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Over the past few decades, the primary cilium, an inconspicuous cell organelle, has increasingly become the focus of current research. The primary cilium is a microtubule-based, nonmotile, antenna-like structure that is present in almost all mammalian cells. The ciliary membrane incorporates a large number of receptor molecules, which further characterize this cellular organelle. These include receptors of the Sonic hedgehog (Shh)-, Wnt-, or platelet-derived growth factor (PDGF) signaling pathways. For this reason, as well as due to the fact that extracellular signaling molecules can bind to the ciliary membrane, primary cilia have been named "the antenna of the cell." In addition to their signaling function, the association of ciliary dysfunctions with a variety of diseases, so-called ciliopathies, underscores the importance of this functional cellular structure. Recent studies have also implicated primary cilia in the adaptation to low-oxygen conditions, which are characteristic of ischemia, such as in stroke or myocardial infarction, or tumor entities. The aim of this review is to provide an overview of these multiple facets and to take a closer look at the evolution of an inconspicuous cell organelle to a major player in hypoxia.

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of motile or non-motile (primary) cilia that are microtubule-based cell protrusions. Primary cilia (PC) are present in most human epithelial and endothelial tissues and serve as a hub for physiological signal transduction, including Sonic Hedgehog (SHH) signaling. Various situations demand examining the function of PC, which may be achieved by measuring the canonical SHH signaling pathway that is almost exclusively mediated by PC. Here, a quantitative PCR (qPCR) based technique is developed to measure the transcriptional level of SHH pathway genes in quiescent hTERT-RPE1 (or RPE1) cells that mostly contain PC. Quiescence in RPE1 cells is achieved by serum starvation for 48 h, while activation of the SHH pathway is promoted by a specific agonist. This cell culture-based assay is easy to follow and sensitive. Successful demonstration of this assay in ciliated RPE1 cells indicates its immense potential as an in vitro assay to examine the proper function of PC upon genetic or epigenetic alteration of one or multiple genes, which may be associated with various ciliopathies.

NME7 (nucleoside diphosphate kinase 7), a lesser studied member of the non-metastatic expressed (NME) family, has been reported as a potential subunit of the γ-tubulin ring complex (γTuRC). However, its role in the cilium assembly and function remains unclear. Our research demonstrated that NME7 is located at the centrosome, including at the spindle poles during metaphase and at the basal bodies during cilium assembly. Notably, a small fraction of NME7 localizes within the cilium. Detailed analysis of cilium assembly after NME7 knockdown and knockout revealed that NME7 is required for this process. NME7 knockout cells exhibited sensitivity to nocodazole, indicating its role in ciliary microtubule stability. In addition, NME7 deficiency impacted the Hedgehog signaling pathway, evident from reduced smoothened (Smo) fluorescence within primary cilia. This role of NME7 in Hedgehog signaling may depend on its nucleoside diphosphate kinase activity and γTuRC association. In conclusion, these findings enhance our understanding of the γTuRC roles in primary cilia in mammalian cells, highlighting the importance of NME7 in ciliary functions and signaling pathways., (© 2025 Ji et al.)

Primary cilia are sensory organelles that regulate various signaling pathways. When microtubules are compared to a highway, motor proteins carry and transport cargo proteins, which are tuned by post-translational modifications, such as acetylation. However, the role of acetylation in primary cilia regulation remains unclear. In this study, histone K (lysine) acetyltransferase 2 B (Kat2b) was identified as a novel regulator of primary cilia. Kat2b, which mainly regulates transcription as a p300/CBP associated factor, is localized to the cytosol, centrosome, and cilium basal body. In addition, basal Kat2b expression gradually increased during ciliogenesis. Kat2b regulates the rate of cilia assembly, particularly in the early stages, and loss of Kat2b reduces the recruitment of intraflagellar transport (IFT) components to the ciliary axoneme and impairs Hedgehog (Hh) signaling activation. In addition, Kat2b-knockout mice showed mild abnormalities and ciliary IFT defects in the kidneys. These results establish a link between acetylation regulated by Kat2b and its relevance to ciliary assembly and function., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Advanced maternal age (AMA) negatively influences the development and cognitive functions of offspring. However, the underlying mechanism remains to be elucidated. As hippocampal autophagy and primary cilia play a crucial role in learning and memory abilities, this study aimed to investigate the effects of AMA on hippocampal autophagy and primary cilia, and to explore their relationship with the changes of LKB1/AMPK signaling pathway in offspring rats. The whole brains and hippocampus of offspring born to 12-month-old (AMA) and 3-month-old (control) Sprague-Dawley (SD) female rats were collected on post-natal days (P) 14, 28, and 60. Transmission electron microscopy was employed to count the number of autophagosomes. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to quantify gene expression, and immunofluorescence was used to measure primary cilia. The results revealed that autophagic activity was inhibited from childhood to adulthood in the AMA group. Furthermore, in the early developmental stage, primary ciliogenesis and growth in the hippocampus in the AMA group were impaired, with astrocytes being more severely affected. In addition, the AMA group exhibited an abnormal activation of the LKB1/AMPK signaling pathway. Thus, in offspring born to mothers of AMA, impaired hippocampal primary ciliary function and aberrant activation of the LKB1/AMPK signaling pathway are associated with inhibited autophagic activity., (© 2024 Wiley Periodicals LLC.)

