Motile Cilia Research Articles

Ordered deployment of distinct ciliary beating machines in growing axonemes of vertebrate multiciliated cells

The beating of motile cilia requires the coordinated action of diverse machineries that include not only the axonemal dynein arms, but also the central apparatus, the radial spokes, and the microtubule inner proteins. These machines exhibit complex radial and proximodistal patterns in mature axonemes, but little is known about the interplay between them during motile ciliogenesis. Here, we describe and quantify the relative rates of axonemal deployment for these diverse cilia beating machineries during the final stages of differentiation of Xenopus epidermal multiciliated cells.

Morphological changes and two Nodal paralogs drive left-right asymmetry in the squamate veiled chameleon (C. calyptratus).

The ancestral mode of left-right (L-R) patterning involves cilia in the L-R organizer. However, the mechanisms regulating L-R patterning in non-avian reptiles remains an enigma, since most squamate embryos are undergoing organogenesis at oviposition. In contrast, veiled chameleon (Chamaeleo calyptratus) embryos are pre-gastrula at oviposition, making them an excellent organism for studying L-R patterning evolution. Here we show that veiled chameleon embryos lack motile cilia at the time of L-R asymmetry establishment. Thus, the loss of motile cilia in the L-R organizers is a synapomorphy of all reptiles. Furthermore, in contrast to avians, geckos and turtles, which have one Nodal gene, veiled chameleon exhibits expression of two paralogs of Nodal in the left lateral plate mesoderm, albeit in non-identical patterns. Using live imaging, we observed asymmetric morphological changes that precede, and likely trigger, asymmetric expression of the Nodal cascade. Thus, veiled chameleons are a new and unique model for studying the evolution of L-R patterning.

Evidence for intraflagellar transport in butterfly spermatocyte cilia.

In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A unique feature of these cilia/flagella is their lack of dependence on intraflagellar transport (IFT) for their assembly. Here, we show that in the common butterfly Pieris brassicae, the spermatocyte cilia are exceptionally long: about 40 μm compared to less than 1 μm in Drosophila. By transmission electron microscopy, we show that P. brassicae spermatocytes display several features not found in melanogaster, including compelling evidence of IFT structures and features of motile cilia.

A case study for heat and mass transfer of viscous fluid flow in double layer due to ciliated channel

The present study is a measurement of the thermal and mass transfer of a two-layered flow of viscous fluid induced by cilia motion under the effect of buoyancy forces through a channel. Cilia are mostly of two classes: motile and non-motile cilia for locomotion and sensory processing. Cilia tissue cells are epithelia lining the lungs that sweep away liquids or solids, and organisms that have cilia are protozoans that use them for movements are the examples. The ciliary apparatus is related to cell cycle movement and proliferation, and cilia display an energetic fragment in human and animal development and in ordinary life. The fluid is considered to be incompressible, and layers of fluid do not mix with each other. The fluid flow for mass and thermal transfer is modelled in wave and fixed frame. Solutions for temperature, velocity of fluid, and concentration profile are obtained using a well-known method, namely the Homotopy Perturbation Method (HPM), by the computation software "MATHEMATICA". The behavior of emerging parameters is shown graphically in the results section. Graphical results can be analyzed for the temperature and velocity profile, which are extreme in the inner layer for both fluid and concentration distribution and are highest at the external layers of fluid. It is the first study not examined in the literature.

Replicating cilia movement and mucus interactions through detailed modeling

Computational framework can measure behavior and efficiency of cilia movement in healthy and diseased tissue.

