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 7

ARMC9- and TOGARAM1-mutant cilia display reduced tubulin PTMs in both patient fibroblasts and zebrafish ventricular cells.

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ARMC9- and TOGARAM1-mutant cilia display reduced tubulin PTMs in both pa...
(A and B) Immunofluorescence images and immunoblots of (A) acetylated and (B) polyglutamylated tubulin in ARMC9 patient fibroblasts versus control. In the immunoblots, GIANTIN and β-actin were used as loading controls. Scale bars: 3 μm. (C and D) Representative immunofluorescence images of 3-dpf zebrafish hindbrain cilia marked with Arl13b (red) and acetylated (green in C) or polyglutamylated (green in D) tubulin. Scale bars: 10 μm. Original magnification, ×3.5 (insets). Note that acetylated tubulin also marks axons in the developing brain, visible at the edges of the image in C. (E) Normalized relative fluorescence intensity for acetylated tubulin signal in human fibroblast cilia (yellow panel: control n = 1106, ARMC9 n = 532, and TOGARAM1 n = 131) and zebrafish hindbrain cilia (pink panel: pooled data from 2 experiments; 10 cilia measured per larva; each data point represents 1 larva; armc9 control [gray] n = 20, armc9–/– [green] n = 21, togaram1 control [gray] n = 20, togaram1–/– [blue] n = 20). (F) Normalized relative fluorescence intensity for polyglutamylated tubulin assessed in human fibroblast cilia (yellow panel: pooled from 3 experiments; control n = 602, ARMC9 n = 298, and TOGARAM1 n = 58) and zebrafish hindbrain cilia (pink panel: pooled data from 2 experiments; 10 cilia measured per larva; armc9 control [gray] n = 22, armc9–/– [green] n = 20, togaram1 control [gray] n = 25, togaram1–/– [blue] n = 20). Zebrafish controls were WT, +/+, or +/– siblings of –/–. In E and F, data points greater than 4 and less than or equal to 2 are not displayed but were included in the statistical analysis. For a complete graph of all data points and a graphical summary of all ARMC9 lines, see Supplemental Figure 8, A and B and C and D, respectively. Statistical significance (adjusted P < 0.025) was assessed using a Bonferroni-corrected Student’s t test for both fibroblast and zebrafish experiments. **P ≤ 0.01 and ****P ≤ 0.0001.