Vangl2, a core component of the PCP signaling pathway, serves as a scaffold protein on the cell membrane, playing a crucial role in organizing protein complexes. Cilia, dynamic structures on the cell surface, carry out a wide range of functions. Research has highlighted a bidirectional regulatory interaction between Vangl2 and cilia, underscoring their interconnected roles in cellular processes. This relationship is demonstrated by the localization of Vangl2 at the base and proximal regions of cilia, where it plays essential roles in ciliary positioning, asymmetric distribution, and ciliogenesis. In contrast, the absence of cilia can disrupt Vangl2-mediated signal transduction processes. This review offers a narrative review of recent research on Vangl2's function in cilia and examines the regulatory effects of cilia on Vangl2-mediated signaling., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed to compensate defective cargo retrieval, its molecular basis remains unclear, especially in relation to BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Activation of the Sonic Hedgehog pathway further enhances CDC42 activity specifically in BBSome-deficient cilia. Inhibition of CDC42 in BBSome-deficient cells decreases the frequency and duration of ciliary actin polymerization events, causing buildup of G protein coupled receptor 161 (GPR161) in bulges along the axoneme during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Hyperactive ciliary CDC42 and ectocytosis and the resulting loss of ciliary material might contribute to BBS disease severity., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The cerebral cortex is critical for advanced cognitive functions and relies on a vast network of neurons to carry out its highly intricate neural tasks. Generating cortical neurons in accurate numbers hinges on cell signaling orchestrated by primary cilia to coordinate the proliferation and differentiation of cortical stem cells. While recent research has shed light on multiple ciliary roles in corticogenesis, specific mechanisms downstream of cilia signaling remain largely unexplored. We previously showed that an excess of early-born cortical neurons in mice mutant for the ciliary gene Inpp5e was rescued by re-introducing Gli3 repressor. By comparing expression profiles between Inpp5e and Gli3 mutants, we here identified novel Gli3 target genes. This approach highlighted the transcription factor gene Sall3 and Pappalysin1 (Pappa), a metalloproteinase involved in IGF signaling, as upregulated genes in both mutants. Further examination revealed that Gli3 directly binds to Sall3 and Pappa enhancers and suppresses their activity in the dorsal telencephalon. Collectively, our analyses provide important mechanistic insights into how primary cilia govern the behavior of neural stem cells, ultimately ensuring the production of adequate numbers of neurons during corticogenesis., (© The Author(s) 2024. Published by Oxford University Press.)

Age-related deterioration of condylar cartilage is an etiological factor in temporomandibular joint-osteoarthritis (TMJ-OA). However, its underlying mechanism remains unknown. Therefore, we examined age-related changes and the relationship between mTOR signaling and primary cilia in condylar cartilage to determine the intrinsic mechanisms of age-related TMJ-OA. Age-related morphological changes were analyzed using micro-computed tomography and safranin O-stained histological samples of the mandibular condyle of C57BL/6J mice (up to 78 weeks old). Immunohistochemistry was used to assess the activity of mTOR signaling, primary cilia frequency, and Golgi size of condylar chondrocytes. Four-week-old mice receiving an 11-week series of intraperitoneal injections of rapamycin, a potent mTOR signaling inhibitor, were used for the histological evaluation of the condylar cartilage. The condylar cartilage demonstrated an age-related reduction in cartilage area, including chondrocyte size, cell density, and cell size distribution. The Golgi size, primary cilia frequency, and mTOR signaling also decreased with age. Rapamycin injections resulted in both diminished cartilage area and cell size, resembling the phenotypes observed in aged mice. Rapamycin-injected mice also exhibited a smaller Golgi size and lower primary cilia frequency in condylar cartilage. We demonstrated that a loss of primary cilia due to a decline in mTOR signaling was correlated with age-related deteriorations in condylar cartilage. Our findings provide new insights into the tissue homeostasis of condylar cartilage, contributing to understanding the etiology of age-related TMJ-OA., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

The Hedgehog (Hh) signaling pathway is critical for regulating cell growth, survival, fate determination, and the overall patterning of both vertebrate and invertebrate body plans. Aberrations in Hh signaling are associated with congenital abnormalities and tumorigenesis. In vertebrates, Hh signaling depends uniquely on primary cilia, microtubule-based organelles that extend from the cell surface. Over the last 2 decades, studies have demonstrated that key molecules regulating Hh signaling dynamically accumulate in primary cilia via intraflagellar transport systems. Moreover, through the primary cilia, extracellular signals are converted to stabilize GLI2 and GLI3 that are transcription factors that play a central role in regulating Hh signaling at the post-translational modification level. Recent in vivo and anatomical studies have uncovered crucial molecules that facilitate the conversion of extracellular signals into the intracellular stabilization of GLI2/GLI3 via primary cilia, emphasizing their essential roles in tissue development and tumorigenesis. This review explores the regulatory mechanisms of GLI2/GLI3 with a focus on mammalian tissue development., Competing Interests: Declarations. Conflict of interest: The author declares no conflict of interest., (© 2024. The Author(s), under exclusive licence to Japanese Association of Anatomists.)

