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

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine

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

The impact of soil organism composition and activated carbon on grass-legume competition

Belowground mechanisms involved in plant competition are still poorly understood. Since plant species are differently affected by soil organisms, changes in soil community composition might affect interspecific competition with consequences for plant community structure. We studied whether soil community composition affects competition between the grass Holcus lanatus L. and the legume Lotus corniculatus L. We established three different soil communities by adding no soil organisms (control), microorganisms 30 μm, AMF and nematodes to gamma-sterilized soil. Nodulation and aboveground biomass of Lotus was decreased in the sterilized control soil and in the presence of Holcus. Contrastingly, the grass grew better in the presence of the legume than in monoculture and was not affected by soil community composition. Legume monocultures tended to produce the greatest aboveground biomass of the plant combinations when soil microorganisms were present, while the root biomass in legume monocultures was the lowest. Then, in a second experiment, we used natural (not sterilized) soil and added activated carbon to test whether the reduced nodulation of Lotus in interspecific competition is caused by allelopathic compounds of Holcus. In the natural soil, nodulation and flowering of Lotus was reduced, but the aboveground biomass was not affected by the competition with Holcus. Contrary to our expectations, activated carbon had a strong negative effect on the nodulation, growth and flowering of Lotus and shifted the interspecific competition in favour of Holcus. Probably, activated carbon impeded the nodulation by disrupting the communication between the legume and N2-fixing bacteria. We suggest that interruption of plant-microbe communications by activated carbon might be widespread and will confound interpretations on the role of allelopathy. Generally, we observed that the symbiosis of the legume with N2-fixing bacteria plays a crucial role in the grass-legume competition. When the symbiosis was deterred, the legume was outcompeted by the grass

Plant defence against nematodes is not mediated by changes in the soil microbial community

1. Indirect plant defence, the recruitment of antagonists of herbivores, is well-known above the ground. In spite of various soil microorganisms acting as antagonists to root herbivores, it is still largely unknown whether plants can promote antagonistic microorganisms as an indirect defence mechanism. 2. In a greenhouse study we examined whether soil microorganisms could mediate plant defence against plant-feeding nematodes. Growth, nutrient contents and root exudation of three plant species (Plantago lanceolata, Holcus lanatus, Lotus corniculatus) and the performance of nematodes and fungal communities in the rhizospheres were measured. 3. The plant species differed in their effects on plant-feeding nematodes; however, the addition of soil microorganisms did not enhance nematode control. Nematode addition changed root exudation patterns and rhizosphere fungal community structure in a plant species-specific manner. Glucose levels in the root exudates of all three examined plant species were enhanced, and P. lanceolata root exudates contained higher levels of fumaric acid when nematodes had been added. 4. We conclude that nematodes have plant species-specific effects on root exudate chemistry and rhizosphere fungal community composition, but these effects do not necessarily enhance indirect control of nematodes by antagonistic microorganisms. More studies on below-ground plant defence are definitely needed

Study on the gene and phenotypic characterisation of autosomal recessive demyelinating motor and sensory neuropathy (Charcot-Marie-Tooth disease) with a gene locus on chromosome 5q23-q33

OBJECTIVES—To report the occurrence of the autosomal recessive form of demyelinating Charcot-Marie-Tooth disease (CMT) with a locus on chromosome 5q23-33 in six non-related European families, to refine gene mapping, and to define the disease phenotype.
METHODS—In an Algerian patient with autosomal recessive demyelinating CMT mapped to chromosome 5q23-q33 the same unique nerve pathology was established as previously described in families with a special form of autosomal recessive demyelinating CMT. Subsequently, the DNA of patients with this phenotype was tested from five Dutch families and one Turkish family for the 5q23-q33 locus.
RESULTS—These patients and the Algerian families showed a similar and highly typical combination of clinical and morphological features, suggesting a common genetic defect. A complete cosegregation for markers D5S413, D5S434, D5S636, and D5S410 was found in the families. Haplotype construction located the gene to a 7 cM region between D5S643 and D5S670. In the present Dutch families linkage disequilibrium could be shown for various risk alleles and haplotypes indicating that most of these families may have inherited the underlying genetic defect form a common distant ancestor.
CONCLUSIONS—This study refines the gene localisation of autosomal recessive demyelinating CMT, mapping to chromosome 5q23-33 and defines the phenotype characterised by a precocious and rapidly progressive scoliosis in combination with a relatively mild neuropathy and a unique pathology. Morphological alterations in Schwann cells of the myelinated and unmyelinated type suggest the involvement of a protein present in both Schwann cell types or an extracellular matrix protein rather than a myelin protein. The combination of pathological features possibly discerns autosomal recessive demyelinating CMT with a gene locus on chromosome 5q23-33 from other demyelinating forms of CMT disease.


Analysis of the p63 gene in classical EEC syndrome, related syndromes, and non-syndromic orofacial clefts

EEC syndrome is an autosomal dominant disorder with the cardinal signs of ectrodactyly, ectodermal dysplasia, and orofacial clefts. EEC syndrome has been linked to chromosome 3q27 and heterozygous p63 mutations were detected in unrelated EEC families. In addition, homozygous p63 null mice exhibit craniofacial abnormalities, limb truncations, and absence of epidermal appendages, such as hair follicles and tooth primordia. In this study, we screened 39 syndromic patients, including four with EEC syndrome, five with syndromes closely related to EEC syndrome, and 30 with other syndromic orofacial clefts and/or limb anomalies. We identified heterozygous p63 mutations in three unrelated cases of EEC syndrome, two Iowa white families and one sporadic case in a Filipino boy. One family is atypical for EEC and has features consistent with Hay-Wells syndrome. In this family, the mutation ablates a splice acceptor site and, in the other two, mutations produce amino acid substitutions, R280C and R304Q, which alter conserved DNA binding sites. Germline mosaicism was detected in the founder of the mutation in one case. These three cases show significant interfamilial and intrafamilial variability in expressivity. We also screened p63 in 62 patients with non-syndromic orofacial clefts, identifying an intronic single nucleotide polymorphism but finding no evidence of mutations that would explain even a subset of non-syndromic orofacial clefts. This study supports a common role for p63 in classical EEC syndrome, both familial and sporadic, but not in other related or non-syndromic forms of orofacial clefts