Activity Of Cilia Research Articles

Region-specific roles of oviductal motile cilia in oocyte/embryo transport and fertility

Abstract The physiological and clinical importance of motile cilia in reproduction is well recognized, however, the specific role they play in transport through the oviduct and how ciliopathies lead to subfertility and infertility is still unclear. The contribution of cilia beating, fluid flow, and smooth muscle contraction to overall progressive transport within the oviduct remains under debate. Therefore, we investigated the role of cilia in the oviduct transport of preimplantation eggs and embryos using a combination of genetic and advanced imaging approaches. We show that the region of the oviduct where cumulus-oocyte complex circling occurs, around the time of fertilization, is correlated with asymmetrical mucosal fold arrangement and non-radially distributed ciliated epithelium. Our results suggest that motile cilia, as well as mucosal fold asymmetry, may contribute to the local flow fields that help steer luminal contents away from the epithelial walls. We also present in vivo, volumetric evidence of delayed egg transport in a genetic mouse model with disrupted motile cilia function in the female reproductive system. Females with Dnah5 deleted in the oviduct epithelium are subfertile and demonstrate disrupted motile cilia activity within the oviduct mucosa. 50% of Dnah5 mutant females have delayed egg transport where COCs did not progress to the ampulla at the expected time-point and remained within the ovarian bursa. The integration of advanced imaging with genetic dysfunction of motile cilia activity provides valuable insights into oviductal transport processes. Potentially, these data could be valuable for better understanding and management of tubal pathologies and human infertility.

Breaking Left-Right Symmetry by the Interplay of Planar Cell Polarity, Calcium Signaling and Cilia.

The formation of the embryonic left-right axis is a fundamental process in animals, which subsequently conditions both the shape and the correct positioning of internal organs. During vertebrate early development, a transient structure, known as the left-right organizer, breaks the bilateral symmetry in a manner that is critically dependent on the activity of motile and immotile cilia or asymmetric cell migration. Extensive studies have partially elucidated the molecular pathways that initiate left-right asymmetric patterning and morphogenesis. Wnt/planar cell polarity signaling plays an important role in the biased orientation and rotational motion of motile cilia. The leftward fluid flow generated in the cavity of the left-right organizer is sensed by immotile cilia through complex mechanisms to trigger left-sided calcium signaling and lateralized gene expression pattern. Disrupted asymmetric positioning or impaired structure and function of cilia leads to randomized left-right axis determination, which is closely linked to laterality defects, particularly congenital heart disease. Despite of the formidable progress made in deciphering the critical contribution of cilia to establishing the left-right asymmetry, a strong challenge remains to understand how cilia generate and sense fluid flow to differentially activate gene expression across the left-right axis. This review analyzes mechanisms underlying the asymmetric morphogenesis and function of the left-right organizer in left-right axis formation. It also aims to identify important questions that are open for future investigations.

Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia.

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

Role of cilia activity and surrounding viscous fluid in properties of metachronal waves.

Large groups of active cilia collectively beat in a fluid medium as metachronal waves, essential for some microorganisms motility and for flow generation in mucociliary clearance. Several models can predict the emergence of metachronal waves, but what controls the properties of metachronal waves is still unclear. Here, we numerically investigate the respective impacts of active beating and viscous dissipation on the properties of metachronal waves in a collection of oscillators, using a simple model for cilia in the presence of noise on regular lattices in one and two dimensions. We characterize the wave using spatial correlation and the frequency of collective beating. Our results clearly show that the viscosity of the fluid medium does not affect the wavelength; the activity of the cilia does. These numerical results are supported by a dimensional analysis, which shows that the result of wavelength invariance is robust against the model taken for sustained beating and the structure of hydrodynamic coupling. Interestingly, the enhancement of cilia activity increases the wavelength and decreases the beating frequency, keeping the wave velocity almost unchanged. These results might have significance in understanding paramecium locomotion and mucociliary clearance diseases.

Collagen VI Deficiency Impairs Tendon Fibroblasts Mechanoresponse in Ullrich Congenital Muscular Dystrophy.

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.

Recording Cilia Activity in Ctenophores.