Primary cilia (PC) are essential signaling hubs for proper epithelial formation and the maintenance of skin homeostasis. Found on most cells in the human body, including skin cells, PC facilitate signal transduction that allows ciliated cells to interact with the immune system via multiple pathways, helping to maintain immune system homeostasis. PC can be altered by various microenvironmental stimuli to develop corresponding regulatory functions. Both PC and ciliary signaling pathways have been shown to be involved in the immune processes of various skin lesions. However, the mechanisms by which PC regulate cellular functions and maintain immune homeostasis in tissues are highly complex, and our understanding of them in the skin remains limited. In this paper, we discuss key ciliary signaling pathways and ciliated cells in the skin, with a focus on their immunomodulatory functions. We have compiled evidence from various cells, tissues and disease models to help explore the potential immunomodulatory effects of PC in the skin and their molecular mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Yuan, Yu, Li, Zhao, Tang and Zheng.)

The establishment of CRISPR/Cas9 gene editing in Anolis sagrei has positioned this species as a powerful model for studies of reptilian gene function. To enhance this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this cell line by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Using CRISPR/Cas9 mutagenesis, we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We determined that loss of itf88 from lizard cells leads to an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei are also SAG responsive in Mus musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Primary cilia are thin hair-like organelles that protrude from the surface of most mammalian cells. They act as specialized cell antennas that can vary widely in response to specific stimuli. However, the effect of changes in cilia length on cellular signaling and behavior remains unclear. Therefore, we aimed to characterize the elongated primary cilia induced by different chemical agents, lithium chloride (LiCl), cobalt chloride (CoCl2) and rotenone, using human retinal pigmented epithelial 1 (hRPE1) cells expressing ciliary G protein-coupled receptor (GPCR), melanin-concentrating hormone (MCH) receptor 1 (MCHR1). MCH induces cilia shortening mainly via MCHR1-mediated Akt phosphorylation. Therefore, we verified the proper functioning of the MCH-MCHR1 axis in elongated cilia. Although MCH shortened cilia that were elongated by LiCl and rotenone, it did not shorten CoCl2-induced elongated cilia, which exhibited lesser Akt phosphorylation. Furthermore, serum readdition was found to delay cilia shortening in CoCl2-induced elongated cilia. In contrast, rotenone-induced elongated cilia rapidly shortened via a chopping mechanism at the tip of the cilia. Conclusively, we found that each chemical exerted different effects on ciliary GPCR signaling and serum-mediated ciliary structure dynamics in cells with elongated cilia. These results provide a basis for understanding the functional consequences of changes in ciliary length., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The primary cilium is a characteristic feature of most non-immune cells and functions as an environmental signal transduction sensor. The defects in primary cilium have profound effects on the developmental program, including the maturation of retinal epithelium. The ciliary length is tightly regulated during ciliogenesis, but the impact of inflammation on ciliary length remains elusive. The current study investigates the outcome of inflammatory stimuli for the primary cilium length in retinal epithelium cells and mouse embryonic fibroblasts. Here, we report that exposure to the pro-inflammatory cytokine TNF-alpha elongates cilia in a mixed-lineage kinase (MLK)-dependent manner. Pro-inflammatory stimuli such as bacterial LPS and interferon-gamma have similar effects on ciliary length. In contrast, febrile condition-mimicking heat stress dramatically reduced the number of ciliated cells regardless of TNF-alpha exposure but did not shorten TNF-induced elongation, suggesting distinct but rapid effects of inflammatory stresses on ciliogenesis., (© 2024 Wiley Periodicals LLC.)