Abstract 3459: Drug persistence pathways in ovarian cancer identified by single-cell analysis of patient samples and cell line models

Abstract Drug resistance and disease recurrence represent a major obstacle in the treatment of high-grade serous ovarian cancer (HGSOC). Here, we performed a comprehensive characterization, at the single-cell level, of residual (persister) cancer cells that survive neoadjuvant treatment in HGSOC and can give rise to future recurrences. We aim to understand the adaptive responses that enable persistence and the key pathways that underlie them to find new targets for combination therapy with the ultimate goal of eradicating all tumor cells. We collected 27 tumor samples (16 pre-treatment and 11 post-neoadjuvant carboplatin/taxol samples, with multi-site sampling per patient) for single-cell RNA sequencing (scRNA-Seq). To complement the patient-derived dataset and model the time course of drug persistence, we performed scRNA-Seq of 13 distinct ovarian cancer cell lines treated with chemotherapy in vitro for a series of time points up to 28 days. We performed single-cell gene expression and copy number analyses of patient samples, annotated detailed cell types, and detected multiple immune cell populations, the majority of which maintained a highly immunosuppressive program both in untreated and treated samples. Remarkably, we detected a rare population of cancer cells that differed dramatically in their transcriptional program. These cells exhibited high expression of components of motile cilia and the transcription factor FOXJ1, a master regulator of ciliogenesis. We validated the presence of FOXJ1+ cells in patient samples by immunohistochemistry and immunofluorescence. Ciliated cells were enriched in the post-treatment vs. pre-treatment samples, suggesting a possible role in drug persistence. We are now experimentally testing the role of the FOXJ1-driven transcriptional program in mediating drug resistance. To systematically identify transcriptional programs activated in persister cells, we performed differential expression analysis on pre- vs. post-treatment samples. We identified previously implicated mechanisms of drug resistance, such as the Epithelial-Mesenchymal Transition and the antiapoptotic response driven by MCL1. We additionally detected upregulation of the metallothionein family of genes, which may represent a mechanism of resistance to platinum-based compounds. We detected potentially novel mechanisms representing a stress response signature driven by JUND, an interferon gamma signature, and a pro-stem cell signature characterized by LGR5 expression. Independently, our analysis of the ovarian cancer cell lines treated with taxol in vitro confirmed the JUND transcriptional program as potentially driving ovarian cancer persistence. Our study will help reveal pathways enabling ovarian cancer cells to persist through treatment and, ultimately, could lead to novel approaches for targeting persistence and achieving deeper therapeutic responses. Citation Format: Elizaveta Leshchiner, Richard Panayiotou, Anay Gupta, Thomas Zhang, Kayli Neil, Nomeda Girnius, Aylin Henstridge, Brian P. Danysh, Ignaty Leshchiner, Sarah J. Hill, Laxmi Parida, Ursula A. Matulonis, Joan S. Brugge, Gad Getz. Drug persistence pathways in ovarian cancer identified by single-cell analysis of patient samples and cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3459.

Plastic loss of motile cilia in the gills of Polypterus in response to high CO2 or terrestrial environments.

The evolutionary transition of vertebrates from water to land during the Devonian period was accompanied by major changes in animal respiratory systems in terms of physiology and morphology. Indeed, the fossil record of the early tetrapods has revealed the existence of internal gills, which are vestigial fish-like traits used underwater. However, the fossil record provides only limited data on the process of the evolutionary transition of gills from fish to early tetrapods. This study investigated the gills of Polypterus senegalus, a basal ray-finned/amphibious fish which shows many ancestral features of stem Osteichthyes. Based on scanning electron microscopy observations and transcriptome analysis, the existence of motile cilia in the gills was revealed which may create a flow on the gill surface leading to efficient ventilation or remove particles from the surface. Interestingly, these cilia were observed to disappear after rearing in terrestrial or high CO2 environments, which mimics the environmental changes in the Devonian period. The cilia re-appeared after being returned to the original aquatic environment. The ability of plastic changes of gills in Polypterus revealed in this study may allow them to survive in fluctuating environments, such as shallow swamps. The ancestor of Osteichthyes is expected to have possessed such plasticity in the gills, which may be one of the driving forces behind the transition of vertebrates from water to land.

Development's Inaugural Outstanding Paper Prize.

Development's Inaugural Outstanding Paper Prize.

Phase-resolved dynamic wavefront imaging of cilia metachronal waves.

