Structure of the ciliogenesis-associated CPLANE complex
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Abstract:
Dysfunctional cilia cause pleiotropic human diseases termed ciliopathies. These hereditary maladies are often caused by defects in cilia assembly, a complex event that is regulated by the ciliogenesis and planar polarity effector (CPLANE) proteins Wdpcp, Inturned, and Fuzzy. CPLANE proteins are essential for building the cilium and are mutated in multiple ciliopathies, yet their structure and molecular functions remain elusive. Here, we show that mammalian CPLANE proteins comprise a bona fide complex and report the near-atomic resolution structures of the human Wdpcp-Inturned-Fuzzy complex and of the mouse Wdpcp-Inturned-Fuzzy complex bound to the small guanosine triphosphatase Rsg1. Notably, the crescent-shaped CPLANE complex binds phospholipids such as phosphatidylinositol 3-phosphate via multiple modules and a CPLANE ciliopathy mutant exhibits aberrant lipid binding. Our study provides critical structural and functional insights into an enigmatic ciliogenesis-associated complex as well as unexpected molecular rationales for ciliopathies.Keywords:
Ciliogenesis
Ciliopathies
Ciliopathy
Motile cilium
The primary cilium is a single immotile microtubule-based organelle that protrudes into the extracellular space. Malformations and dysfunctions of the cilia have been associated with various forms of syndromic and non-syndromic diseases, termed ciliopathies. The primary cilium is therefore gaining attention due to its potential as a therapeutic target. In this review, we examine ciliary receptors, ciliogenesis, and ciliary trafficking as possible therapeutic targets. We first discuss the mechanisms of selective distribution, signal transduction, and physiological roles of ciliary receptors. Next, pathways that regulate ciliogenesis, specifically the Aurora A kinase, mammalian target of rapamycin, and ubiquitin-proteasome pathways are examined as therapeutic targets to regulate ciliogenesis. Then, in the photoreceptors, the mechanism of ciliary trafficking which takes place at the transition zone involving the ciliary membrane proteins is reviewed. Finally, some of the current therapeutic advancements highlighting the role of large animal models of photoreceptor ciliopathy are discussed.
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Bardet–Biedl Syndrome
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Ciliopathies
Ciliopathy
Joubert syndrome
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Nephronophthisis
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Ciliogenesis
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Cilia are highly conserved, microtubule-based organelles. The growing numbers of human diseases due to cilia dysfunction, termed ciliopathies, highlights the functional importance of cilia. Primary ciliary dyskinesia (PCD) is a genetic disorder of the motile cilia, displaying diverse clinical manifestations. With many molecular and genetic tools, zebrafish have emerged as a useful organism model, which allows the investigation of human diseases. Here, we describe the utility of zebrafish in the study of cilia and PCD. With forward and reverse genetic approaches, several novel mutations encoding essential components in motile cilia had been identified in zebrafish. These discoveries will increase our understanding toward cilia biology and have the potential to improve the diagnosis and treatment for affected individuals.
Ciliopathies
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Joubert syndrome
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Primary cilia are microtubule-based “antennae-like” organelles extending from the apical surface of most mammalian cells. They integrate mechanical or chemical signals essential for cell homeostasis and differentiation through several cooperating compartments. Mutations in genes that encode ciliary proteins or proteins essential for correct ciliary function are the cause of a major group of inherited and variable developmental disorders known as ciliopathies. Functional interactions between ciliary compartments, the molecular basis of variable clinical phenotypes, and the mechanisms of cilia formation are all still poorly understood.
The presence or absence of cilia can be easily imaged and quantitated. This lends itself to high-throughput, high-content imaging in reverse genetic screens of cellular phenotypes. siRNA reverse genetic screens were performed to assess increased cilia incidence and identified a key negative regulator of ciliogenesis. Analysis showed this regulator acts through a mechanism involving actin remodelling and acto-myosin contraction. Pharmacological inhibition of this target may therefore comprise a novel therapeutic approach for treatment of a broad group of ciliopathy disease classes. Further screening of a data-set for supernumerary primary cilia, added to the evidence that cytokinesis is not a prerequisite for ciliogenesis. Disruption of the centralspindilin complex caused mitotic failure and maturation of supernumerary centrosomes, leading to the formation of the supernumerary cilia, a known cellular phenotype of severe ciliopathies.
Finally, a combinatorial screening approach to generate double genetic perturbations of ciliary genes identified reciprocal synthetic genetic interactions between anterograde intraflagellar transport (the IFT B complex) and the transition zone.
Reverse genetics screening techniques have identified novel regulators and pathways of ciliogenesis, and a potential therapeutic target for ciliopathies. Furthermore, combinatorial screening has highlighted a novel and complex interaction in ciliary biology, that may provide potential new insights into ciliary organisation and disease pathomechanisms.
Ciliogenesis
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Ciliopathy
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Ciliopathies
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The cilia are microtubule-based organelles that protrude from the cell surface. Abnormalities in cilia result in various ciliopathies, including polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS), and oral-facial-digital syndrome type I (OFD1), which show genetic defects associated with cilia formation. Although an increasing number of human diseases is attributed to ciliary defects, the functions or regulatory mechanisms of several ciliopathy genes remain unclear. Because multi ciliated cells (MCCs) are especially deep in vivo, studying ciliogenesis is challenging. Here, we demonstrate that ik is essential for ciliogenesis in vivo.In the absence of ik, zebrafish embryos showed various ciliopathy phenotypes, such as body curvature, abnormal otoliths, and cyst formation in the kidney. RNA sequencing analysis revealed that ik positively regulated ofd1 expression required for cilium assembly. In fact, depletion of ik resulted in the downregulation of ofd1 expression with ciliary defects, and these ciliary defects in ik mutants were rescued by restoring ofd1 expression. Interestingly, ik affected ciliogenesis particularly in the proximal tubule but not in the distal tubule in the kidney.This study demonstrates the role of ik in ciliogenesis in vivo for the first time. Loss of ik in zebrafish embryos displays various ciliopathy phenotypes with abnormal ciliary morphology in ciliary tissues. Our findings on the ik-ofd1 axis provide new insights into the biological function of ik in clinical ciliopathy studies in humans.
Ciliopathy
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Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert-syndrome and related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from induced pluripotent stem cells (iPSCs) of CEP290-LCA patients displayed less developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defects in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium.
Ciliogenesis
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The primary cilium is found in most mammalian cells and plays a functional role in tissue homeostasis and organ development by modulating key signaling pathways. Ciliopathies are a group of genetically heterogeneous disorders resulting from defects in cilia development and function. Patients with ciliopathic disorders exhibit a range of phenotypes that include nephronophthisis (NPHP), a progressive tubulointerstitial kidney disease that commonly results in end-stage renal disease (ESRD). In recent years, distal appendages (DAPs), which radially project from the distal end of the mother centriole, have been shown to play a vital role in primary ciliary vesicle docking and the initiation of ciliogenesis. Mutations in the genes encoding these proteins can result in either a complete loss of the primary cilium, abnormal ciliary formation, or defective ciliary signaling. DAPs deficiency in humans or mice commonly results in NPHP. In this review, we outline recent advances in our understanding of the molecular functions of DAPs and how they participate in nephronophthisis development.
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Cystic kidney disease
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Abstract Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans , CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.
Ciliopathy
Ciliopathies
Motile cilium
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Joubert syndrome
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