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Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Airway ciliary function analysis underpins PCD diagnostics and ex vivo/in vitro mucociliary clearance studies. It is an important measure of airway culture model integrity in health and after microbial/viral infections or airway drug therapies.

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., Author summary Primary ciliary dyskinesia (PCD) is a rare genetic disease that affects the function of motile cilia. The estimated incidence is about 1 in 15,000 births, but diagnosis can be difficult since it is genetically heterogenous. At present, about 30% of PCD patients lack a genetic diagnosis. By applying whole exome sequencing and bioinformatic analysis, we identified a variant in an uncharacterized gene, CFAP57, in a subject previously diagnosed with PCD. This is the first reported example of PCD caused by a mutation that likely only affects a subset of the inner dynein arms. These findings demonstrate the effectiveness of whole exome sequencing and bioinformatic analysis for identifying pathogenic variants in rare genetic diseases like PCD, expand our understanding of how dynein arms are positioned during cilia assembly, and identify a variant in CFAP57 that causes PCD.

BackgroundPrimary ciliary dyskinesia (PCD) is a rare genetic disorder resulting in abnormal ciliary motility/structure, extremely heterogeneous at genetic and ultrastructural levels. We aimed, in light of extensive genotyping, to identify specific and quantitative ciliary beating anomalies, according to the ultrastructural phenotype.MethodsWe prospectively included 75 patients with PCD exhibiting the main five ultrastructural phenotypes (n=15/group), screened all corresponding PCD genes and measured quantitative beating parameters by high-speed video-microscopy (HSV).ResultsSixty-eight (91%) patients carried biallelic mutations. Combined outer/inner dynein arms (ODA/IDA) defect induces total ciliary immotility, regardless of the gene involved. ODA defect induces a residual beating with dramatically low ciliary beat frequency (CBF) related to increased recovery stroke and pause durations, especially in case of DNAI1 mutations. IDA defect with microtubular disorganisation induces a low percentage of beating cilia with decreased beating angle and, in case of CCDC39 mutations, a relatively conserved mean CBF with a high maximal CBF. Central complex defect induces nearly normal beating parameters, regardless of the gene involved, and a gyrating motion in a minority of ciliated edges, especially in case of RSPH1 mutations. PCD with normal ultrastructure exhibits heterogeneous HSV values, but mostly an increased CBF with an extremely high maximal CBF.ConclusionQuantitative HSV analysis in PCD objectives beating anomalies associated with specific ciliary ultrastructures and genotypes. It represents a promising approach to guide the molecular analyses towards the best candidate gene(s) to be analysed or to assess the pathogenicity of the numerous sequence variants identified by next-generation-sequencing.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterizedCFAP57gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown ofCFAP57in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, andCFAP57is allelic with theBOP2gene identified previously inChlamydomonas. Two independent, insertionalfap57 Chlamydomonasmutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the “g” inner dyneins (DHC7 and DHC3) and the “d” inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations inCFAP57as a cause of PCD with a specific defect in the inner dynein arm assembly process.SignificanceMotile cilia are found throughout eukaryotic organisms and performs essential functions. Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia. By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene,CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations inCFAP57as a cause of PCD.

Functional cilia and flagella are crucial to the propulsion of physiological fluids, motile cells, and microorganisms. Motility assessment of individual cells allows discrimination of normal from dysfunctional behavior, but cell-scale analysis of individual trajectories to represent a population is laborious and impractical for clinical, industrial, and even research applications. We introduce an assay that quantifies swimming capability as a function of the variation in polar moment of inertia of cells released from an acoustic trap. Acoustic confinement eliminates the need to trace discrete trajectories and enables automated analysis of hundreds of cells in minutes. The approach closely approximates the average speed estimated from the mean squared displacement of individual cells for wild-type Chlamydomonas reinhardtii and two mutants (ida3 and oda5) that display aberrant swimming behaviors. Large-population acoustic trap-and-release rapidly differentiates these cell types based on intrinsic motility, which provides a highly sensitive and efficient alternative to conventional particle tracing.

