ATR promotes cilia signalling: links to developmental impacts

Mutations in ATR (ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development

Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of origin recognition complex factors

Meier–Gorlin syndrome (MGS) is a rare, congenital primordial microcephalic dwarfism disorder. MGS is caused by genetic variants of components of the origin recognition complex (ORC) consisting of ORC1–6 and the pre-replication complex, which together enable origin firing and hence genome replication. In addition, ORC1 has previously been shown to play a role in ciliogenesis. Here, we extend this work and investigate the function of ORC1 and two other members of the complex on cilia at an organismal level. Knockdown experiments in zebrafish confirmed the impact of ORC1 on cilia. ORC1-deficiency confers defects anticipated to arise from impaired cilia function such as formation of oedema, kidney cysts, curved bodies and left–right asymmetry defects. We found ORC1 furthermore required for cilium formation in zebrafish and demonstrate that ciliopathy phenotypes in ORC1-depleted zebrafish could not be rescued by reconstitution with ORC1 bearing a genetic variant previously identified in MGS patients. Loss-of-function of Orc4 and Orc6, respectively, conferred similar ciliopathy phenotypes and cilium shortening in zebrafish, suggesting that several, if not all, components of the ORC regulate ciliogenesis downstream to or in addition to their canonical function in replication initiation. This study presents the first in vivo evidence of an influence of the MGS genes of the ORC family on cilia, and consolidates the possibility that cilia dysfunction could contribute to the clinical manifestation of ORC-deficient MGS

The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5

G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5

Resting cells rely on the DNA helicase component MCM2 to build cilia

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues

Centrobin controls primary ciliogenesis in vertebrates

The BRCA2 interactor, centrobin, is a centrosomal protein that has been implicated in centriole duplication and microtubule stability. We used genome editing to ablate CNTROB in hTERT-RPE1 cells and observed an increased frequency of monocentriolar and acentriolar cells. Using a novel monoclonal antibody, we found that centrobin primarily localizes to daughter centrioles but also associates with mother centrioles upon serum starvation. Strikingly, centrobin loss abrogated primary ciliation upon serum starvation. Ultrastructural analysis of centrobin nulls revealed defective axonemal extension after mother centriole docking. Ciliogenesis required a C-terminal portion of centrobin that interacts with CP110 and tubulin. We also depleted centrobin in zebrafish embryos to explore its roles in an entire organism. Centrobin-depleted embryos showed microcephaly, with curved and shorter bodies, along with marked defects in laterality control, morphological features that indicate ciliary dysfunction. Our data identify new roles for centrobin as a positive regulator of vertebrate ciliogenesis