Kif9 is an active kinesin motor required for ciliary beating and proximodistal patterning of motile axonemes.

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair of microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate ortholog, Kif9, is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility and the proper distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro reveal that Xenopus Kif9 is a long-range processive motor, although it does not mediate long-range movement in ciliary axonemes in vivo. Together, our data suggest that Kif9 is integral for ciliary beating and is necessary for proper axonemal distal end integrity.

Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.

Establishment of the early cilia preassembly protein complex during motile ciliogenesis.

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

Genetics and biology of primary ciliary dyskinesia.

Ciliopathies are a growing class of disorders caused by abnormal ciliary axonemal structure and function. Our understanding of the complex genetic and functional phenotypes of these conditions has rapidly progressed. Primary ciliary dyskinesia (PCD) remains the sole genetic disorder of motile cilia dysfunction. However, unlike many Mendelian genetic disorders, PCD is not caused by mutations in a single gene or locus, but rather, autosomal recessive mutation in one of many genes that lead to a similar phenotype. The first reported PCD mutations, more than a decade ago, identified genes encoding known structural components of the ciliary axoneme. In recent years, mutations in genes encoding novel cytoplasmic and regulatory proteins have been discovered. These findings have provided new insights into the functions of the motile cilia, and a better understanding of motile cilia disease. Advances in genetic tools will soon allow more precise genetic testing, mandating that clinicians must understand the genetic basis of PCD. Here, we review genetic mutations, their biological impact on cilia structure and function, and the implication of emerging genetic diagnostic tools.

Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia.

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.

NKp46 regulates allergic responses.

Natural killer (NK) cells are cytotoxic cells that are able to rapidly kill viruses, tumor cells, parasites, bacteria, and even cells considered "self". The activity of NK cells is controlled by a fine balance of inhibitory and activating signals mediated by a complex set of different receptors. However, the function of NK cells is not restricted only to the killing of target cells, NK cells also possess other properties such as the secretion of proangiogenic factors during pregnancy. Here, we demonstrate another unique NK-cell activity, namely the regulation of T-cell mediated allergic responses, which is dependent on the NK-cell specific receptor NKp46 (Ncr1 in mice). Using mice in which the Ncr1 gene has been replaced with a green fluorescent protein, we demonstrate reduced delayed-type hypersensitivity and airway hypersensitivity. Interestingly, we show that this reduction in airway hypersensitivity is due to differences in the stimulation of T cells resulting in an altered cytokine profile.

Picking up speed: advances in the genetics of primary ciliary dyskinesia.

Abnormal ciliary axonemal structure and function are linked to the growing class of genetic disorders collectively known as ciliopathies, and our understanding of the complex genetics and functional phenotypes of these conditions has rapidly expanded. While progress in genetics and biology has uncovered numerous cilia-related syndromes, primary ciliary dyskinesia (PCD) remains the sole genetic disorder of motile cilia dysfunction. The first disease-causing mutation was described just 13 y ago, and since that time, the pace of gene discovery has quickened. These mutations separate into genes that encode axonemal motor proteins, structural and regulatory elements, and cytoplasmic proteins that are involved in assembly and preassembly of ciliary elements. These findings have yielded novel insights into the processes involved in ciliary assembly, structure, and function, which will allow us to better understand the clinical manifestations of PCD. Moreover, advances in techniques for genetic screening and sequencing are improving diagnostic approaches. In this article, we will describe the structure, function, and emerging genetics of respiratory cilia, review the genotype-phenotype relationships of motor ciliopathies, and explore the implications of recent discoveries for diagnostic testing for PCD.

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy.

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.

Transcriptional analysis of primary ciliary dyskinesia airway cells reveals a dedicated cilia glutathione pathway.

Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA-Seq, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, and healthy individuals, and we induced pluripotent stem cells (iPScs) generated from a patient with PCD. Transcriptomic analysis revealed unique signatures in PCD airway cells compared with their mothers' cells and the cells of healthy individuals. Gene expression in heterozygous mothers' cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism Chlamydomonas reinhardtii demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility, pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.

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.

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.

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 storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction.

The identification of factors that regulate airway epithelial cell proliferation and differentiation are essential for understanding the pathophysiology of airway diseases. Rho-associated protein kinases (ROCKs) are downstream effector proteins of RhoA GTPase that direct the functions of cell cytoskeletal proteins. ROCK inhibition with Y27632 has been shown to enhance the survival and cloning of human embryonic stem cells and pluripotent cells in other tissues. We hypothesized that Y27632 treatment exerts a similar effect on airway epithelial basal cells, which function as airway epithelial progenitor cells. Treatment with Y27632 enhanced basal-cell proliferation in cultured human tracheobronchial and mouse tracheal epithelial cells. ROCK inhibition accelerated the maturation of basal cells, characterized by a diminution of the cell size associated with cell compaction and the expression of E-cadherin at cell-cell junctions. Transient treatment of cultured basal cells with Y27632 did not affect subsequent ciliated or mucous cell differentiation under air-liquid interface conditions, and allowed for the initial use of lower numbers of human or mouse primary airway epithelial cells than otherwise possible. Moreover, the use of Y27632 during lentivirus-mediated transduction significantly improved posttransduction efficiency and the selection of a transduced cell population, as determined by reporter gene expression. These findings suggest an important role for ROCKs in the regulation of proliferation and maturation of epithelial basal cells, and demonstrate that the inhibition of ROCK pathways using Y27632 provides an adjunctive tool for the in vitro genetic manipulation of airway epithelial cells by lentivirus vectors.

One person can make a difference: identification of people with a rare genetic lung disease.

To improve access to care for rare conditions in resource-restricted regions, a concerted effort to establish centres of excellence and training of local physicians is needed https://bit.ly/3ZTBvaj.

Magnify is a universal molecular anchoring strategy for expansion microscopy.

Expansion microscopy enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a crosslinked water-swellable hydrogel. Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. We describe a strategy called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11 times and facilitates imaging of cells and tissues with effectively around 25-nm resolution using a diffraction-limited objective lens of about 280 nm on conventional optical microscopes or with around 15 nm effective resolution if combined with super-resolution optical fluctuation imaging. We demonstrate Magnify on a broad range of biological specimens, providing insight into nanoscopic subcellular structures, including synaptic proteins from mouse brain, podocyte foot processes in formalin-fixed paraffin-embedded human kidney and defects in cilia and basal bodies in drug-treated human lung organoids.

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

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 partial 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. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, 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. Brief Summary: A mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.