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Primary ciliary dyskinesia is a rare monogenic syndrome that is associated with chronic respiratory disease, infertility, and laterality defects. Although more than 50 genes causative of primary ciliary dyskinesia have been identified, variants in the genes encoding coiled-coil domain-containing 39 (CCDC39) and CCDC40 in particular cause severe disease that is not explained by loss of ciliary motility alone. Here, we sought to understand the consequences of these variants on cellular functions beyond impaired motility. We used human cells with pathogenic variants in CCDC39 and CCDC40 , Chlamydomonas reinhardtii genetics, cryo-electron microscopy, and proteomics to define perturbations in ciliary assembly and cilia stability, as well as multiple motility-independent pathways. Analysis of proteomics of cilia from patient cells identified that the absence of the axonemal CCDC39/CCDC40 heterodimer resulted in the loss of a network of more than 90 ciliary structural proteins, including 14 that were defined as ciliary address recognition proteins, which provide docking for the missing structures. The absence of the network impaired microtubule architecture, activated cell quality control pathways, switched multiciliated cell fate to mucus-producing cells and resulted in a defective periciliary barrier. In CCDC39 variant cells, these phenotypes were reversed through expression of a normal CCDC39 transgene. These findings indicate that the CCDC39/CCDC40 heterodimer functions as a scaffold to support the assembly of an extensive network of ciliary proteins, whose loss results in both motility-dependent and motility-independent phenotypes that may explain the severity of disease. Gene therapy might be a potential treatment option to be explored in future studies.

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy., (© 2024 Wiley Periodicals LLC.)

Mutations in CCDC11 (cfap53) have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, explaining the multiciliation phenotype observed in Ccdc11-depleted cells. The small GTPase RhoA is critical for cytokinesis, and our Ccdc11 depletion phenotypes are reminiscent of RhoA loss of function. Here, we demonstrate that during cytokinesis CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA, and CCDC11 regulates total RhoA protein levels. Our results connect CCDC11 to cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis., (© 2024 The Author(s). Cytoskeleton published by Wiley Periodicals LLC.)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to kidney failure. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single-nucleus multimodal atlas of an orthologous mouse PKD model at early, mid, and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalog differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single-nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution., Competing Interests: Competing interests statement:B.D.H. is a consultant for Janssen Research & Development, LLC, Pfizer and Chinook Therapeutics. B.D.H holds equity in Chinook Therapeutics. B.D.H holds grant funding from Chinook Therapeutics and Janssen Research & Development, LLC. O.M.W has received grants from AstraZeneca unrelated to the current work.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT., (© 2024. The Author(s).)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution., Competing Interests: Competing Interest Statement: B.D.H. is a consultant for Janssen Research & Development, LLC, Pfizer and Chinook Therapeutics, holds equity in Chinook Therapeutics and grant funding from Chinook Therapeutics and Janssen Research & Development, LLC. O.M.W has received grants from AstraZeneca unrelated to the current work. The remaining authors declare no competing interests.

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies., Competing Interests: Declaration of Interest The authors declare no competing interests

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

Mutations in genes that disrupt centrosome structure or function can cause congenital kidney developmental defects and lead to fibrocystic pathologies. Yet, it is unclear how defective centrosome biogenesis impacts renal progenitor cell physiology. Here, we examined the consequences of impaired centrosome duplication on kidney stromal progenitor cell growth, differentiation, and fate. Conditional deletion of the ciliopathy gene Cep120, which is essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of interstitial lineages including pericytes, fibroblasts and mesangial cells. These phenotypes were caused by a combination of delayed mitosis, activation of the mitotic surveillance pathway leading to apoptosis, and changes in both Wnt and Hedgehog signaling that are key for differentiation of stromal cells. Cep120 ablation resulted in small hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, Cep120 and centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis after renal injury via enhanced TGF-β/Smad3-Gli2 signaling. Our study defines the cellular and developmental defects caused by loss of Cep120 and aberrant centrosome biogenesis in the embryonic kidney stroma., (© 2023. The Author(s).)