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Cilia loss and impaired motile ciliary functions are among the typical pathological features of chronic rhinosinusitis with nasal polyps (CRSwNP). IL17A and IL22 are the canonical cytokines of type 3 inflammation, exhibiting similar functional effects on epithelial cells. In this study, we sought to examine the effects of IL17A and IL22 on ciliated cells and investigate the potential involvement of Hippo-YAP signaling in their influence on ciliogenesis., Methods: We assessed both the mRNA and protein expression levels of IL17A and IL22 in nasal tissues obtained from patients with CRSwNP and compared them to those from healthy controls. To further explore the impact of IL17A and IL22, we established a primary human nasal epithelial cell model using different concentrations (2 ng/mL, 10 ng/mL, 50 ng/mL) for a duration of 28 days in an air-liquid interface culture. Additionally, we employed the inhibitor verteporfin to investigate whether IL17A and IL22 exert their effects on ciliated cells via the Hippo-YAP pathway., Results: The mRNA and protein levels of IL17A and IL22 in CRSwNP were significantly higher than those in healthy controls, revealing a robust correlation between IL17A and IL22. YAP was highly expressed in the nucleus of ciliated cells in CRSwNP and displayed a positive correlation with clinical symptoms. Both IL17A and IL22 were found to reduce the number of ciliated cells. IL17A, but not IL22, suppressed ciliogenesis by disrupting the proper development and docking of the basal body of ciliated cells, resulting in motile ciliary dysfunctions. Furthermore, the expression of YAP within the nucleus of ciliated cells gradually declined as these cells reached the final stage of differentiation. However, this process was obstructed by IL17A only. YAP inhibitors, such as verteporfin, markedly reversed the effects of IL17A by increasing the proportion of ciliated cells, suppressing nuclear YAP expression in these cells, and enhancing ciliary beating frequency., Conclusions: Both IL17A and IL22 are overexpressed in nasal epithelium of CRSwNP, which is associated with the impairment of epithelial cell differentiation. Furthermore, IL17A has been shown to exert a disruptive effect on morphogenesis of motile cilia via activation of YAP., Competing Interests: Disclosure statement Supported by National Key R&D Program of China (2022YFC2504100); the National Natural Science Foundation of China (U20A20399, 82071019, 82271148, 82000958, 82271191, 82101197); the Key-area Research and Development Program of Guangdong Province (2020B0101130015); the General Program of Natural Science Foundation of Guangdong Province (2021A1515011764); Guangdong Basic and Applied Basic Research Foundation (2021A1515110739); Young Talent Support Project of Guangzhou Association for Science and Technology, Science and Technology Program of Guangzhou, China (2023A04J1093); China Postdoctoral Science Foundation (2022M713614); Sun Yat-sen University Clinical Research 5010 Program (2019006); and the Third Affiliated Hospital of Sun Yat-sen University "Five by Five" Project (2023ww601). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptor was selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease., (Copyright © 2024 International Society of Nephrology. All rights reserved.)

Primary cilia are present on renal tubules and are implicated to play a pivotal role in transducing signals during development; however, the oncogenic role of cilia in clear cell renal cell carcinoma (ccRCC) has not been examined. Here we show that VHL wild-type ccRCC cell lines have a high incidence of primary cilia, and a high frequency of primary cilia is positively correlated with VHL expression and poor prognosis. Besides, the depletion of KIF3A and IFT88, genes required for ciliogenesis, significantly inhibited tumor proliferation and metastasis in vitro and in vivo. Further analysis found that mutations of key genes in hedgehog signaling are enriched in VHL wild ccRCC, its downstream signaling activation depends on ciliogenesis. Moreover, depletion of primary cilia or suppression of hedgehog pathway activation with inhibitor-induced robust autophagic cell death. Collectively, our findings revealed that primary cilia could serve as a diagnostic tool and provide new insights into the mechanism of VHL wild-type ccRCC progression. Targeting the primary cilia-hedgehog pathway may represent an effective therapeutic strategy for VHL wild-type ccRCC., (© 2024. The Author(s).)

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing the primary cilium, the cell's antenna, which acts as a signaling hub. Fuz, an effector of planar cell polarity signaling, regulates Shh signaling by facilitating cilia formation, and the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of the genes encoding these proteins is similar; however, their functional relationship has not been previously explored. This study identified the genetic and biochemical linkage between Fuz and Gpr161 in mouse neural tube development. Fuz was found to be genetically epistatic to Gpr161 with respect to regulation of Shh signaling in mouse neural tube development. The Fuz protein biochemically interacts with Gpr161, and Fuz regulates Gpr161-mediated ciliary localization, a process that might utilize β-arrestin 2. Our study characterizes a previously unappreciated Gpr161-Fuz axis that regulates Shh signaling during mouse neural tube development., Competing Interests: Competing interests R.H.F. and B.J.W. participated in TeratOmic Consulting LLC, a now-defunct consulting company. R.H.F. serves on the editorial board for the journal Reproductive and Developmental Medicine and receives travel funds to attend editorial board meetings. All other authors have no conflict of interest to declare., (© 2024. Published by The Company of Biologists Ltd.)

