The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus.

BACKGROUND: Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA-sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality. OBJECTIVES: We sought to comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (ie, eosinophilic esophagitis [EoE]). METHODS: We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings. RESULTS: In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n = 6 individuals] and 398 from esophagus [n = 6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably IFN-γ, IL-10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and the hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DNAX-activation protein-12 (also known as TYROBP) interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling. CONCLUSIONS: In EoE, esophageal eosinophils exist as 2 populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types.

A novel homozygous mutation in the DNAAF3 gene leads to severe asthenozoospermia and teratospermia.

Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder characterized by ultrastructural defects in the cilia or flagella of cells, causing respiratory abnormalities, sinusitis, visceral transposition, and male infertility. DNAAF3 plays an important role in the assembly and transportation of axonemal dynein complexes in cilia or flagella and has been shown to be associated with PCD. To date, only two cases of PCD with infertility associated with DNAAF3 mutations have been reported, and no mouse models for this gene have been successfully constructed. This study was conducted on an infertile Chinese male patient with a history of bronchitis. Examination of the patient's semen revealed severe asthenozoospermia and teratospermia. Whole exome sequencing revealed a new homozygous loss-of-function DNAAF3 mutation. CRISPR-Cas9 gene-editing technology was used to construct the same mutation in C57/B6 mice, revealing that homozygous C57/B6 mice were characterized by severe hydrocephalus and early death. The results of this study expand the mutation spectrum of DNAAF3 and confirm its correlation with PCD pathogenesis. This study provides new insights on the mechanisms underlying male infertility related to DNAAF3 mutation and PCD.

A novel mutation in PCD-associated gene DNAAF3 causes male infertility due to asthenozoospermia.

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive disease manifested with recurrent infections of respiratory tract and infertility. DNAAF3 is identified as a novel gene associated with PCD and different mutations in DNAAF3 results in different clinical features of PCD patients, such as situs inversus, sinusitis and bronchiectasis. However, the sperm phenotypic characteristics of PCD males are generally poorly investigated. Our reproductive medicine centre received a case of PCD patient with infertility, who presented with sinusitis, recurrent infections of the lower airway and severe asthenozoospermia; However, no situs inversus was found in the patient. A novel homozygous mutation in DNAAF3(c.551T>A; p.V184E) was identified in the PCD patient by whole-exome sequencing. Subsequent Sanger sequencing further confirmed that the DNAAF3 had a homozygous missense variant in the fifth exon. Transmission electron microscopy and immunostaining analysis of the sperms from the patient showed a complete absence of outer dynein arms and partial absence of inner dynein arms, which resulted in the reduction in sperm motility. However, this infertility was overcome by intracytoplasmic sperm injections, as his wife achieved successful pregnancy. These findings showed that the PCD-associated pathogenic mutation within DNAAF3 also causes severe asthenozoospermia and male infertility ultimately due to sperm flagella axoneme defect in humans. Our study not only contributes to understand the sperm phenotypic characteristics of patients with DNAAF3 mutations but also expands the spectrum of DNAAF3 mutations and may contribute to the genetic diagnosis and therapy for infertile patient with PCD.

Pathogenic variants identified using whole-exome sequencing in Chinese patients with primary ciliary dyskinesia.

The genetic factors contributing to primary ciliary dyskinesia (PCD), a rare autosomal recessive disorder, remain elusive for ~20%-35% of patients with complex and abnormal clinical phenotypes. Our study aimed to identify causative variants of PCD-associated pathogenic candidate genes using whole-exome sequencing (WES). All patients were diagnosed with PCD based on clinical phenotype or transmission electron microscopy images of cilia. WES and bioinformatic analysis were then conducted on patients with PCD. Identified candidate variants were validated by Sanger sequencing. Pathogenicity of candidate variants was then evaluated using in silico software and the American College of Medical Genetics and Genomics (ACMG) database. In total, 13 rare variants were identified in patients with PCD, among which were three homozygous causative variants (including one splicing variant) in the PCD-associated genes CCDC40 and DNAI1. Moreover, two stop-gain heterozygous variants of DNAAF3 and DNAH1 were classified as pathogenic variants based on the ACMG criteria. This study identified novel potential pathogenic genetic factors associated with PCD. Noteworthy, the patients with PCD carried multiple rare causative gene variants, thereby suggesting that known causative genes along with other functional genes should be considered for such heterogeneous genetic disorders.