Mutations that disrupt centrosome biogenesis or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet how centrosome dysfunction results in the kidney disease phenotypes remains unknown. Here, we examined the consequences of conditional knockout of the ciliopathy gene Cep120, essential for centrosome duplication, in the nephron and collecting duct progenitor niches of the mouse embryonic kidney. Cep120 loss led to reduced abundance of both cap mesenchyme and ureteric bud populations, due to a combination of delayed mitosis, increased apoptosis and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis and decline in kidney function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in the pathways essential for development, fibrosis and cystogenesis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney morphogenesis and identifies new therapeutic targets for patients with renal centrosomopathies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

Defective centrosome function can disrupt embryonic kidney development, by causing changes to the renal interstitium that leads to fibrocystic disease pathologies. Yet, it remains unknown how mutations in centrosome genes impact kidney interstitial cells. Here, we examined the consequences of defective centrosome biogenesis on stromal progenitor cell growth, differentiation and fate. Conditional deletion of Cep120 , a ciliopathy gene essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of pericytes, interstitial fibroblasts and mesangial cells. This was due to delayed mitosis, increased apoptosis, and changes in Wnt and Hedgehog signaling essential for differentiation of stromal lineages. Cep120 ablation resulted in hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis via enhanced TGF-β/Smad3-Gli2 signaling after renal injury. Our study defines the cellular and developmental defects caused by centrosome dysfunction in embryonic kidney stroma., Highlights: Defective centrosome biogenesis in kidney stroma causes:Reduced abundance of stromal progenitors, interstitial and mesangial cell populationsDefects in cell-autonomous and paracrine signalingAbnormal/delayed nephrogenesis and tubular dilationsAccelerates injury-induced fibrosis via defective TGF-β/Smad3-Gli2 signaling axis.

Mutations that disrupt centrosome structure or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet, it remains unclear how mutations in proteins essential for centrosome biogenesis impact embryonic kidney development. Here, we examined the consequences of conditional deletion of a ciliopathy gene, Cep120 , in the two nephron progenitor niches of the embryonic kidney. Cep120 loss led to reduced abundance of both metanephric mesenchyme and ureteric bud progenitor populations. This was due to a combination of delayed mitosis, increased apoptosis, and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis, and decline in filtration function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in pathways essential for branching morphogenesis, cystogenesis and fibrosis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney development, and identifies new therapeutic targets for renal centrosomopathies., Highlights: Defective centrosome biogenesis in nephron progenitors causes:Reduced abundance of metanephric mesenchyme and premature differentiation into tubular structuresAbnormal branching morphogenesis leading to reduced nephron endowment and smaller kidneysChanges in cell-autonomous and paracrine signaling that drive cystogenesis and fibrosisUnique cellular and developmental defects when compared to Pkd1 knockout models.

Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Multiciliated cells (MCC) are evolutionary conserved, highly specialized cell types that contain dozens to hundreds of motile cilia that they use to propel fluid directionally. To template these cilia, each MCC produces between 30 and 500 basal bodies via a process termed centriole amplification. Much progress has been made in recent years in understanding the pathways involved in MCC fate determination, differentiation, and ciliogenesis. Recent studies using mammalian cell culture systems, mice, Xenopus, and other model organisms have started to uncover the mechanisms involved in centriole and cilia biogenesis. Yet, how MCC progenitor cells regulate the precise number of centrioles and cilia during their differentiation remains largely unknown. In this review, we will examine recent findings that address this fundamental question., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment., Competing Interests: Declarations of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Centrioles are vital cellular structures that organise centrosomes and cilia. Due to their subresolutional size, centriole ultrastructural features have been traditionally analysed by electron microscopy. Here we present an adaptation of magnified analysis of the proteome expansion microscopy method, to be used for a robust analysis of centriole number, duplication status, length, structural abnormalities and ciliation by conventional optical microscopes. The method allows the analysis of centriole's structural features from large populations of adherent and nonadherent cells and multiciliated cultures. We validate the method using EM and superresolution microscopy and show that it can be used as an affordable and reliable alternative to electron microscopy in the analysis of centrioles and cilia in various cell cultures. LAY DESCRIPTION: Centrioles are microtubule-based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 nm wide. Centrioles assemble dozens of proteins around them forming centrosomes, which nucleate microtubules and organise spindle poles in mitosis. Centrioles, in addition, assemble cilia and flagella, two critically important organelles for signalling and motility. Due to centriole small size, electron microscopy has been a major imaging technique for the analysis of their ultrastructural features. However, being technically demanding, electron microscopy it is not easily available to the researchers and it is rarely used to collect large datasets. Expansion microscopy is an emerging approach in which biological specimens are embedded in a swellable polymer and isotopically expanded several fold. Physical separation of cellular structures allows the analysis of, otherwise unresolvable, structures by conventional optical microscopes. We present an adaptation of expansion microscopy approach, specifically developed for a robust analysis of centrioles and cilia. Our protocol can be used for the analysis of centriole number, duplication status, length, localisation of various centrosomal components and ciliation from large populations of cultured adherent and nonadherent cells and multiciliated cultures. We validate the method against electron microscopy and superresolution microscopy and demonstrate that it can be used as an accessible and reliable alternative to electron microscopy., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells., Competing Interests: RN, DK, KS, TS, SB, JL, MM No competing interests declared, (© 2019, Nanjundappa et al.)

Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole's distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of distal appendage assembly. We further show that distal appendages undergo a dramatic ultra-structural reorganization before mitosis, during which they temporarily lose outer components, while inner components maintain a nine-fold organization. Finally, using electron tomography we reveal that mammalian distal appendages associate with two centriole microtubule triplets via an elaborate filamentous base and that they appear as almost radial finger-like protrusions. Our findings challenge the traditional portrayal of mammalian distal appendage as a pinwheel-like structure that is maintained throughout mitosis.

In an ancillary analysis of cross-sectional observational studies of bone health in end-stage kidney disease (ESKD), Evenepoel et al. reported that subjects with autosomal-dominant polycystic kidney disease (ADPKD) had a unique phenotype in their renal osteodystrophy. ADPKD caused resistance to parathyroid hormone (PTH) producing lower turnover states and preservation of cortical bone mineral density. PTH resistance was probably produced by increased osteocyte sclerostin levels, which is regulated by mechanical loading sensed through primary cilia sensory function affected by mutation in PKD1 and PKD2., (Copyright © 2019. Published by Elsevier Inc.)

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Background: Biallelic deleterious variants in RTTN, which encodes rotatin, are associated with primary microcephaly, polymicrogyria, seizures, intellectual disability, and primordial dwarfism in human infants., Methods and Results: We performed exome sequencing of an infant with primary microcephaly, pontocerebellar hypoplasia, and intractable seizures and his healthy, unrelated parents. We cultured the infant's fibroblasts to determine primary ciliary phenotype., Results: We identified biallelic variants in RTTN in the affected infant: a novel missense variant and a rare, intronic variant that results in aberrant transcript splicing. Cultured fibroblasts from the infant demonstrated reduced length and number of primary cilia., Conclusion: Biallelic variants in RTTN cause primary microcephaly in infants. Functional characterization of primary cilia length and number can be used to determine pathogenicity of RTTN variants.