Monoclonal antibodies (mAbs) are promising therapies to treat airway chronic inflammatory disease (asthma or nasal polyps). To date, no study has specifically assessed, in vitro, the potential function of neonatal Fc receptor (FcRn) in IgG transcytosis through the human nasal airway epithelium. The objective of this study was to report the in vitro expression and function of FcRn in nasal human epithelium. FcRn expression was studied in an air&ndash, liquid interface (ALI) primary culture model of human nasal epithelial cells (HNEC) from polyps. FcRn expression was characterized by quantitative RT-PCR, western blot, and immunolabeling. The ability of HNECs to support mAb transcytosis via FcRn was assessed by transcytosis assay. This study demonstrates the expression of FcRn mRNA and protein in HNEC. We report a high expression of FcRn in the cytosol of ciliated, mucus, and basal cells by immunohistochemistry with a higher level of FcRn proteins in differentiated HNEC. We also proved in vitro transepithelial delivery of an IgG1 therapeutic mAb with a dose&ndash, response curve. This is the first time that FcRn expression and mAb transcytosis has been shown in a model of human nasal respiratory epithelium in vitro. This study is a prerequisite for FcRn-dependent nasal administration of mAbs.

The effects of cilium length on the dynamics of cilia motion were investigated by high-speed video microscopy of uniciliate mutants of the swimming alga,Chlamydomonas reinhardtii.Cells with short cilia were obtained by deciliating cells via pH shock and allowing cilia to reassemble for limited times. The frequency of cilia beating was estimated from motion of the cell body and of the cilium. Key features of the ciliary waveform were quantified from polynomial curves fitted to the cilium in each image frame. Most notably, periodic beating did not emerge until the cilium reached a critical length between 2-4 µm. Surprisingly, in cells that exhibited periodic beating, the frequency of beating was similar for all lengths with only a slight decrease in frequency as length increased from 4 µm to the normal length of 10-12 µm. The waveform average curvature (rad/µm) was also conserved as the cilium grew. The mechanical metrics of ciliary propulsion: force, torque, and power all increased in proportion to length. Mechanical efficiency of beating appeared to be maximal at the normal wild-type length of 10-12 μm. These quantitative features of ciliary behavior illuminate the biophysics of cilia motion and, in future studies, may help distinguish competing hypotheses of the underlying mechanism of oscillation.

Mucociliary clearance is one of the major lines of defense of the human respiratory system. The mucus layer coating the airways is constantly moved along and out of the lung by the activity of motile cilia, expelling at the same time particles trapped in it. The efficiency of the cilia motion can experimentally be assessed by measuring the velocity of micro-beads traveling through the fluid surrounding the cilia. Here we present a mathematical model of the fluid flow and of the micro-beads motion. The coordinated movement of the ciliated edge is represented as a continuous envelope imposing a periodic moving velocity boundary condition on the surrounding fluid. Vanishing velocity and vanishing shear stress boundary conditions are applied to the fluid at a finite distance above the ciliated edge. The flow field is expanded in powers of the amplitude of the individual cilium movement. It is found that the continuous component of the horizontal velocity at the ciliated edge generates a 2D fluid velocity field with a parabolic profile in the vertical direction, in agreement with the experimental measurements. Conversely, we show than this model can be used to extract microscopic properties of the cilia motion by extrapolating the micro-bead velocity measurement at the ciliated edge. Finally, we derive from these measurements a scalar index providing a direct assessment of the cilia beating efficiency. This index can easily be measured in patients without any modification of the current clinical procedures., Author summary Mucociliary clearance is the first line of defense mechanisms of the human airways. The mucus transporting debris, particles, microorganisms and pollutants is carried away by the coordinated motion of cilia beating at the surface of the airway epithelium. We present here a mathematical and numerical model aiming at defining a global index for assessing the efficiency of this beating. Numerical simulations show that the bead velocity parallel to the wall varies according a parabolic profile with the distance to the wall. The velocity extrapolated at the wall is demonstrated to be a measurement of the momentum transfer between cilia and the surrounding fluid. This model allows us to interpret experimental measurements performed in a companion article and to propose a universal index characterizing the beating efficiency, which can be extracted in the current clinical setting.

Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivo measurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow, presented in greater detail in a second companion article. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. The obtained experimental data are used to feed a 2D mathematical and numerical model of the coupling between cilia, fluid, and micro-bead motion. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress, which can easily be measured in the clinical setting, is proposed as a new index for characterizing the efficiency of ciliary beating., Author summary Mucociliary clearance is the first line of defense of the human pulmonary airways. Mucus transporting debris, particles, microorganisms and pollutants is carried away by the coordinated motion of cilia beating at the surface of the airway epithelium. We present here an experimental, mathematical and numerical study aiming at defining a global index for assessing the efficiency of this beating. We measure experimentally the ciliary beat frequency, ciliary beat amplitude, and metachronal wavelength on ciliated edges obtained from nasal brushing. Properties of fluid motion are simultaneously extracted from micro-bead tracking next to the ciliated edge. A mathematical and numerical model is developed to describe the fluid motion induced by the cilia tips considered as a moving wall. Experimental and numerical results show that the bead velocity is a parabolic function of the distance to the wall. It allows us to infer the shear stress exerted by the cilia on fluid from micro-bead tracking. This quantity is proposed as a universal index characterizing the beating efficiency, which can be extracted in the current clinical setting.

High speed videomicroscopy (HSV) becomes a key test in primary ciliary dyskinesia (PCD), providing a qualitative evaluation and recently a quantitative analysis of ciliary beating. We aimed to correlate ultrastructural phenotype and ciliary beating Method: 75 PCD patients were prospectively included to reach 15 patients per main ultrastructural phenotypes (combined outer/inner dynein arm defect, outer dynein arm defect, inner dynein arm defect with microtubular disorganization, central complex defect and PCD with normal ultrastructure). Ciliary beating was assessed by the proportion of beating ciliated edges, the ciliary beat frequency (CBF), a qualitative evaluation and a quantitative analysis allowing measuring objective parameters Result: Combined outer/inner dynein arm defect induced a total ciliary immotility, whatever the involved gene; (ii) Outer dynein arm defect induced a minimal residual beating with dramatically low CBF related to increased pause and recovery duration, especially in case of DNAI1 mutations; (iii) Inner dynein arm defect induced a weak proportion of ciliated edges still beating, a decreased beating angle and furthermore a relatively conserved CBF in case of CCDC39 mutations; (iv) Central complex defect induced nearly normal quantitative parameters with a circular gyrating motion in a minority of ciliated edges, whatever the involved gene; (v) PCD with normal ultrastructure induced a wide discrepancy in beating parameters but a frequent increased CBF Conclusion: Quantitative analysis by HSV allows objectively detecting abnormal values of ciliary beating parameters characteristic of each of the main ultrastructural phenotypes reported in PCD.

International audience; Introduction. Cilia motility is an important diagnostic feature for various nasal diseases, both of acquired and genetic origin. So far this assessment has been performed ex-vivo in nasal samples. Sampling is invasive and may damage cilia, and ex-vivo measurements may not always reflect the in-vivo cilia function. In this work we investigated the possibility of assessing cilia motility in vivo in humans with a preliminary study using confocal micro-endoscopy.Materials and methods. We used the high-power laser confocal endoscope developed by Mauna Kea Technology (MKT). This device has a 1µm spatial resolution and a temporal resolution varying between 8 and 90 Hz. Ex-vivo pig trachea samples and human nasal biopsy samples were labelled with fluorescent marker Octadecyl Rhodamine B Chloride (R18). Beating cilia were easily identified allowing the acquisition of 40 videos ready for analysis. Using in-house software that estimates frequencies based on luminance variation in a small window and FFT analysis, we evaluated cilia and compared the beat frequency with ground truth.Results. We validated our estimations on all sequences acquired between 30 and 90Hz. Videos acquired at less than 30Hz did not offer sufficient temporal resolution We only observed occasional errors when the software identified a harmonic oscillation instead of the fundamental frequency.

