Molecular basis promoting centriole triplet microtubule assembly.

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the ß-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years.

Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models.

The human photoreceptor function is dependent on a highly specialised cilium. Perturbation of cilial function can often lead to death of the photoreceptor and loss of vision. Retinal ciliopathies are a genetically diverse range of inherited retinal disorders affecting aspects of the photoreceptor cilium. Despite advances in the understanding of retinal ciliopathies utilising animal disease models, they can often lack the ability to accurately mimic the observed patient phenotype, possibly due to structural and functional deviations from the human retina. Human-induced pluripotent stem cells (hiPSCs) can be utilised to generate an alternative disease model, the 3D retinal organoid, which contains all major retinal cell types including photoreceptors complete with cilial structures. These retinal organoids facilitate the study of disease mechanisms and potential therapies in a human-derived system. Three-dimensional retinal organoids are still a developing technology, and despite impressive progress, several limitations remain. This review will discuss the state of hiPSC-derived retinal organoid technology for accurately modelling prominent retinal ciliopathies related to genes, including RPGR, CEP290, MYO7A, and USH2A. Additionally, we will discuss the development of novel gene therapy approaches targeting retinal ciliopathies, including the delivery of large genes and gene-editing techniques.

The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies.

Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.

Transport and barrier mechanisms that regulate ciliary compartmentalization and ciliopathies.

Primary cilia act as cell surface antennae, coordinating cellular responses to sensory inputs and signalling molecules that regulate developmental and homeostatic pathways. Cilia are therefore critical to physiological processes, and defects in ciliary components are associated with a large group of inherited pleiotropic disorders - known collectively as ciliopathies - that have a broad spectrum of phenotypes and affect many or most tissues, including the kidney. A central feature of the cilium is its compartmentalized structure, which imparts its unique molecular composition and signalling environment despite its membrane and cytosol being contiguous with those of the cell. Such compartmentalization is achieved via active transport pathways that bring protein cargoes to and from the cilium, as well as gating pathways at the ciliary base that establish diffusion barriers to protein exchange into and out of the organelle. Many ciliopathy-linked proteins, including those involved in kidney development and homeostasis, are components of the compartmentalizing machinery. New insights into the major compartmentalizing pathways at the cilium, namely, ciliary gating, intraflagellar transport, lipidated protein flagellar transport and ciliary extracellular vesicle release pathways, have improved our understanding of the mechanisms that underpin ciliary disease and associated renal disorders.

The miR-669a-5p/G3BP/HDAC6/AKAP12 Axis Regulates Primary Cilia Length.

Primary cilia are conserved organelles in most mammalian cells, acting as "antennae" to sense external signals. Maintaining a physiological cilium length is required for cilium function. MicroRNAs (miRNAs) are potent gene expression regulators, and aberrant miRNA expression is closely associated with ciliopathies. However, how miRNAs modulate cilium length remains elusive. Here, using the calcium-shock method and small RNA sequencing, a miRNA is identified, namely, miR-669a-5p, that is highly expressed in the cilia-enriched noncellular fraction. It is shown that miR-669a-5p promotes cilium elongation but not cilium formation in cultured cells. Mechanistically, it is demonstrated that miR-669a-5p represses ras-GTPase-activating protein SH3-domain-binding protein (G3BP) expression to inhibit histone deacetylase 6 (HDAC6) expression, which further upregulates A-kinase anchor protein 12 (AKAP12) expression. This effect ultimately blocks cilia disassembly and leads to greater cilium length, which can be restored to wild-type lengths by either upregulating HDAC6 or downregulating AKAP12. Collectively, these results elucidate a previously unidentified miR-669a-5p/G3BP/HDAC6/AKAP12 signaling pathway that regulates cilium length, providing potential pharmaceutical targets for treating ciliopathies.

Patient stem cell-derived in vitro disease models for developing novel therapies of retinal ciliopathies.

Primary cilia are specialized organelles on the surface of almost all cells in vertebrate tissues and are primarily involved in the detection of extracellular stimuli. In retinal photoreceptors, cilia are uniquely modified to form outer segments containing components required for the detection of light in stacks of membrane discs. Not surprisingly, vision impairment is a frequent phenotype associated with ciliopathies, a heterogeneous class of conditions caused by mutations in proteins required for formation, maintenance and/or function of primary cilia. Traditionally, immortalized cell lines and model organisms have been used to provide insights into the biology of ciliopathies. The advent of methods for reprogramming human somatic cells into pluripotent stem cells has enabled the generation of in vitro disease models directly from patients suffering from ciliopathies. Such models help us in investigating pathological mechanisms specific to human physiology and in developing novel therapeutic approaches. In this article, we review current protocols to differentiate human pluripotent stem cells into retinal cell types, and discuss how these cellular and/or organoid models can be utilized to interrogate pathobiology of ciliopathies affecting the retina and for testing prospective treatments.