Novel compound heterozygous DNAAF2 mutations cause primary ciliary dyskinesia in a Han Chinese family.

PURPOSE: Primary ciliary dyskinesia (PCD), which commonly causes male infertility, is an inherited autosomal recessive disorder. This study aimed to investigate the clinical manifestations and screen mutations associated with the dynein axonemal assembly factor 2 (DNAAF2) gene in a Han Chinese family with PCD. METHODS: A three-generation family with PCD was recruited in this study. Eight family members underwent comprehensive medical examinations. Genomic DNA was extracted from the participants' peripheral blood, and targeted next-generation sequencing technology was used to perform the mutation screening. The DNAAF2 expression was analyzed by immunostaining and Western blot. RESULTS: The proband exhibited the typical clinical features of PCD. Spermatozoa from the proband showed complete immotility but relatively high viability. Two novel compound heterozygous mutations in the DNAAF2 gene, c.C156A [p.Y52X] and c.C26A [p.S9X], were identified. Both nonsense mutations were detected in the proband, whereas the other unaffected family members carried either none or only one of the two mutations. The two nonsense heterozygous mutations were not detected in the 600 ethnically matched normal controls or in the Genome Aggregation Database. The defect of the DNAAF2 and the outer dynein arms and inner dynein arms were notably observed in the spermatozoa from the proband by immunostaining. CONCLUSION: This study identified two novel compound heterozygous mutations of DNAAF2 leading to male infertility as a result of PCD in a Han Chinese family. The findings may enhance the understanding of the pathogenesis of PCD and improve reproductive genetic counseling in China.

Novel DNAAF6 variants identified by whole-exome sequencing cause male infertility and primary ciliary dyskinesia.

PURPOSE: To identify the genetic cause of patients with primary ciliary dyskinesia (PCD) and male infertility from two unrelated Han Chinese families. METHODS: We conducted whole-exome sequencing of three individuals with PCD and male infertility from two unrelated Chinese families, and performed a targeted look-up for DNAAF6 variants in our previously reported cohort of 442 individuals (219 with isolated oligoasthenospermia and 223 fertile controls). Ultrastructural and immunostaining analyses of patients' spermatozoa were performed. The pathogenicity of the variants was validated using patient's spermatozoa and HEK293T cells. Intracytoplasmic sperm injection (ICSI) treatment was conducted in two patients. RESULTS: We identified one novel hemizygous frameshift variant (NM_173494, c.319_329del: p.R107fs) of DNAAF6 gene (previously named PIH1D3) in family 1 and one novel hemizygous missense variant (c.290G>T: p.G97V) in family 2. No hemizygous deleterious variants in DNAAF6 were detected in the control cohort of 442 individuals. Ultrastructural and immunostaining analyses of patients' spermatozoa showed the absence of outer and inner dynein arms in sperm flagella. Both variants were proven to lead to DNAAF6 protein degradation in HEK293T cells. Both patients carrying DNAAF6 variants underwent one ICSI cycle and delivered one healthy child each. CONCLUSION: We identified novel DNAAF6 variants causing male infertility and PCD in Han Chinese patients. This finding extended the spectrum of variants in DNAAF6 and revealed new light on the impact of DNAAF6 variants in sperm flagella.

Exploring the Role of Fallopian Ciliated Cells in the Pathogenesis of High-Grade Serous Ovarian Cancer.