Pelagic ctenophores swim in the water with the help of eight rows of long fused cilia. Their entire behavioral repertoire is dependent to a large degree on coordinated cilia activity. Therefore, recording cilia beating is paramount to understanding and registering the behavioral responses and investigating its neural and hormonal control. Here, we present a simple protocol to monitor and quantify cilia activity in semi-intact ctenophore preparations (using Pleurobrachia and Bolinopsis as models), which includes a standard electrophysiological setup for intracellular recording.

Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells

Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)—(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air-liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA.

Recording cilia activity in ctenophores: effects of nitric oxide and low molecular weight transmitters.

Cilia are the major effectors in Ctenophores, but very little is known about their transmitter control and integration. Here, we present a simple protocol to monitor and quantify cilia activity and provide evidence for polysynaptic control of cilia coordination in ctenophores. We also screened the effects of several classical bilaterian neurotransmitters (acetylcholine, dopamine, L-DOPA, serotonin, octopamine, histamine, gamma-aminobutyric acid (GABA), L-aspartate, L-glutamate, glycine), neuropeptide (FMRFamide), and nitric oxide (NO) on cilia beating in Pleurobrachia bachei and Bolinopsis infundibulum. NO and FMRFamide produced noticeable inhibitory effects on cilia activity, whereas other tested transmitters were ineffective. These findings further suggest that ctenophore-specific neuropeptides could be major candidates for signal molecules controlling cilia activity in representatives of this early-branching metazoan lineage.

Ependyma in Neurodegenerative Diseases, Radiation-Induced Brain Injury and as a Therapeutic Target for Neurotrophic Factors.

The neuron loss caused by the progressive damage to the nervous system is proposed to be the main pathogenesis of neurodegenerative diseases. Ependyma is a layer of ciliated ependymal cells that participates in the formation of the brain-cerebrospinal fluid barrier (BCB). It functions to promotes the circulation of cerebrospinal fluid (CSF) and the material exchange between CSF and brain interstitial fluid. Radiation-induced brain injury (RIBI) shows obvious impairments of the blood-brain barrier (BBB). In the neuroinflammatory processes after acute brain injury, a large amount of complement proteins and infiltrated immune cells are circulated in the CSF to resist brain damage and promote substance exchange through the BCB. However, as the protective barrier lining the brain ventricles, the ependyma is extremely vulnerable to cytotoxic and cytolytic immune responses. When the ependyma is damaged, the integrity of BCB is destroyed, and the CSF flow and material exchange is affected, leading to brain microenvironment imbalance, which plays a vital role in the pathogenesis of neurodegenerative diseases. Epidermal growth factor (EGF) and other neurotrophic factors promote the differentiation and maturation of ependymal cells to maintain the integrity of the ependyma and the activity of ependymal cilia, and may have therapeutic potential in restoring the homeostasis of the brain microenvironment after RIBI or during the pathogenesis of neurodegenerative diseases.

Primary Cilium Identifies a Quiescent Cell Population in the Human Intestinal Crypt.

Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt-villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics.

Primary ciliary dyskinesia: two different clinical cases

Primary ciliary dyskinesia (PSD) is a clinically and genetically heterogeneous condition characterized by defective motile cilia activity. It is a rare disease with a frequency of 1 in 15,000-20,000 people and is generally inherited in an autosomal recessive and X-linked disease pattern. The diagnosis of PSD becomes easier when the patient has situs inverus anomaly accompanying recurrent lower and upper respiratory tract infections, as in our second case. However, it becomes vice versa in the absence of a situs anomaly. In this case report, we present two PSD patients with diverse clinical characteristics, one with Kartagener’s syndrome and the other without these features

Surface crawling and pedal surface collecting in aquatic gastropods: A case of scientific amnesia

Aquatic gastropods, both freshwater and marine, often crawl along the water surface with the sole of the foot facing upward. Differential regulation of the activity of cilia on the sole turns the gliding mechanism into pedal surface collecting, by which food particles floating on the water are collected on the sole. Over the last 300 years, surface crawling and pedal surface collecting have been studied in numerous species, but the accumulated knowledge has fallen victim to scientific amnesia. Today, pedal surface collecting usually is considered a unique behaviour that evolved in the family Ampullariidae (Caenogastropoda), which includes the genus Pomacea with some of the globally worst invasive pests. Consequently it may appear feasible to tackle invasive Pomacea species specifically by delivering molluscicides via the water surface. Based on a review of our forgotten literature, I here argue that such an approach would have potentially devastating, unintended effects on native gastropod faunas.

