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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.

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.

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.