The coordination and the directional order of ciliary metachronal waves are the major factors that determine the effectiveness of mucociliary clearance (MCC). Even though metachronal waves play an essential part in immune response, clinical diagnostic tools and imaging techniques that can reliably and efficiently capture their spatial distribution and function are currently limited. We present label-free high-speed visualization of ciliary metachronal wave propagations in freshly-excised tracheal explants using a spectrally-encoded interferometric microscope over a two-dimensional (2D) plane of 0.5 mm × 0.5 mm at an acquisition rate of 50 frame-per-second. Furthermore, phase-resolved enhanced dynamic (PHRED) analysis of time-series doppler images was performed, where spatial-temporal characteristics of cilia metachronal wave motions are revealed through frequency component analysis and spatial filtering. The PHRED analysis of phase-resolved Doppler (PRD) images offers a capability to distinguish the propagation direction of metachronal waves, and quantitatively assess amplitude and dominant frequency of cilia beating at each spatial location. Compared to the raw PRD images, the phase-resolved dynamic wavefront imaging (PRDWI) method showed the direction and coordination of collective cilia movement more distinctively. The PRDWI technique can have broad application prospects for the diagnosis of human respiratory diseases and evaluation of the curative effect of treatments and open new perspectives in biomedical sciences.

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia.

Localized and chronic hypoxia of airway mucosa is a common feature of progressive respiratory diseases, including cystic fibrosis (CF). However, the impact of prolonged hypoxia on airway stem cell function and differentiated epithelium is not well elucidated. Acute hypoxia alters the transcription and translation of many genes, including the CF transmembrane conductance regulator (CFTR). CFTR-targeted therapies (modulators) have not been investigated in vitro under chronic hypoxic conditions found in CF airways in vivo. Nasal epithelial cells (hNECs) derived from eight CF and three non-CF participants were expanded and differentiated at the air-liquid interface (26-30 days) at ambient and 2% oxygen tension (hypoxia). Morphology, global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility and ion transport) of basal stem cells and differentiated cultures were assessed. hNECs expanded at chronic hypoxia, demonstrating epithelial cobblestone morphology and a similar proliferation rate to hNECs expanded at normoxia. Hypoxia-inducible proteins and pathways in stem cells and differentiated cultures were identified. Despite the stem cells' plasticity and adaptation to chronic hypoxia, the differentiated epithelium was significantly thinner with reduced barrier integrity. Stem cell lineage commitment shifted to a more secretory epithelial phenotype. Motile cilia abundance, length, beat frequency and coordination were significantly negatively modulated. Chronic hypoxia reduces the activity of epithelial sodium and CFTR ion channels. CFTR modulator drug response was diminished. Our findings shed light on the molecular pathophysiology of hypoxia and its implications in CF. Targeting hypoxia can be a strategy to augment mucosal function and may provide a means to enhance the efficacy of CFTR modulators.

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum.

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.

GEMC1 and MCIDAS interactions with SWI/SNF complexes regulate the multiciliated cell-specific transcriptional program

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.

Mucociliary Wnt signaling promotes cilia biogenesis and beating

It is widely thought that Wnt/Lrp6 signaling proceeds through the cytoplasm and that motile cilia are signaling-inert nanomotors. Contrasting both views, we here show in the mucociliary epidermis of X. tropicalis embryos that motile cilia transduce a ciliary Wnt signal that is distinct from canonical β-catenin signaling. Instead, it engages a Wnt-Gsk3-Ppp1r11-Pp1 signaling axis. Mucociliary Wnt signaling is essential for ciliogenesis and it engages Lrp6 co-receptors that localize to cilia via a VxP ciliary targeting sequence. Live-cell imaging using a ciliary Gsk3 biosensor reveals an immediate response of motile cilia to Wnt ligand. Wnt treatment stimulates ciliary beating in X. tropicalis embryos and primary human airway mucociliary epithelia. Moreover, Wnt treatment improves ciliary function in X. tropicalis ciliopathy models of male infertility and primary ciliary dyskinesia (ccdc108, gas2l2). We conclude that X. tropicalis motile cilia are Wnt signaling organelles that transduce a distinct Wnt-Pp1 response.