The sense of smell is still considered a fuzzy sensation. Softly wafting aromas can stimulate the appetite and trigger memories; however, there are many unexplored aspects of its underlying mechanisms, and not all of these have been elucidated. Although the final sense of smell takes place in the brain, it is greatly affected during the preliminary stage, when odorants are converted into electrical signals. After signal conversion through ion channels in olfactory cilia, action potentials are generated through other types of ion channels located in the cell body. Spike trains through axons transmit this information as digital signals to the brain, however, before odorants are converted into digital electric signals, such as an action potential, modification of the transduction signal has already occurred. This review focuses on the early stages of olfactory signaling. Modification of signal transduction mechanisms and their effect on the human sense of smell through three characteristics (signal amplification, olfactory adaptation, and olfactory masking) produced by olfactory cilia, which is the site of signal transduction are being addressed in this review., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Primary cilia are microtubule-based sensory organelles whose dysfunction causes ciliopathies in humans. The formation, function, and maintenance of primary cilia depend crucially on intraflagellar transport (IFT); however, the regulatory mechanisms of IFT at ciliary tips are poorly understood. Here, we identified that the ciliopathy kinase Mak is a ciliary tip-localized IFT regulator that cooperatively acts with the ciliopathy kinase Ick, an IFT regulator. Simultaneous disruption of Mak and Ick resulted in loss of photoreceptor ciliary axonemes and severe retinal degeneration. Gene delivery of Ick and pharmacological inhibition of FGF receptors, Ick negative regulators, ameliorated retinal degeneration in Mak -/- mice. We also identified that Ccrk kinase is an upstream activator of Mak and Ick in retinal photoreceptor cells. Furthermore, the overexpression of Mak, Ick, and Ccrk and pharmacological inhibition of FGF receptors suppressed ciliopathy-related phenotypes caused by cytoplasmic dynein inhibition in cultured cells. Collectively, our results show that the Ccrk-Mak/Ick axis is an IFT regulator essential for retinal photoreceptor maintenance and present activation of Ick as a potential therapeutic approach for retinitis pigmentosa caused by MAK mutations., (© 2024 Chaya et al.)

Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling., (© 2024 Ansari et al.)

Astrocyte diversity is greatly influenced by local environmental modulation. Here we report that the majority of astrocytes across the mouse brain possess a singular primary cilium localized to the cell soma. Comparative single-cell transcriptomics reveals that primary cilia mediate canonical SHH signaling to modulate astrocyte subtype-specific core features in synaptic regulation, intracellular transport, energy and metabolism. Independent of canonical SHH signaling, primary cilia are important regulators of astrocyte morphology and intracellular signaling balance. Dendritic spine analysis and transcriptomics reveal that perturbation of astrocytic cilia leads to disruption of neuronal development and global intercellular connectomes in the brain. Mice with primary ciliary-deficient astrocytes show behavioral deficits in sensorimotor function, sociability, learning and memory. Our results uncover a critical role for primary cilia in transmitting local cues that drive the region-specific diversification of astrocytes within the developing brain., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ciliopathies are characterized by the absence or dysfunction of primary cilia. Despite the fact that cognitive impairments are a common feature of ciliopathies, how cilia dysfunction affects neuronal development has not been characterized in detail. Here, we show that primary cilium-mediated signaling is required cell-autonomously by neurons during neural circuit formation. In particular, a functional primary cilium is crucial during axonal pathfinding for the switch in responsiveness of axons at a choice point or intermediate target. Using different animal models and in vivo, ex vivo and in vitro experiments, we provide evidence for a crucial role of primary cilium-mediated signaling in long-range axon guidance. The primary cilium on the cell body of commissural neurons transduces long-range guidance signals sensed by growth cones navigating an intermediate target. In extension of our finding that Shh is required for the rostral turn of post-crossing commissural axons, we suggest a model implicating the primary cilium in Shh signaling upstream of a transcriptional change of axon guidance receptors, which in turn mediate the repulsive response to floorplate-derived Shh shown by post-crossing commissural axons., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld-Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes., (© 2024. The Author(s).)

The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling., (© 2024 Larsen et al.)

Primary cilia are antenna-like organelles which sense extracellular cues and act as signalling hubs. Cilia dysfunction causes a heterogeneous group of disorders known as ciliopathy syndromes affecting most organs. Cilia disassembly, the process by which cells lose their cilium, is poorly understood but frequently observed in disease and upon cell transformation. Here, we uncover a role for the PI3Kα signalling enzyme in cilia disassembly. Genetic PI3Kα-hyperactivation, as observed in PIK3CA-related overgrowth spectrum (PROS) and cancer, induced a ciliopathy-like phenotype during mouse development. Mechanistically, PI3Kα and PI3Kβ produce the PIP 3 lipid at the cilia transition zone upon disassembly stimulation. PI3Kα activation initiates cilia disassembly through a kinase signalling axis via the PDK1/PKCι kinases, the CEP170 centrosomal protein and the KIF2A microtubule-depolymerising kinesin. Our data suggest diseases caused by PI3Kα-activation may be considered 'Disorders with Ciliary Contributions', a recently-defined subset of ciliopathies in which some, but not all, of the clinical manifestations result from cilia dysfunction., (© 2024. The Author(s).)

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: S.S. is the founder and scientific advisor of 7TM Antibodies GmbH, Jena, Germany. F.N. is an employee of 7TM Antibodies. All other authors declare no competing interests., (Copyright: © 2024 Walker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The primary cilium behaves as a platform for sensing and integrating extracellular cues to control a plethora of cellular activities. However, the functional interaction of this sensory organelle with epithelial-mesenchymal transition (EMT) during pulmonary fibrosis remains unclear. Here, we reveal a critical role for cylindromatosis (CYLD) in reciprocally linking the EMT program and ciliary homeostasis during pulmonary fibrosis. A close correlation between the EMT program and primary cilia is observed in bleomycin-induced pulmonary fibrosis as well as TGF-β-induced EMT model. Mechanistic study reveals that downregulation of CYLD underlies the crosstalk between EMT and ciliary homeostasis by inactivating histone deacetylase 6 (HDAC6) during pulmonary fibrosis. Moreover, manipulation of primary cilia is an effective means to modulate the EMT program. Collectively, these results identify a pivotal role for the CYLD/HDAC6 signaling in regulating the reciprocal interplay between the EMT program and ciliary homeostasis during pulmonary fibrosis., (© 2024. The Author(s).)