High-grade serous epithelial ovarian cancer (HGSOC) is the fifth leading cause of cancer death in women and the first among gynecological malignancies. Despite an initial response to standard chemotherapy, most HGSOC patients relapse. To improve treatment options, we must continue investigating tumor biology. Tumor characteristics (e.g., risk factors and epidemiology) are valuable clues to accomplish this task. The two most frequent risk factors for HGSOC are the lifetime number of ovulations, which is associated with increased oxidative stress in the pelvic area caused by ovulation fluid, and a positive family history due to genetic factors. In the attempt to identify novel genetic factors (i.e., genes) associated with HGSOC, we observed that several genes in linkage with HGSOC are expressed in the ciliated cells of the fallopian tube. This finding made us hypothesize that ciliated cells, despite not being the cell of origin for HGSOC, may take part in HGSOC tumor initiation. Specifically, malfunction of the ciliary beat impairs the laminar fluid flow above the fallopian tube epithelia, thus likely reducing the clearance of oxidative stress caused by follicular fluid. Herein, we review the up-to-date findings dealing with HGSOC predisposition with the hypothesis that fallopian ciliated cells take part in HGSOC onset. Finally, we review the up-to-date literature concerning genes that are located in genomic loci associated with epithelial ovarian cancer (EOC) predisposition that are expressed by the fallopian ciliated cells.

Primary Ciliary Dyskinesia Caused by Homozygous DNAAF1 Mutations Resulting from a Consanguineous Marriage: A Case Report from Japan.

We present the case of a 58-year-old female patient with primary ciliary dyskinesia (PCD). She was born to parents with a consanguineous marriage. Chest computed tomography conducted at age 41 years indicated no situs inversus, and findings of bronchiectasis were limited to the middle and lingular lobes. Despite long-term macrolide therapy, bronchiectasis exacerbations frequently occurred. PCD was suspected because of the low nasal nitric oxide level (20.7 nL/min). Electron microscopy revealed outer and inner dynein arm defects, and a genetic analysis identified a homozygous single-nucleotide deletion in the DNAAF1 gene. Based on these results, the patient was diagnosed with PCD due to a biallelic DNAAF1 mutation.

DNAAF5 promotes hepatocellular carcinoma malignant progression by recruiting USP39 to improve PFKL protein stability.

Purposes: Dynein axonemal assembly factor 5 (DNAAF5) is the transcription factor of regulating the cytoskeleton and hydrodynamic protein complex assembly, however, it was not well elucidated in the malignant progression of hepatocellular carcinoma (HCC). Methods: We investigated the role of DNAAF5 in hepatocellular carcinoma by using multiple groups of clinical tissues combined with data from the TCGA database. Then we overexpressed DNAAF5 in hepatocellular carcinoma tumor tissues, which correlates with poor patient survival outcomes. Furthermore, we constructed stable cell lines of HCC cells to confirm the cancer-promoting effects of DNAAF5 in hepatocellular carcinoma. To explore the mechanisms of DNAAF5, transcriptome sequencing combined with mass spectrometry was also performed, which showed that DNAAF5 affects its downstream signaling pathway by interacting with PFKL and that DNAAF5 regulates PFKL protein stability by recruiting the deubiquitination protein, USP39. To corroborate these findings, the same series of tissue microarrays were used to confirm correlations between DNAAF5 and PFKL expressions. In animal experiments, DNAAF5 also promoted the proliferation of HCC cells. Results: We found that DNAAF5 expressions were markedly higher in HCC tissues, compared to the adjacent normal tissues. Increased levels of DNAAF5 were associated with significantly worse prognostic outcomes for HCC patients. Cell function experiments showed that HCC cells of overexpressing DNAAF5 exhibited faster proliferation rates, stronger clone formation abilities and higher drug resistance rates. However, tumor cell proliferation rates and colony formation were significantly decreased after DNAAF5 knockout, accompanied by an increase in sensitivity to sorafenib. In addition, the results of our study showed that DNAAF5 accelerates PFKL protein deubiquitination by recruiting USP39 in HCC cells. Furthermore, The overexpression of DNAAF5 could promote HCC cell proliferation in vivo and in vitro, whereas USP39 knockdown inhibited this effect. Overall, DNAAF5 serves as a scaffold protein to recruit USP39 to form a ternary complex by directly binding the PFKL protein, thereby improving the stability of the latter, which promotes the malignant process of hepatocellular carcinoma. Conclusions: These findings revealed DNAAF5 was negatively correlated with the prognosis of patients with hepatocellular carcinoma. It underlying mechanism showed that DNAAF5 directly binds PFKL and recruits the deubiquitinated protein (USP39) to improve the stability of the PFKL protein, thus enhancing abnormal glycolysis in HCC cells.