The multifaceted roles of microtubule-associated proteins in the primary cilium and ciliopathies.

The primary cilium is a conserved microtubule-based organelle that is critical for transducing developmental, sensory and homeostatic signaling pathways. It comprises an axoneme with nine parallel doublet microtubules extending from the basal body, surrounded by the ciliary membrane. The axoneme exhibits remarkable stability, serving as the skeleton of the cilium in order to maintain its shape and provide tracks to ciliary trafficking complexes. Although ciliary trafficking and signaling have been exhaustively characterized over the years, less is known about the unique structural and functional complexities of the axoneme. Recent work has yielded new insights into the mechanisms by which the axoneme is built with its proper length and architecture, particularly regarding the activity of microtubule-associated proteins (MAPs). In this Review, we first summarize current knowledge about the architecture, composition and specialized compartments of the primary cilium. Next, we discuss the mechanistic underpinnings of how a functional cilium is assembled, maintained and disassembled through the regulation of its axonemal microtubules. We conclude by examining the diverse localizations and functions of ciliary MAPs for the pathobiology of ciliary diseases.

Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Effects of Sesquiterpene Lactones on Primary Cilia Formation (Ciliogenesis).

Sesquiterpene lactones (SLs), plant-derived metabolites with broad spectra of biological effects, including anti-tumor and anti-inflammatory, hold promise for drug development. Primary cilia, organelles extending from cell surfaces, are crucial for sensing and transducing extracellular signals essential for cell differentiation and proliferation. Their life cycle is linked to the cell cycle, as cilia assemble in non-dividing cells of G0/G1 phases and disassemble before entering mitosis. Abnormalities in both primary cilia (non-motile cilia) and motile cilia structure or function are associated with developmental disorders (ciliopathies), heart disease, and cancer. However, the impact of SLs on primary cilia remains unknown. This study evaluated the effects of selected SLs (grosheimin, costunolide, and three cyclocostunolides) on primary cilia biogenesis and stability in human retinal pigment epithelial (RPE) cells. Confocal fluorescence microscopy was employed to analyze the effects on primary cilia formation (ciliogenesis), primary cilia length, and stability. The effects on cell proliferation were evaluated by flow cytometry. All SLs disrupted primary cilia formation in the early stages of ciliogenesis, irrespective of starvation conditions or cytochalasin-D treatment, with no effect on cilia length or cell cycle progression. Interestingly, grosheimin stabilized and promoted primary cilia formation under cilia homeostasis and elongation treatment conditions. Thus, SLs have potential as novel drugs for ciliopathies and tumor treatment.

CiliaMiner: an integrated database for ciliopathy genes and ciliopathies.

Cilia are found in eukaryotic species ranging from single-celled organisms, such as Chlamydomonas reinhardtii, to humans, but not in plants. The ability to respond to repellents and/or attractants, regulate cell proliferation and differentiation and provide cellular mobility are just a few examples of how crucial cilia are to cells and organisms. Over 30 distinct rare disorders generally known as ciliopathy are caused by abnormalities or functional impairments in cilia and cilia-related compartments. Because of the complexity of ciliopathies and the rising number of ciliopathies and ciliopathy genes, a ciliopathy-oriented and up-to-date database is required. Here, we present CiliaMiner, a manually curated ciliopathy database that includes ciliopathy lists collected from articles and databases. Analysis reveals that there are 55 distinct disorders likely related to ciliopathy, with over 4000 clinical manifestations. Based on comparative symptom analysis and subcellular localization data, diseases are classified as primary, secondary or atypical ciliopathies. CiliaMiner provides easy access to all of these diseases and disease genes, as well as clinical features and gene-specific clinical features, as well as subcellular localization of each protein. Additionally, the orthologs of disease genes are also provided for mice, zebrafish, Xenopus, Drosophila, Caenorhabditis elegans and Chlamydomonas reinhardtii. CiliaMiner (https://kaplanlab.shinyapps.io/ciliaminer) aims to serve the cilia community with its comprehensive content and highly enriched interactive heatmaps, and will be continually updated. Database URL: https://kaplanlab.shinyapps.io/ciliaminer/.

[Regulatory role and mechanism of primary cilia in craniofacial and dental development].