Entropic analysis of cilia-modulated slip flow of trimetallic nanofluid through electroosmotic corrugated pump in the presence of inclined magnetic field

An incredible eradication of thermal indulgence is required to enhance the flow and heat transfer enhancement in micro/nanofluidic devices. In addition, the rapid transport and instantaneous mixing of colloidal suspensions of metallic particles at nanoscale are exceptionally crucial at ascendency of inertial and surface forces. To address these challenges, the present work is intended to investigate the role of trimetallic nanofluid comprising of three kinds of nano-sized granules (titanium oxide, Silica and Aluminium dioxide) with pure blood through a heated micropump in the presence of inclined magnetic field and axially implemented electric field. To ensure rapid mixing in unidirectional flow, the pump internal surface is lined-up with mimetic motile cilia with slip boundary. The embedded cilia whip in pattern due to dynein molecular motion controlled by time and produce a set of metachronal waves along the pump wall. The shooting technique is executed to compute the numerical solution. In a comparative glance it is revealed that the trimetallic nanofluid exhibits 10% higher heat transfer efficiency as compared to bi-hybrid and mono nanofluids. Moreover, the involvement of electroosmosis results in almost 17% decrease in the heat transfer rate if it values jumps from 1 to 5. The fluid temperature in case of trimetallic nanofluid is higher and thus keeps the heat transfer entropy and the total entropy lower. Furthermore, involvement of thermal radiated and momentum slip significantly contribute in reducing heat losses.

Characteristic genetic spectrum of primary ciliary dyskinesia in Japanese patients and global ethnic heterogeneity: population-based genomic variation database analysis.

Primary ciliary dyskinesia (PCD) is a hereditary disease caused by pathogenic variants in genes associated with motile cilia. Some variants responsible for PCD are reported to be ethnic-specific or geographical-specific. To identify the responsible PCD variants of Japanese PCD patients, we performed next-generation sequencing of a panel of 32 PCD genes or whole-exome sequencing in 26 newly identified Japanese PCD families. We then combined their genetic data with those from 40 Japanese PCD families reported previously, for an overall analysis of 66 unrelated Japanese PCD families. We conducted Genome Aggregation Database and TogoVar database analyses to reveal the PCD genetic spectrum of the Japanese population and compare with other ethnic groups worldwide. We identified 22 unreported variants among the 31 patients in the 26 newly identified PCD families, including 17 deleterious variants estimated to cause lack of transcription or nonsense-mediated mRNA decay and 5 missense mutations. In all 76 PCD patients from the 66 Japanese families, we identified 53 variants on 141 alleles in total. Copy number variation in DRC1 is the most frequent variant in Japanese PCD patients, followed by DNAH5 c.9018C>T. We found 30 variants specific to the Japanese population, of which 22 are novel. Furthermore, 11 responsible variants in the Japanese PCD patients are common in East Asian populations, while some variants are more frequent in other ethnic groups. In conclusion, PCD is genetically heterogeneous between different ethnicities, and Japanese PCD patients have a characteristic genetic spectrum.

An operative framework to model mucus clearance in silico by coupling cilia motion with the liquid environment.

Mucociliary clearance is the first defense mechanism of the respiratory tract against inhaled particles. This mechanism is based on the collective beating motion of cilia at the surface of epithelial cells. Impaired clearance, either caused by malfunctioning or absent cilia, or mucus defects, is a symptom of many respiratory diseases. Here, by exploiting the lattice Boltzmann particle dynamics technique, we develop a model to simulate the dynamics of multiciliated cells in a two-layer fluid. First, we tuned our model to reproduce the characteristic length- and time-scales of the cilia beating. We then check for the emergence of the metachronal wave as a consequence of hydrodynamic mediated correlations between beating cilia. Finally, we tune the viscosity of the top fluid layer to simulate the mucus flow upon cilia beating, and evaluate the pushing efficiency of a carpet of cilia. With this work, we build a realistic framework that can be used to explore several important physiological aspects of mucociliary clearance.

Profiling of differentially expressed proteins between fresh and frozen-thawed Duroc boar semen using ProteinChip CM10.