Spontaneous abortion is the most common complication in early pregnancy, the exact etiology of most cases cannot be determined. Emerging studies suggest that mutations in ciliary genes may be associated with progression of pregnancy loss. However, the involvement of primary cilia on spontaneous abortion and the underlying molecular mechanisms remains poorly understood. We observed the number and length of primary cilia were significantly decreased in decidua of spontaneous abortion in human and lipopolysaccharide (LPS)-induced abortion mice model, accompanied with increased expression of proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. The length of primary cilia in human endometrial stromal cell (hESC) was significantly shortened after TNF-α treatment. Knocking down intraflagellar transport 88 (IFT88), involved in cilia formation and maintenance, promoted the expression of TNF-α. There was a reverse regulatory relationship between cilia shortening and TNF-α expression. Further research found that shortened cilia impair decidualization in hESC through transforming growth factor (TGF)-β/SMAD2/3 signaling. Primary cilia were impaired in decidua tissue of spontaneous abortion, which might be mainly caused by inflammatory injury. Primary cilia abnormalities resulted in dysregulation of TGF-β/SMAD2/3 signaling transduction and decidualization impairment, which led to spontaneous abortion., (© 2024 Wiley Periodicals LLC.)

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling.

Heterotopic ossification (HO), defined as the formation of extraskeletal bone in muscle and soft tissues, is a diverse pathological process caused by either genetic mutations or inciting trauma. Fibrodysplasia ossificans progressiva (FOP) is a genetic form of HO caused by mutations in the bone morphogenetic protein (BMP) type I receptor gene activin A receptor type 1 ( ACVR1 ). These mutations make ACVR1 hypersensitive to BMP and responsive to activin A. Hedgehog (Hh) signaling also contributes to HO development. However, the exact pathophysiology of how skeletogenic cells contribute to endochondral ossification in FOP remains unknown. Here, we showed that the wild-type or FOP-mutant ACVR1 localized in the cilia of stem cells from human exfoliated deciduous teeth with key FOP signaling components, including activin A receptor type 2A/2B, SMAD family member 1/5, and FK506-binding protein 12kD. Cilia suppression by deletion of intraflagellar transport 88 or ADP ribosylation factor like GTPase 3 effectively inhibited pathological BMP and Hh signaling, subdued aberrant chondro-osteogenic differentiation in primary mouse or human FOP cells, and diminished in vivo extraskeletal ossification in Acvr1 Q207D , Sox2-Cre ; Acvr1 R206H/+ FOP mice and in burn tenotomy-treated wild-type mice. Our results provide a rationale for early and localized suppression of cilia in affected tissues after injury as a therapeutic strategy against either genetic or acquired HO.

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1 + cells in the cranial sutures play an important role in bone growth. However, whether Gli1 + cells in facial sutures differentiate into bone under distraction force is unknown., Methods: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1 + cell lineage tracing model was used to observe the distribution of Gli1 + cells and explore the role of Gli1 + cells in facial bone remodeling., Results: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1 + cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2 + cells. Hedgehog signaling was upregulated in Gli1 + cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1 + cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1 + cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1 + cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1 + cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force., Conclusions: Our study indicates that the primary cilia of Gli1 + cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1 + cells in zygomaticomaxillary sutures., (© 2024. The Author(s).)

Primary cilia are solitary, immotile sensory organelles present on most cells in the body that participate broadly in human health, physiology and disease. Cilia generate a unique environment for signal transduction with tight control of protein, lipid and second messenger concentrations within a relatively small compartment, enabling reception, transmission and integration of biological information. In this Review, we discuss how cilia function as signalling hubs in cell-cell communication using three signalling pathways as examples: ciliary G-protein-coupled receptors (GPCRs), the Hedgehog (Hh) pathway and polycystin ion channels. We review how defects in these ciliary signalling pathways lead to a heterogeneous group of conditions known as 'ciliopathies', including metabolic syndromes, birth defects and polycystic kidney disease. Emerging understanding of these pathways' transduction mechanisms reveals common themes between these cilia-based signalling pathways that may apply to other pathways as well. These mechanistic insights reveal how cilia orchestrate normal and pathophysiological signalling outputs broadly throughout human biology., (© 2024. Springer Nature Limited.)