Case Report: DNAAF4 Variants Cause Primary Ciliary Dyskinesia and Infertility in Two Han Chinese Families.

Background : Primary ciliary dyskinesia (PCD) is a rare genetic disorder, predominantly autosomal recessive. The dynein axonemal assembly factor 4 (DNAAF4) is mainly involved in the preassembly of multisubunit dynein protein, which is fundamental to the proper functioning of cilia and flagella. There are few reports of PCD-related pathogenic variants of DNAAF4, and almost no DNAAF4-related articles focused on sperm phenotype. Moreover, the association between DNAAF4 and scoliosis has never been reported, to the best of our knowledge. Materials and Methods: We recruited two patients with a clinical diagnosis of PCD. One came from a consanguineous and another from a non-consanguineous family. Clinical data, laboratory test results, and imaging data were analyzed. Through whole exome sequencing, immunofluorescence, electron microscopy, high-speed video microscopy analysis, and hematoxylin-eosin (HE) staining, we identified the disease-associated variants and validated the pathogenicity. Results: Proband 1 (P1, F1: II-1), a 19-year-old man, comes from a non-consanguineous family-I, and proband 2 (P2, F2: II-1), a 37-year-old woman, comes from a consanguineous family-II. Both had sinusitis, bronchiectasis, situs inversus, and scoliosis. P1 also had asthenoteratozoospermia, and P2 had an immature uterus. Two homozygous pathogenic variants in DNAAF4 (NM_130810.4), c.988C > T, p.(Arg330Trp), and DNAAF4 (NM_130810.4), c.733 C > T, p.(Arg245*), were identified through whole exome sequencing. High-speed microscopy analysis showed that most of the cilia were static in P1, with complete static of the respiratory cilia in P2. Immunofluorescence showed that the outer dynein arms (ODA) and inner dynein arms (IDA) were absent in the respiratory cilia of both probands, as well as in the sperm flagellum of P1. Transmission electron microscopy revealed the absence of ODA and IDA of respiratory cilia of P2, and HE staining showed irregular, short, absent, coiled, and bent flagella. Conclusion: Our study identified a novel variant c.733C > T, which expanded the spectrum of DNAAF4 variants. Furthermore, we linked DNAAF4 to asthenoteratozoospermia and likely scoliosis in patients with PCD. This study will contribute to a better understanding of PCD.

Homozygous mutation in DNAAF4 causes primary ciliary dyskinesia in a Chinese family.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder that affects the structure and function of motile cilia, leading to classic clinical phenotypes, such as situs inversus, chronic sinusitis, bronchiectasis, repeated pneumonia and infertility. In this study, we diagnosed a female patient with PCD who was born in a consanguineous family through classic clinical manifestations, transmission electron microscopy and immunofluorescence staining. A novel DNAAF4 variant NM_130810: c.1118G>A (p. G373E) was filtered through Whole-exome sequencing. Subsequently, we explored the effect of the mutation on DNAAF4 protein from three aspects: protein expression, stability and interaction with downstream DNAAF2 protein through a series of experiments, such as transfection of plasmids and Co-immunoprecipitation. Finally, we confirmed that the mutation of DNAAF4 lead to PCD by reducing the stability of DNAAF4 protein, but the expression and function of DNAAF4 protein were not affected.