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

Altered cell function and increased replication of rhinoviruses and EV-D68 in airway epithelia of asthma patients.

Rhinovirus (RV) infections constitute one of the main triggers of asthma exacerbations and an important burden in pediatric yard. However, the mechanisms underlying this association remain poorly understood. In the present study, we compared infections of in vitro reconstituted airway epithelia originating from asthmatic versus healthy donors with representative strains of RV-A major group and minor groups, RV-C, RV-B, and the respiratory enterovirus EV-D68. We found that viral replication was higher in tissues derived from asthmatic donors for all tested viruses. Viral receptor expression was comparable in non-infected tissues from both groups. After infection, ICAM1 and LDLR were upregulated, while CDHR3 was downregulated. Overall, these variations were related to viral replication levels. The presence of the CDHR3 asthma susceptibility allele (rs6967330) was not associated with increased RV-C replication. Regarding the tissue response, a significantly higher interferon (IFN) induction was demonstrated in infected tissues derived from asthmatic donors, which excludes a defect in IFN-response. Unbiased transcriptomic comparison of asthmatic versus control tissues revealed significant modifications, such as alterations of cilia structure and motility, in both infected and non-infected tissues. These observations were supported by a reduced mucociliary clearance and increased mucus secretion in non-infected tissues from asthmatic donors. Altogether, we demonstrated an increased permissiveness and susceptibility to RV and respiratory EV infections in HAE derived from asthmatic patients, which was associated with a global alteration in epithelial cell functions. These results unveil the mechanisms underlying the pathogenesis of asthma exacerbation and suggest interesting therapeutic targets.

Issue Information

Front cover: The node forming transiently in mammalian embryos plays a key role in left‐right axis determination. The node is a cavity, in which crown cells with immotile cilia surround pit cells that rotate their motile cilia to generate a leftward fl uid fl ow (nodal fl ow). Experiments using GCaMP6, which emits green fl uorescence in response to Ca2+, and optical tweezers revealed that immotile cilia sense mechanical force generated by nodal fl ow (Katoh et al. (2023) Science 379: 66–71, DOI: 10.1126/science.abq8148). A similar mechanism was shown to exist in zebrafi sh (Djenoune et al. (2023) Science 379: 71–78, DOI: 10.1126/science.abq7317). This picture shows the application of this mechanism to ‘hiburi‐ryo’ (fishing by using flames to drive fish). The fishermen in the pond are creating a leftward current to be sensed by the men on the left bank, who are activated, and their torch flames intensify. Designed by TRAIS Co., Ltd. (Kobe, Japan).image

Characterization of a DRC1 null variant associated with primary ciliary dyskinesia and female infertility

ProposeWe here present a female case with primary ciliary dyskinesia (PCD) and infertility. In this report, we also present the evaluation of the patient family, including her twin sister, also with PCD and infertility.MethodsConfirmation of the PCD clinical diagnosis was performed through assessment of cilia motility, by high-speed video microscopy (HSVM), axoneme ultrastructure, by transmission electron microscopy (TEM), and genetic characterization, by whole-exome sequence (WES). Gene expression studies used qPCR for mRNA expression and immunofluorescence to determine cell protein localization.ResultsWe identified a homozygous nonsense variant in the DRC1 gene (NM 145038.5:c.352C>T (p.Gln118Ter)) in the female patient with PCD and infertility that fit the model of autosomal recessive genetic transmission. This variant eventually results in a dyskinetic ciliary beat with a lower frequency and a partial lack of both dynein arms as revealed by TEM analysis. Moreover, this variant implies a decrease in the expression of DRC1 mRNA and protein. Additionally, expression analysis suggested that DRC1 may interact with other DRC elements.ConclusionsOur findings suggest that the DRC1 null variant leads to PCD associated with infertility, likely caused by defects in axoneme from Fallopian tube cilia. Overall, our outcomes contribute to a better understanding of the genetic factors involved in the pathophysiology of PCD and infertility, and they highlight the interaction of different genes in the patient phenotype, which should be investigated further because it may explain the high heterogeneity observed in PCD patients.