The primary cilium, an antenna-like sensory organelle that protrudes from the surface of most eukaryotic cell types, has become a signaling hub of growing interest given that defects in its structure and/or function are associated with human diseases and syndromes, known as ciliopathies. With the continuously expanding role of primary cilia in health and diseases, identifying new players in ciliogenesis will lead to a better understanding of the function of this organelle. It has been shown that the primary cilium shares similarities with the immune synapse, a highly organized structure at the interface between an antigen-presenting or target cell and a lymphocyte. Studies have demonstrated a role for known cilia regulators in immune synapse formation. However, whether immune synapse regulators modulate ciliogenesis remains elusive. Here, we find that programmed death ligand 1 (PD-L1), an immune checkpoint protein and regulator of immune synapse formation, plays a role in the regulation of ciliogenesis. We found that PD-L1 is enriched at the centrosome/basal body and Golgi apparatus of ciliated cells and depleting PD-L1 enhanced ciliogenesis and increased the accumulation of ciliary membrane trafficking proteins Rab8a, BBS5, and sensory receptor protein PC-2. Moreover, PD-L1 formed a complex with BBS5 and PC-2. In addition, we found that depletion of PD-L1 resulted in the ciliary accumulation of Gli3 and the downregulation of Gli1. Our results suggest that PD-L1 is a new player in ciliogenesis, contributing to PC-2-mediated sensory signaling and the Hh signaling cascade.

Epithelial-to-mesenchymal transition (EMT), which plays an essential role in development, tissue repair and fibrosis, and cancer progression, is a reversible cellular program that converts epithelial cells to mesenchymal cell states characterized by motility-invasive properties. The mostly signaling pathways that initiated and controlled the EMT program are regulated by a solitary, non-motile organelle named primary cilium. Acting as a signaling nexus, primary cilium dynamically concentrates signaling molecules to respond to extracellular cues. Recent research has provided direct evidence of connection between EMT and primary ciliogenesis in multiple contexts, but the mechanistic understanding of this relationship is complicated and still undergoing. In this review, we describe the current knowledge about the ciliary signaling pathways involved in EMT and list the direct evidence that shows the link between them, trying to figure out the intricate relationship between EMT and primary ciliogenesis, which may aid the future development of primary cilium as a novel therapeutic approach targeted to EMT., (© 2023. The Author(s).)

We recently reported that growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous sonic hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, Gdf15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors., (© 2024 Baur et al.)

Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of polycystin-1. The signaling pathways underlying this process, termed cilia-dependent cyst activation (CDCA), remain unknown. Using translating ribosome affinity purification RNASeq on mouse kidneys with polycystin-1 and cilia inactivation before cyst formation, we identify the differential 'CDCA pattern' translatome specifically dysregulated in kidney tubule cells destined to form cysts. From this, Glis2 emerges as a candidate functional effector of polycystin signaling and CDCA. In vitro changes in Glis2 expression mirror the polycystin- and cilia-dependent changes observed in kidney tissue, validating Glis2 as a cell culture-based indicator of polycystin function related to cyst formation. Inactivation of Glis2 suppresses polycystic kidney disease in mouse models of ADPKD, and pharmacological targeting of Glis2 with antisense oligonucleotides slows disease progression. Glis2 transcript and protein is a functional target of CDCA and a potential therapeutic target for treating ADPKD., (© 2024. The Author(s).)

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling., (© 2024. The Author(s).)

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric G s . This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for G s coupling. Mutations that prevent sterol binding to GPR161 suppress G s -mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The efficient transport of proteins into the primary cilium is a crucial step for many signaling pathways. Dysfunction of this process can lead to the disruption of signaling cascades or cilium assembly, resulting in developmental disorders and cancer. Previous studies on the protein delivery to the cilium were mostly focused on the membrane-embedded receptors. In contrast, how soluble proteins are delivered into the cilium is poorly understood. In our work, we identify the exocyst complex as a key player in the ciliary trafficking of soluble Gli transcription factors. In line with the known function of the exocyst in intracellular vesicle transport, we demonstrate that soluble proteins, including Gli2/3 and Lkb1, can use the endosome recycling machinery for their delivery to the primary cilium. Finally, we identify GTPases: Rab14, Rab18, Rab23, and Arf4 that are involved in vesicle-mediated Gli protein ciliary trafficking. Our data pave the way for a better understanding of ciliary transport and uncover transport mechanisms inside the cell., (© 2024. The Author(s).)

Establishment of the left-right (LR, sinistral, dextral) body axis in many vertebrate embryos relies on cilia-driven leftward fluid flow within an LR organizer (LRO). A cardinal question is how leftward flow triggers symmetry breakage. The chemosensation model posits that ciliary flow enriches a signaling molecule on the left side of the LRO that promotes sinistral cell fate. However, the nature of this sinistralizing signal has remained elusive. In the Xenopus LRO, we identified the stem cell growth factor R-Spondin 2 (Rspo2) as a symmetrically expressed, sinistralizing signal. As predicted for a flow-mediated signal, Rspo2 operates downstream of leftward flow but upstream of the asymmetrically expressed gene dand5. Unexpectedly, in LR patterning, Rspo2 acts as an FGF receptor antagonist: Rspo2 via its TSP1 domain binds Fgfr4 and promotes its membrane clearance by Znrf3-mediated endocytosis. Concordantly, we find that at flow-stage, FGF signaling is dextralizing and forms a gradient across the LRO, high on the dextral- and low on the sinistral side. Rspo2 gain- and loss-of function equalize this FGF signaling gradient and sinistralize and dextralize development, respectively. We propose that leftward flow of Rspo2 produces an FGF signaling gradient that governs LR-symmetry breakage., (© 2024. The Author(s).)