Cellular adhesion forces depend on local biological conditions meaning that adhesion characterization must be performed while preserving cellular integrity. We presently postulate that magnetic bead twisting provides an appropriate stress, i.e., basically a clamp, for assessment in living cells of both cellular adhesion and mechanical properties of the cytoskeleton. A global dissociation rate obeying a Bell-type model was used to determine the natural dissociation rate ([Formula: see text]) and a reference stress ([Formula: see text]). These adhesion parameters were determined in parallel to the mechanical properties for a variety of biological conditions in which either adhesion or cytoskeleton was selectively weakened or strengthened by changing successively ligand concentration, actin polymerization level (by treating with cytochalasin D), level of exerted stress (by increasing magnetic torque), and cell environment (by using rigid and soft 3D matrices). On the whole, this multiscale evaluation of the cellular and molecular responses to a controlled stress reveals an evolution which is consistent with stochastic multiple bond theories and with literature results obtained with other molecular techniques. Present results confirm the validity of the proposed bead-twisting approach for its capability to probe cellular and molecular responses in a variety of biological conditions.

Introduction Les dyskinesies ciliaires primitives (DCP) sont un groupe de maladies genetiques rares responsable d’une anomalie du mouvement des cils respiratoires. Le diagnostic est classiquement confirme grâce a l’identification d’une anomalie de l’ultra-structure ciliaire par microscopie electronique a transmission. L’analyse genetique ne peut etre proposee en premiere intention, etant donne le nombre (> 30 genes) et la taille (> 80 exons) des genes impliques. Nous avons souhaite etudier si la mesure objective du mouvement ciliaire par video-microscopie a haute vitesse pourrait predire le phenotype ultra-structural et ainsi guider l’analyse genetique. Methodes Nous avons prospectivement inclus 75 patients ayant un diagnostic certain de DCP, presentant un des 5 principaux phenotypes ultra-structuraux identifie par microscopie electronique (15 patients par groupe), et pour lesquels tous les genes correspondants ont ete sequences. L’etude du mouvement ciliaire a inclus le pourcentage de cil battant, la frequence de battement ciliaire, une evaluation qualitative et la mesure quantitative de 12 parametres du mouvement ciliaire. Resultats L’absence combinee des bras de dyneine externe et interne induit une immobilite ciliaire totale, quel que soit le gene implique. L’absence de bras de dyneine externe permet un rare mouvement ciliaire residuel avec une frequence de battement effondre en rapport avec un allongement du temps de pause, notamment en cas de mutation de DNAH1. L’absence de bras de dyneine interne avec desorganisation axonemale induit un faible pourcentage de cil battant avec une diminution de l’angle de battement, associe en cas de mutation de CCDC39 a une frequence maximale de battement augmentee. L’absence de complexe central permet un mouvement ciliaire relativement conserve, malgre une minorite de cil presentant un mouvement ciliaire circulaire, quel que soit le gene implique. Dans les DCP avec ultra-structure normale, les parametres de mouvement ciliaires ont des valeurs particulierement heterogenes, malgre une frequence de battement ciliaire augmentee avec une frequence maximale extreme. Conclusion L’analyse quantitative du mouvement ciliaire par video-microscopie a haute vitesse permet l’identification de parametres objectifs caracteristiques du phenotype ultra-structural des DCP.

Methods Ciliated cells issued from nasal brushing (controls and primary ciliary dyskinesia patients) were recorded by high-speed video-microscopy (350 frames s). We have performed an original quantitative analysis of ciliary beat dynamics (CBD) by following cilium tips. It allows to describe different parameters including ciliary beat frequency also measured by Fast-Fourier-Transform and Video-Kymography. We have also developed the microbeads tracking method (MBT) to get an index of the global efficiency of ciliary beat. Here, micro-beads (4.5 μm) have been used as markers of the flow generated by beating cilia.