Centrosome number is tightly controlled to ensure proper ciliogenesis, mitotic spindle assembly, and cellular homeostasis. Centrosome amplification (the formation of excess centrosomes) has been noted in renal cells of patients and animal models of various types of cystic kidney disease. Whether this defect plays a causal role in cystogenesis remains unknown. Here, we investigate the consequences of centrosome amplification during kidney development, homeostasis, and after injury. Increasing centrosome number in vivo perturbed proliferation and differentiation of renal progenitors, resulting in defective branching morphogenesis and renal hypoplasia. Centrosome amplification disrupted mitotic spindle morphology, ciliary assembly, and signaling pathways essential for the function of renal progenitors, highlighting the mechanisms underlying the developmental defects. Importantly, centrosome amplification was sufficient to induce rapid cystogenesis shortly after birth. Finally, we discovered that centrosome amplification sensitized kidneys in adult mice, causing cystogenesis after ischemic renal injury. Our study defines a new mechanism underlying the pathogenesis of renal cystogenesis, and identifies a potentially new cellular target for therapy., (© 2018 Dionne et al.)

Biallelic nonsense mutations of SYNE1 underlie a variable array of cerebellar and non-cerebellar pathologies of unknown molecular etiology. SYNE1 encodes multiple isoforms of Nesprin1 that associate with the nuclear envelope, with large cerebellar synapses and with ciliary rootlets of photoreceptors. Using two novel mouse models, we determined the expression pattern of Nesprin1 isoforms in the cerebellum whose integrity and functions are invariably affected by SYNE1 mutations. We further show that a giant isoform of Nesprin1 associates with the ciliary rootlets of ependymal cells that line brain ventricles and establish that this giant ciliary isoform of Nesprin1 harbors a KASH domain. Whereas cerebellar phenotypes are not recapitulated in Nes1g STOP/STOP mice, these mice display a significant increase of ventricular volume. Together, these data fuel novel hypotheses about the molecular pathogenesis of SYNE1 mutations and support that KASH proteins may localize beyond the nuclear envelope in vivo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The two centrioles of the centrosome in quiescent cells are inherently asymmetric structures that differ in age, morphology and function. How these asymmetric properties are established and maintained during quiescence remains unknown. Here, we show that a daughter centriole-associated ciliopathy protein, Cep120, plays a critical inhibitory role at daughter centrioles. Depletion of Cep120 in quiescent mouse and human cells causes accumulation of pericentriolar material (PCM) components including pericentrin, Cdk5Rap2, ninein and Cep170. The elevated PCM levels result in increased microtubule-nucleation activity at the centrosome. Consequently, loss of Cep120 leads to aberrant dynein-dependent trafficking of centrosomal proteins, dispersal of centriolar satellites, and defective ciliary assembly and signaling. Our results indicate that Cep120 helps to maintain centrosome homeostasis by inhibiting untimely maturation of the daughter centriole, and defines a potentially new molecular defect underlying the pathogenesis of ciliopathies such as Jeune Asphyxiating Thoracic Dystrophy and Joubert syndrome., Competing Interests: EB, RN, TC, MM No competing interests declared, (© 2018, Betleja et al.)

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between β-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates β-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and β-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1 flox/flox :Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD., (Published by Elsevier Inc.)

SYNE1 (synaptic nuclear envelope 1) encodes multiple isoforms of Nesprin1 (nuclear envelope spectrin 1) that associate with the nuclear envelope (NE) through a C-terminal KASH (Klarsicht/Anc1/Syne homology) domain (Figure 1A) [1-4]. This domain interacts directly with the SUN (Sad1/Unc84) domain of Sun proteins [5-7], a family of transmembrane proteins of the inner nuclear membrane (INM) [8, 9], to form the so-called LINC complexes (linkers of the nucleoskeleton and cytoskeleton) that span the entire NE and mediate nuclear positioning [10-12]. In a stark departure from this classical depiction of Nesprin1 in the context of the NE, we report here that rootletin recruits Nesprin1α at the ciliary rootlets of photoreceptors and identify asymmetric NE aggregates of Nesprin1α and Sun2 that dock filaments of rootletin at the nuclear surface. In NIH 3T3 cells, we show that recombinant rootletin filaments also dock to the NE through the specific recruitment of an ∼600-kDa endogenous isoform of Nesprin1 (Nes1 600kDa ) and of Sun2. In agreement with the association of Nesprin1α with photoreceptor ciliary rootlets and the functional interaction between rootletin and Nesprin1 in fibroblasts, we demonstrate that multiple isoforms of Nesprin1 are integral components of ciliary rootlets of multiciliated ependymal and tracheal cells. Together, these data provide a novel functional paradigm for Nesprin1 at ciliary rootlets and suggest that the wide spectrum of human pathologies linked to truncating mutations of SYNE1 [13-15] may originate in part from ciliary defects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The establishment of left-right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites-cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer's vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite-associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease., (© 2016 Silva, Betleja, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The centrosome and cilium are evolutionarily conserved components of the microtubule cytoskeleton, and act as a cellular signaling center that regulates the activity of numerous developmental signaling pathways. Several genetic syndromes, called the ciliopathies, are associated with defects in the structure or function of the centrosome-cilium complex. In the mammalian kidney, these organelles are found at the apical surface of renal epithelial cells lining the various segments of the nephron, where they relay information from the extracellular environment to the interior of the cell. Cilium-based signaling plays an important role in the development and homeostasis of mammalian kidneys, and ciliary dysfunction is implicated in the pathogenesis of cystic kidney disease. Given the importance of centrosomes and cilia in renal function, techniques used to visualize these organelles, analyze their composition, and test their functionality have become essential in many studies of kidney development and disease. Fluorescence microscopy is a powerful, widely used technique that has enhanced our understanding of molecular mechanisms that regulate the assembly, maintenance, and function of these organelles in various organs. Here, we present detailed steps for the isolation of kidneys from adult and embryonic mice, describe protocols to label centrosomes and cilia in renal tissues, and methods used to culture and image kidneys ex vivo., (Copyright © 2015. Published by Elsevier Inc.)

The centrosome is the main microtubule-organizing center in animal cells, and helps to influence the morphology of the microtubule cytoskeleton in interphase and mitosis. The centrosome also templates the assembly of the primary cilium, and together they serve as a nexus of cell signaling that provide cells with diverse organization, motility, and sensory functions. The majority of cells in the human body contain a solitary centrosome and cilium, and cells have evolved regulatory mechanisms to precisely control the numbers of these essential organelles. Defects in the structure and function of cilia lead to a variety of complex disease phenotypes termed ciliopathies, while dysregulation of centrosome number has long been proposed to induce genome instability and tumor formation. Here, we review recent findings that link centrosome amplification to changes in cilium number and signaling capacity, and discuss how supernumerary centrosomes may be an important aspect of a set of cilia-related disease phenotypes.

Competing Interests: The authors have declared that no competing interests exist.

The primary cilium is a nexus of cell signaling, and ciliary dysfunction is associated with polycystic kidney disease, retinal degeneration, polydactyly, neural tube defects, and obesity (ciliopathies). Signaling molecules for cilium-associated pathways are concentrated in the cilium, and this is essential for efficient signaling. Cilia are nucleated from centrioles, and aberrant centriole numbers are seen in many cancers and in some ciliopathies. We tested the effect of supernumerary centrioles on cilium function and found that cells with extra centrioles often formed more than one cilium, had reduced ciliary concentration of Smoothened in response to Sonic hedgehog stimulation, and reduced Shh pathway transcriptional activation. This ciliary dilution phenotype was also observed with the serotonin receptor Htr6, fibrocystin PKHD1, and Arl13b. The presence of extra centrioles and cilia disrupted epithelial organization in 3D spheroid culture. Cells mutant for the tuberous sclerosis gene Tsc2 also had extra cilia and diluted ciliary protein. In most cells, extra cilia were clustered and shared the same ciliary pocket, suggesting that the ciliary pocket is the rate-limiting structure for trafficking of ciliary proteins. Thus, extra centrioles and cilia disrupt signaling and may contribute to disease phenotypes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase-arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.

Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes., (2010 Wiley-Liss, Inc.)

A key feature of the polycystic kidney diseases is aberrant cell proliferation, a consequence of dysfunctional ciliary signaling. The NIMA-related kinases (Nek) Nek1 and Nek8 carry the causal mutations of two of the eight established mouse models of polycystic kidneys. Nek proteins have roles in cell cycle and may contribute to coordinate regulation of cilia and cell-cycle progression. Herein is reported that in a mouse kidney epithelial cell line, mNek1 localizes to centrosomes in interphase and remains associated with the mitotic spindle pole during mitosis. In contrast, mNek8 localizes to the proximal region of the primary cilium and is not observed in dividing cells. Knockdown of mNek8 by siRNA does not affect ciliary assembly. Taken together with the phenotypes of the mutant mice, these data suggest that mNek1 and mNek8 provide links between cilia, centrosomes, and cell-cycle regulation.

The Nek family of cell-cycle kinases is widely represented in eukaryotes and includes numerous proteins that were described only recently and remain poorly characterized. Comparing Neks in the context of clades allows us to examine the question of whether microbial eukaryotic Neks, although not strictly orthologs of their vertebrate counterparts, can provide clues to ancestral functions that might be retained in the vertebrate Neks. Relatives of the Nek2/NIMA proteins play important roles at the G2-M transition in nuclear envelope breakdown and centromere separation. Nek6, Nek7 and Nek9 also seem to regulate mitosis. By contrast, Nek1 and Nek8 have been linked with polycystic kidney disease. Results of statistical analysis indicate that the family coevolved with centrioles that function as both microtubule-organizing centers and the basal bodies of cilia. This evolutionary perspective, taken together with functional studies of microbial Neks, provides new insights into the cellular roles of the proteins and disease with which some of them have been linked.

Polycystic kidney disease and related syndromes involve dysregulation of cell proliferation in conjunction with ciliary defects. The relationship between cilia and cell cycle is enigmatic, but it may involve regulation by the NIMA-family of kinases (Neks). We previously showed that the Nek Fa2p is important for ciliary function and cell cycle in Chlamydomonas. We now show that Fa2p localizes to an important regulatory site at the proximal end of cilia in both Chlamydomonas and a mouse kidney cell line. Fa2p also is associated with the proximal end of centrioles. Its localization is dynamic during the cell cycle, following a similar pattern in both cell types. The cell cycle function of Fa2p is kinase independent, whereas its ciliary function is kinase dependent. Mice with mutations in Nek1 or Nek8 have cystic kidneys; therefore, our discovery that a member of this phylogenetic group of Nek proteins is localized to the same sites in Chlamydomonas and kidney epithelial cells suggests that Neks play conserved roles in the coordination of cilia and cell cycle progression.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Importantly, we demonstrate that BBS-7 and BBS-8 are required for the normal localization/motility of the IFT proteins OSM-5/Polaris and CHE-11, and to a notably lesser extent, CHE-2. We propose that BBS proteins play important, selective roles in the assembly and/or function of IFT particle components. Our findings also suggest that some of the cardinal and secondary symptoms of BBS, such as obesity, diabetes, cardiomyopathy, and learning defects may result from cilia dysfunction.

The NIMA kinases are one of several families of kinases that participate in driving the eukaryotic cell cycle. NIMA-related kinases have been implicated in G2/M progression, chromatin condensation and regulation of the centrosome cycle. Here we report the identification of a new member of this family, FA2, from Chlamydomonas reinhardtii. FA2 was originally discovered in a genetic screen for deflagellation-defective mutants. We have previously shown that FA2 is essential for basal-body/centriole-associated microtubule severing. We now report that the FA2 NIMA-related kinase also plays a role in cell cycle progression in Chlamydomonas. This is the first indication that members of the NIMA family might exert their effects through the regulation of microtubule severing.