A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome

ARMC9 and TOGARAM1 define a Joubert syndrome-associated protein module that regulates axonemal post-translational modifications and cilium stability

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 use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module composed of ARMC9-TOGARAM1-CCDC66-CEP104- CSPP1. TOGARAM1-variants cause JBTS and disrupt its interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and glutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in both ARMC9 and TOGARAM1 patient cells lines suggest a role for this new JBTS-associated protein complex in ciliary stability

Missense mutations in the WD40 domain of AHI1 cause non-syndromic retinitis pigmentosa

Background: Recent findings suggesting that Abelson helper integration site 1 (AHI1) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We assessed whether AHI1 variants cause non-syndromic retinitis pigmentosa (RP). Methods: Exome sequencing was performed in three probands with RP. The effects of the identified missense variants in AHI1 were predicted by three-dimensional structure homology modelling. Ciliary parameters were evaluated in patient's fibroblasts, and recombinant mutant proteins were expressed in ciliated retinal pigmented epithelium cells. Results: In the three patients with RP, three sets of compound heterozygous variants were detected in AHI1 (c.2174G>A; p.Trp725* and c.2258A>T; p.Asp753Val, c.660delC; p.Ser221Glnfs*10 and c.2090C>T; p.Pro697Leu, c.2087A>G; p.His696Arg and c.2429C>T; p.Pro810Leu). All four missense variants were present in the conserved WD40 domain of Jouberin, the ciliary protein encoded by AHI1, with variable predicted implications for the domain structure. No significant changes in the percentage of ciliated cells, nor in cilium length or intraflagellar transport were detected. However, expression of mutant recombinant Jouberin in ciliated cells showed a significantly decreased enrichment at the ciliary base. Conclusions: This report confirms that mutations in AHI1 can underlie autosomal recessive RP. Moreover, it structurally and functionally validates the effect of the RP-associated AHI1 variants on protein function, thus proposing a new genotype-phenotype correlation for AHI1 mutation associated retinal ciliopathies

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4)

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

Tiina Paunio on työryhmän UK10K jäsen.The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.Peer reviewe

PDE6D Mediates Trafficking of Prenylated Proteins NIM1K and UBL3 to Primary Cilia

Mutations in PDE6D impair the function of its cognate protein, phosphodiesterase 6D (PDE6D), in prenylated protein trafficking towards the ciliary membrane, causing the human ciliopathy Joubert Syndrome (JBTS22) and retinal degeneration in mice. In this study, we purified the prenylated cargo of PDE6D by affinity proteomics to gain insight into PDE6D-associated disease mechanisms. By this approach, we have identified a specific set of PDE6D-interacting proteins that are involved in photoreceptor integrity, GTPase activity, nuclear import, or ubiquitination. Among these interacting proteins, we identified novel ciliary cargo proteins of PDE6D, including FAM219A, serine/threonine-protein kinase NIM1 (NIM1K), and ubiquitin-like protein 3 (UBL3). We show that NIM1K and UBL3 localize inside the cilium in a prenylation-dependent manner. Furthermore, UBL3 also localizes in vesicle-like structures around the base of the cilium. Through affinity proteomics of UBL3, we confirmed its strong interaction with PDE6D and its association with proteins that regulate small extracellular vesicles (sEVs) and ciliogenesis. Moreover, we show that UBL3 localizes in specific photoreceptor cilium compartments in a prenylation-dependent manner. Therefore, we propose that UBL3 may play a role in the sorting of proteins towards the photoreceptor outer segment, further explaining the development of PDE6D-associated retinal degeneration

Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARl 13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain

Contains fulltext : 125199.pdf (publisher's version ) (Open Access

Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility.

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly