Marius Ueffing, Mohammad Keramatipour, John A. Sayer, Fowzan S. Alkuraya, Ruxandra Bachmann-Gagescu, Ronald Roepman, Dan Doherty

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Figure 6

ARMC9 or TOGARAM1 dysfunction does not grossly affect the TZ.

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ARMC9 or TOGARAM1 dysfunction does not grossly affect the TZ. (A) Normal...
(A) Normalized relative fluorescence intensity of ARL13B signal in human fibroblast cilia (yellow panel; data were pooled from 3 experiments; control [gray] n = 1089, ARMC9 [green] n = 582, and TOGARAM1 [blue] n = 126) and in 3-dpf zebrafish hindbrain cilia (pink panel; data were pooled from 4 experiments; 10 cilia were measured per larva; each data point represents 1 larva; control armc9 [gray] n = 42, armc9–/– [green] n = 41, control togaram1 [gray] n = 45, togaram1–/– [blue] n = 40). Bars represent the mean. Controls were WT, +/+, or +/– siblings of –/–. Statistical significance was assessed using a Student’s t test for both human fibroblast (Bonferroni-adjusted P < 0.025) and zebrafish (P < 0.05) experiments. **P ≤ 0.01 and ****P ≤ 0.0001. (B) Normalized relative fluorescence intensity of INPP5E signal in human fibroblast cilia (data were pooled from 3 experiments: control [gray] n = 620, ARMC9 [green] n = 248, TOGARAM1 [blue] n = 62). See Supplemental Figure 7 for ARL13B and INPP5E signal intensity across all ARMC9 fibroblast lines. Results were not significant using an unpaired Student’s t test. (C and D) Western blot analysis of ARL13B (C) and INPP5E (D) in ARMC9 UW132-4 patient fibroblasts. GIANTIN and β-actin served as loading controls, respectively. (E) Representative immunofluorescence signal for Arl13b (red) and polyglutamylated (green) in the 3-dpf zebrafish hindbrain cilia quantified in A. Scale bars: 10 μm. Original magnification, ×3.5 (insets). (F) Single hindbrain cilia stained with Arl13b (red) and Cc2d2a (green) in 3-dpf control, armc9–/–, and togaram1–/– zebrafish. Scale bars: 1 μm. (G) Representative immunofluorescence signal for RPGRIP1L (white) and ARL13B (red) in cilia from control and 2 TOGARAM1-mutant hTERT-RPE1 lines. Scale bars: 2 μm. (H) Representative immunofluorescence for RPGRIP1L (green) and ARL13B (red) in ARMC9 and TOGARAM1 patient fibroblasts. Percentages of cilia with robust RPGRIP1L puncta are indicated. Scale bars: 2 μm.