Primary cilia protruding from cell surface are important cell receptors and exist in most types of vertebrate cells. Primary cilia can sense extracellular mechanical signals, chemical signals as well as optical signals, and transduce them into cells, which is crucial for embryonic development and maintenance of tissue homeostasis. Mutations of gene that are responsible for the structure or function of cilia can lead to abnormal cilia signal transport, which in turn leads to ciliopathies. About 30% of ciliopathies are characterized by craniofacial phenotype. The most common cilia-related craniofacial defects include micrognathia, cleft lip, cleft palate, orbital hypertelorism/hypotelorism, flat nasal bridge, prominent forehead, craniosynostosis, and so on, suggesting that primary cilia plays an important role in the normal development of craniofacial development. This review summarizes the key genes involved in the regulation of craniofacial development in primary cilia and the disease phenotypes caused by important cilia gene mutations, in order to provide a reference for understanding the etiology of primary cilia-related craniofacial congenital developmental defects.

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations.

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD.

Compound heterozygous IFT81 variations in a skeletal ciliopathy patient cause Bardet-Biedl syndrome-like ciliary defects.

Owing to their crucial roles in development and homeostasis, defects in cilia cause ciliopathies with diverse clinical manifestations. The intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes, mediates not only the intraciliary bidirectional trafficking but also import and export of ciliary proteins together with the kinesin-2 and dynein-2 motor complexes. The BBSome, containing eight subunits encoded by causative genes of Bardet-Biedl syndrome (BBS), connects the IFT machinery to ciliary membrane proteins to mediate their export from cilia. Although mutations in subunits of the IFT-A and dynein-2 complexes cause skeletal ciliopathies, mutations in some IFT-B subunits are also known to cause skeletal ciliopathies. We here show that compound heterozygous variations of an IFT-B subunit, IFT81, found in a patient with skeletal ciliopathy cause defects in its interactions with other IFT-B subunits, and in ciliogenesis and ciliary protein trafficking when one of the two variants was expressed in IFT81-knockout (KO) cells. Notably, we found that IFT81-KO cells expressing IFT81(Δ490-519), which lacks the binding site for the IFT25-IFT27 dimer, causes ciliary defects reminiscent of those found in BBS cells and those in IFT74-KO cells expressing a BBS variant of IFT74, which forms a heterodimer with IFT81. In addition, IFT81-KO cells expressing IFT81(Δ490-519) in combination with the other variant, IFT81 (L645*), which mimics the cellular conditions of the above skeletal ciliopathy patient, demonstrated essentially the same phenotype as those expressing only IFT81(Δ490-519). Thus, our data indicate that BBS-like defects can be caused by skeletal ciliopathy variants of IFT81.

Functions of actin-binding proteins in cilia structure remodeling and signaling.

Cilia are microtubule-based organelles found on the surfaces of many types of cells, including cardiac fibroblasts, vascular endothelial cells, human retinal pigmented epithelial-1 (RPE-1) cells, and alveolar epithelial cells. These organelles can be classified as immotile cilia, referred to as primary cilia in mammalian cells, and motile cilia. Primary cilia are cellular sensors that detect extracellular signals; this is a critical function associated with ciliopathies, which are characterized by the typical clinical features of developmental disorders. Cilia are extensively studied organelles of the microtubule cytoskeleton. However, the ciliary actin cytoskeleton has rarely been studied. Clear evidence has shown that highly regulated actin cytoskeleton dynamics contribute to normal ciliary function. Actin-binding proteins (ABPs) play vital roles in filamentous actin (F-actin) morphology. Here, we discuss recent progress in understanding the roles of ABPs in ciliary structural remodeling and further downstream ciliary signaling with a focus on the molecular mechanisms underlying actin cytoskeleton-related ciliopathies.

Organization, functions, and mechanisms of the BBSome in development, ciliopathies, and beyond.

The BBSome is an octameric protein complex that regulates ciliary transport and signaling. Mutations in BBSome subunits are closely associated with ciliary defects and lead to ciliopathies, notably Bardet-Biedl syndrome. Over the past few years, there has been significant progress in elucidating the molecular organization and functions of the BBSome complex. An improved understanding of BBSome-mediated biological events and molecular mechanisms is expected to help advance the development of diagnostic and therapeutic approaches for BBSome-related diseases. Here, we review the current literature on the structural assembly, transport regulation, and molecular functions of the BBSome, emphasizing its roles in cilium-related processes. We also provide perspectives on the pathological role of the BBSome in ciliopathies as well as how these can be exploited for therapeutic benefit.

