Oral-facial-digital syndromes (OFDS) are a group of clinically and genetically heterogeneous disorders characterized by defects in the development of the face and oral cavity along with digit anomalies. Pathogenic variants in over 20 genes encoding ciliary proteins have been found to cause OFDS through deleterious structural or functional impacts on primary cilia. We identified by exome sequencing bi-allelic missense variants in a novel disease-causing ciliary gene RAB34 in four individuals from three unrelated families. Affected individuals presented a novel form of OFDS (OFDS-RAB34) accompanied by cardiac, cerebral, skeletal and anorectal defects. RAB34 encodes a member of the Rab GTPase superfamily and was recently identified as a key mediator of ciliary membrane formation. Unlike many genes required for cilium assembly, RAB34 acts selectively in cell types that use the intracellular ciliogenesis pathway, in which nascent cilia begin to form in the cytoplasm. We find that the protein products of these pathogenic variants, which are clustered near the RAB34 C-terminus, exhibit a strong loss of function. Although some variants retain the ability to be recruited to the mother centriole, cells expressing mutant RAB34 exhibit a significant defect in cilium assembly. While many Rab proteins have been previously linked to ciliogenesis, our studies establish RAB34 as the first small GTPase involved in OFDS and reveal the distinct clinical manifestations caused by impairment of intracellular ciliogenesis.
We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.
- MeSH
- cilie genetika MeSH
- ciliopatie * genetika MeSH
- databáze faktografické MeSH
- genový knockout MeSH
- myši knockoutované MeSH
- myši MeSH
- proteiny buněčného cyklu MeSH
- proteiny nervové tkáně MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Primary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling.
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- aurora kinasa A antagonisté a inhibitory genetika metabolismus MeSH
- bazální tělíska metabolismus MeSH
- buněčný cyklus genetika MeSH
- chromatografie kapalinová MeSH
- cilie genetika metabolismus patologie MeSH
- HEK293 buňky MeSH
- interfáze fyziologie MeSH
- intracelulární signální peptidy a proteiny genetika metabolismus MeSH
- kineziny genetika metabolismus MeSH
- lidé MeSH
- mitóza genetika MeSH
- onkogenní proteiny genetika metabolismus MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny hedgehog metabolismus MeSH
- RNA interference MeSH
- signální transdukce genetika MeSH
- sodíkové kanály metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: The aim of this study was to investigate the potential association and combined prognostic significance of the frequency of primary cilia (PC), programmed cell death protein-1 receptor (PD1) and CD8+ tumor infiltrating lymphocytes (TIL) in patients with clear cell renal cancer (ccRCC). METHODS: The frequency of PC, PD1 expression and the frequency of intratumoral CD8+ TIL were evaluated in 104 ccRCC patients. RESULTS: The median frequency of PC was 0.003. The expression of PD1+ cells were <5% in 52 patients, 5-25% in 34 patients and 26-50% in 13 patients and >50% in 5 patients. Intratumoral CD8+ TIL were evaluable in all patients: negative in 1 patient, <25% in 63, 26-50% in 29 and >50% in 11 patients. Overall survival (OS) according to the frequency of PC was significantly shorter in patients with higher frequency (≥0.002) than in patients with lower frequency (<0.002) (p<0.001). Median OS was significantly shorter in patients with higher (25%) CD8+ TIL and higher (>25%) PD1+ expression than in patients with lower (<25%) expression (4.6 vs. 97. years, p=0.006 and 2.9 vs. 8.9 years, p=0.006, respectively). CONCLUSIONS: The present study provides the first data on the potential association and combined prognostic significance of frequency of PC, PD1+ cells and CD8+ TIL in patients with clear cell renal cancer.
- MeSH
- antigeny CD279 genetika MeSH
- CD8-pozitivní T-lymfocyty patologie MeSH
- cilie genetika patologie MeSH
- dospělí MeSH
- Kaplanův-Meierův odhad MeSH
- karcinom z renálních buněk genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- přežití po terapii bez příznaků nemoci MeSH
- prognóza * MeSH
- regulace genové exprese u nádorů MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- tumor infiltrující lymfocyty patologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.
- MeSH
- buněčná membrána metabolismus MeSH
- cilie genetika fyziologie ultrastruktura MeSH
- delece genu MeSH
- endocytóza MeSH
- flagella genetika fyziologie ultrastruktura MeSH
- interakce hostitele a parazita MeSH
- Leishmania genetika patogenita fyziologie ultrastruktura MeSH
- mezibuněčné spoje MeSH
- myši MeSH
- protozoální proteiny genetika metabolismus MeSH
- Psychodidae parazitologie MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.
- MeSH
- Bardetův-Biedlův syndrom genetika metabolismus MeSH
- bazální tělíska metabolismus ultrastruktura MeSH
- cilie genetika metabolismus MeSH
- ciliopatie genetika metabolismus MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- encefalokéla genetika metabolismus MeSH
- flagella genetika metabolismus ultrastruktura MeSH
- fluorescenční mikroskopie MeSH
- kompartmentace buňky MeSH
- lidé MeSH
- mutace MeSH
- polycystická choroba ledvin genetika metabolismus MeSH
- poruchy ciliární motility genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA interference MeSH
- transmisní elektronová mikroskopie MeSH
- Trypanosoma genetika metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.
- MeSH
- cilie genetika patologie MeSH
- exom genetika MeSH
- kojenec MeSH
- kostra abnormality růst a vývoj MeSH
- lidé MeSH
- MAP kinasový signální systém MeSH
- mnohočetné abnormality genetika patofyziologie MeSH
- protein-serin-threoninkinasy genetika MeSH
- proteiny hedgehog genetika MeSH
- rodokmen MeSH
- sekvenční analýza DNA MeSH
- signální transdukce MeSH
- syndrom krátkého žebra a polydaktylie genetika patologie MeSH
- těhotenství MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.
- MeSH
- cilie genetika metabolismus MeSH
- cytokineze genetika MeSH
- cytoskelet genetika metabolismus MeSH
- flagella genetika metabolismus MeSH
- mikrotubuly genetika MeSH
- protozoální proteiny genetika metabolismus MeSH
- stadia vývoje genetika MeSH
- Trypanosoma brucei brucei genetika růst a vývoj MeSH
- tvar buňky genetika MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits β-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- beta-katenin antagonisté a inhibitory metabolismus MeSH
- buňky NIH 3T3 MeSH
- cilie genetika patologie MeSH
- cystická onemocnění ledvin genetika MeSH
- dánio pruhované genetika MeSH
- exony MeSH
- fenotyp MeSH
- fosfoproteiny genetika metabolismus MeSH
- HEK293 buňky MeSH
- ledviny patologie MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- proteiny asociované s mikrotubuly genetika metabolismus MeSH
- signální dráha Wnt genetika MeSH
- transmisní elektronová mikroskopie MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