Primary cilia protrude from most vertebrate cell bodies and act as specialized 'signalling antennae' that can substantially lengthen or retract in minutes to hours in response to specific stimuli. Here, we review the conditions and mechanisms responsible for regulating primary cilia length (PCL) in mammalian nonsensory neurons, and propose four models of how they could affect ciliary signalling and alter cell state and suggest experiments to distinguish between them. These models include (i) the passive indicator model, where changes in PCL have no consequence; (ii) the rheostat model, in which a longer cilium enhances signalling; (iii) the local concentration model, where ciliary shortening increases the local protein concentration to facilitate signalling; and (iv) the altered composition model where changes in PCL skew signalling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Intraflagellar transport (IFT) trains, the polymers composed of two multi-subunit complexes, IFT-A and IFT-B, carry out bidirectional intracellular transport in cilia, vital for cilia biogenesis and signaling. IFT-A plays crucial roles in the ciliary import of membrane proteins and the retrograde cargo trafficking. However, the molecular architecture of IFT-A and the assembly mechanism of the IFT-A into the IFT trains in vivo remains elusive. Here, we report the cryo-electron microscopic structures of the IFT-A complex from protozoa Tetrahymena thermophila. We find that IFT-A complexes present two distinct, elongated and folded states. Remarkably, comparison with the in situ cryo-electron tomography structure of the anterograde IFT train unveils a series of adjustments of the flexible arms in apo IFT-A when incorporated into the anterograde train. Our results provide an atomic-resolution model for the IFT-A complex and valuable insights into the assembly mechanism of anterograde IFT trains., (© 2023. The Author(s).)

The primary cilium is a central signaling component during embryonic development. Here we focus on CROCCP2, a hominid-specific gene duplicate from ciliary rootlet coiled coil (CROCC), also known as rootletin, that encodes the major component of the ciliary rootlet. We find that CROCCP2 is highly expressed in the human fetal brain and not in other primate species. CROCCP2 gain of function in the mouse embryonic cortex and human cortical cells and organoids results in decreased ciliogenesis and increased cortical progenitor amplification, particularly basal progenitors. CROCCP2 decreases ciliary dynamics by inhibition of the IFT20 ciliary trafficking protein, which then impacts neurogenesis through increased mTOR signaling. Loss of function of CROCCP2 in human cortical cells and organoids leads to increased ciliogenesis, decreased mTOR signaling, and impaired basal progenitor amplification. These data identify CROCCP2 as a human-specific modifier of cortical neurogenesis that acts through modulation of ciliary dynamics and mTOR signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Almost all cell types of mammals have a small protrusion named a primary cilium on their surface. Primary cilia are enriched by cilia-specific ion channels and G-protein-coupled receptors. They are known to regulate various cellular functions that contribute to the development and homeostasis of living organisms by receiving extracellular signals and transfusing them to the cell body. All functions are performed when the structure of the primary cilia is maintained properly. Abnormalities in primary cilia or their signaling can lead to a collection of diseases in various organs called ciliopathies. The primary cilium is dynamic, static, or fixed. The length of primary cilia varies as the cell cycle progresses and is also altered by extracellular stimuli. Ligand binding to cilia-specific receptors is also known to alter the length. Thus, there is a need for a method to study the morphological changes of the primary cilium in a time-dependent manner, especially under stimuli or mechanical shocks. Time-lapse imaging of primary cilia is one of the most powerful methods to capture the time-dependent behavior of primary cilia. Overexpression of ciliary proteins fused to fluorescent proteins is commonly used for the time-lapse imaging of primary cilia. However, overexpression has drawbacks in terms of artifacts. In addition, the time-lapse imaging of the tiny primary cilia requires some technical tricks. Here, we present a detailed description of the methods for time-lapse imaging of primary cilium, from the generation of cell lines that stably express fluorescent protein-labeled cilia-localized proteins at the physiological level to image analysis, including quantification through image acquisition., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cilia are hair-like projections that assemble at the surface of cells in various tissues of multicellular organisms through a complex cell biological process called ciliogenesis. Cilia can assemble as single structures per cell (i.e. non-motile primary cilia), which act as cell signaling centers that dictate cell fate, or can be assembled in distinct cell types as many copies per cell (i.e. motile cilia) that beat to move fluids at the cell surface. The mechanisms that orchestrate formation and function of cilia, which are dysregulated in pathological settings such as ciliopathies, remain incompletely understood. Stem cell-derived organoids represent valuable models to study the mechanisms of ciliogenesis, ciliary signaling, and ciliary beating that collectively promote tissue development and homeostasis. Here, we present a comprehensive protocol for the growth of mammary organoids derived from mouse mammary stem cells and for immunofluorescence staining of primary cilia in these three-dimensional structures., (Copyright © 2023 Elsevier Inc. All rights reserved.)