Identification of Two Novel DNAAF2 Variants in Two Consanguineous Families with Primary Ciliary Dyskinesia.

BACKGROUND: Dynein axonemal assembly factor 2 (DNAAF2) is involved in the early preassembly of dynein in the cytoplasm, which is essential for motile cilia function. Primary ciliary dyskinesia (PCD) associated with DNAAF2 variants has rarely been reported in females with infertility. Moreover, there is no report linking DNAAF2 to scoliosis in human. MATERIALS AND METHODS: We recruited patients from two consanguineous families with a clinical diagnosis of PCD and collected their clinical history, laboratory tests, and radiographic data. Sequencing and bioinformatics analysis were then performed. Immunofluorescence and high-speed microscope analysis were used to support the pathogenicity of the variant. RESULTS: Proband 1, a 26-year-old female from family I, exhibited scoliosis, bronchiectasis, sinusitis, situs inversus, and infertility. We found a novel homozygous missense variant in DNAAF2, c.491T>C, p.(Leu164Pro) in this patient. Subsequent immunofluorescence indicated the absence of outer dynein arm and inner dynein arm of cilia, and high-speed microscopy analysis showed that the most of the cilia are static, which support the pathogenicity of this variant. Proband 2, a 53-year-old female, presented with bronchiectasis, sinusitis, and infertility. In this patient, a new homozygous frameshift variant DNAAF2, c.822del, p.(Ala275Profs*10) was identified. The disease-causing variants mentioned above are not included in the current authorized genetic databases. CONCLUSION: Our findings expand the spectrum of DNAAF2 variants and link DNAAF2 to female infertility and likely scoliosis in patients with PCD.

Reading disability due to an ocular motor disorder: A case of an adolescent girl with a previous diagnosis of dyslexia.

Dyslexia is a reading disability characterized by difficulties with accurate and/or fluent word recognition, which are thought to stem from a phonological processing impairment. Herein we report the case of a 13-year-old girl who received the diagnosis of dyslexia at age 12 years. We considered this diagnosis to be incorrect because her reading difficulty was caused by a spontaneously repeated eye movement toward the vertical direction; the eyes were likely to show slow, upward drifts followed by quick downward movement at the physical examination, and the amplitude of the downward movement was increased when she changed eye positions to look at the upper direction in the evaluation of the eye tracker. Although we considered there was the possibility that the spontaneously repeated eye movement was classified as the spontaneous downbeat nystagmus, the eye tracker showed the transition of the gaze starting from and returning to was inconsistent with nystagmus, and we concluded that the term of nystagmus like abnormal eye movement was appropriate for the expression of the spontaneously repeated eye movement. There was no apparent abnormality on head magnetic resonance imaging (MRI), and whole exome sequencing showed no known candidate genes to explain the cerebellar dysfunction. An accumulation of similar cases in the future should help elucidate the pathomechanism observed in this case, and we should fully pay attention to evaluate the neurological aspects of the patients before settling on the diagnosis of dyslexia.

Chiral Cilia Orientation in the Left-Right Organizer.

Chirality is a property of asymmetry between an object and its mirror image. Most biomolecules and many cell types are chiral. In the left-right organizer (LRO), cilia-driven flows transfer such chirality to the body scale. However, the existence of cellular chirality within tissues remains unknown. Here, we investigate this question in Kupffer's vesicle (KV), the zebrafish LRO. Quantitative live imaging reveals that cilia populating the KV display asymmetric orientation between the right and left sides, resulting in a chiral structure, which is different from the chiral cilia rotation. This KV chirality establishment is dynamic and depends on planar cell polarity. While its impact on left-right (LR) symmetry breaking remains unclear, we show that this asymmetry does not depend on the LR signaling pathway or flow. This work identifies a different type of tissue asymmetry and sheds light on chirality genesis in developing tissues.

Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans.

Neural tube defects (NTDs) are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC). We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1 DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10) resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans.

Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice.

We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48 Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.