Eupatilin Improves Cilia Defects in Human CEP290 Ciliopathy Models.

The photoreceptor outer segment is a highly specialized primary cilium that is essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290, there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290-related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids by modulating the expression of rhodopsin and by targeting cilia and synaptic plasticity pathways. This work sheds light on the mechanism of action of eupatilin and supports its potential as a variant-independent approach for CEP290-associated ciliopathies.

Primary cilia shape hallmarks of health and aging.

Primary cilia are specialized organelles that sense changes in extracellular milieu, and their malfunction is responsible for several disorders (ciliopathies). Increasing evidence shows that primary cilia regulate tissue and cellular aging related features, which led us to review the evidence on their role in potentiating and/or accelerating the aging process. Primary cilia malfunction is associated with some age-related disorders, from cancer to neurodegenerative and metabolic disorders. However, there is limited understanding of molecular pathways underlying primary cilia dysfunction, resulting in scarce ciliary-targeted therapies available. Here, we discuss the findings on primary cilia dysfunction as modulators of the health and aging hallmarks, and the pertinence of ciliary pharmacological targeting to promote healthy aging or treat age-related diseases.

Modeling ciliopathies in patient-derived primary cells.

Ciliopathies comprise a group of inherited diseases caused by mutations in genes encoding proteins that localize to cilia or centrosomes. They afflict multiple organs and are one of the most frequent monogenic causes of kidney failure in adults, adolescents and children. Primary cilia play diverse roles in cell signaling, cell cycle regulation, planar cell polarity and mechanosensing. The use of patient-derived cells possessing endogenous disease causing mutations enables the study of these processes and their dysregulation in disease. Here we describe methods to cultivate patient-derived dermal fibroblast and renal epithelial cells isolated from urine. Fibroblasts are highly robust, long-lived, and easy to culture cells in which ciliary assembly can be easily induced. Similarly, the ability to acquire and culture ciliated renal epithelial cells without patient-invasive-intervention holds great potential to further our understanding of ciliopathies. In addition to monolayer cultures, we also detail the formation of three-dimensional renal-epithelial organoids-so-called tubuloids-that demonstrate epithelial-polarization and transepithelial transport activities like those seen in vivo renal-tubules. These in vitro models are powerful tools to investigate the underlying disease mechanisms of human ciliopathies that can be employed without the need for heavy-handed genetic or molecular manipulations.

Primary cilia as dynamic and diverse signalling hubs in development and disease.

Primary cilia, antenna-like sensory organelles protruding from the surface of most vertebrate cell types, are essential for regulating signalling pathways during development and adult homeostasis. Mutations in genes affecting cilia cause an overlapping spectrum of >30 human diseases and syndromes, the ciliopathies. Given the immense structural and functional diversity of the mammalian cilia repertoire, there is a growing disconnect between patient genotype and associated phenotypes, with variable severity and expressivity characteristic of the ciliopathies as a group. Recent technological developments are rapidly advancing our understanding of the complex mechanisms that control biogenesis and function of primary cilia across a range of cell types and are starting to tackle this diversity. Here, we examine the structural and functional diversity of primary cilia, their dynamic regulation in different cellular and developmental contexts and their disruption in disease.

Studying the morphology, composition and function of the photoreceptor primary cilium in zebrafish.

Vision is one of our dominant senses and its loss has a profound impact on the life quality of affected individuals. Highly specialized neurons in the retina called photoreceptors convert photons into neuronal responses. This conversion of photons is mediated by light sensitive opsin proteins, which are found in the outer segments of the photoreceptors. These outer segments are highly specialized primary cilia, explaining why retinal dystrophy is a key feature of ciliopathies, a group of diseases resulting from abnormal and dysfunctional cilia. Therefore, research on ciliopathies often includes the analysis of the retina with special focus on the photoreceptor and its outer segment. In the last decade, the zebrafish has emerged as an excellent model organism to study human diseases, in particular with respect to the retina. The cone-rich retina of zebrafish resembles the fovea of the human macula and thus represents an excellent model to study human retinal diseases. Here we give detailed guidance on how to analyze the morphological and ultra-structural integrity of photoreceptors in the zebrafish using various histological and imaging techniques. We further describe how to conduct functional analysis of the retina by electroretinography and how to prepare isolated outer segment fractions for different -omic approaches. These different methods allow a comprehensive analysis of photoreceptors, helping to enhance our understanding of the molecular and structural basis of ciliary function in health and of the consequences of its dysfunction in disease.