Post-transcriptional and Post-translational Modifications of Primary Cilia: How to Fine Tune Your Neuronal Antenna.

Primary cilia direct cellular signaling events during brain development and neuronal differentiation. The primary cilium is a dynamic organelle formed in a multistep process termed ciliogenesis that is tightly coordinated with the cell cycle. Genetic alterations, such as ciliary gene mutations, and epigenetic alterations, such as post-translational modifications and RNA processing of cilia related factors, give rise to human neuronal disorders and brain tumors such as glioblastoma and medulloblastoma. This review discusses the important role of genetics/epigenetics, as well as RNA processing and post-translational modifications in primary cilia function during brain development and cancer formation. We summarize mouse and human studies of ciliogenesis and primary cilia activity in the brain, and detail how cilia maintain neuronal progenitor populations and coordinate neuronal differentiation during development, as well as how cilia control different signaling pathways such as WNT, Sonic Hedgehog (SHH) and PDGF that are critical for neurogenesis. Moreover, we describe how post-translational modifications alter cilia formation and activity during development and carcinogenesis, and the impact of missplicing of ciliary genes leading to ciliopathies and cell cycle alterations. Finally, cilia genetic and epigenetic studies bring to light cellular and molecular mechanisms that underlie neurodevelopmental disorders and brain tumors.

Современные подходы к оценке двигательной активности ресничек эпителия верхних дыхательных путей

Violation of the main mechanism of protection of the mucous membrane of the upper respiratory tract mucociliary transport (MCT) is the cause of the development of most diseases of the nasal cavity and paranasal sinuses (PNS). Nevertheless, to assess the MCT in clinical conditions, mainly methods of a subjective nature are used aiming at determining the time of the MCT. At the present stage, the development of a method for an objective assessment of the MCT using new computer and mathematical technologies is an important area of scientific research in otorhinolaryngology. Objective. Based on a review of publications of modern methods for assessing the MCT used in clinical practice and scientific research, to identify promising areas of methods for its study. Materials and methods. The analysis of domestic and foreign literature publications (articles and corresponding abstracts) presented in the databases of the National Library of Russia, including information on methods for assessing mucociliary transport and recording the motor activity of the cilia of the upper respiratory tract (URT) epithelium, was carried out. The materials were chosen according to the keywords: mucociliary transport, motor activity of the ciliated epithelium, rhinology. Results. When studying modern publications, it turned out that in MCT studies high-speed video recording of the work of cilia is increasingly used, in combination with computer and mathematical processing of the data obtained, which makes it possible to give a full assessment of both the mechanism of violation and the functional efficiency of the work of cilia. In the course of the information search, it was found that modern methods used in the study of the MCT tend to automate the receipt of results based on the development of software. The conducted analysis of the literature testifies to the prospects of combining the approaches of related scientific fields of medicine, physics, and biology in the study of the motor function of cilia. One of the promising scientific achievements is the method of Doppler optical coherence tomography (DOCT) using a laser radiation source, which makes it possible to noninvasively and noncontactly assess the nature of cilia movement disorders at the level of spatial planar sections. Most of the modern research methods presented in the article indicate the need for further development and use of new approaches and criteria for assessing the motor activity of cilia.

Simple dynamics underlying the survival behaviors of ciliates.

Ciliates are swimming microorganisms in aquatic environments. Habitats where ciliates accumulate include nutrient-rich solid–liquid interfaces such as pond bottom walls and waterweed surfaces. The ciliates stay near the walls to survive. We investigated the dynamics of the near-wall behavior of ciliates. In experiments, the ciliates were made to slide on a flat wall of glass substrate. When encountering the wall, the wall-side cilia of the cells stop their motion and lose their propelling activity, which indicates that the ciliates have a mechano-sensing system for cilia beating. Based on the experimental results, we hypothesized that the ciliary thrust force that propels the cell body becomes asymmetric, and the asymmetry of the thrust force generates a head-down torque to keep the cell sliding on the wall. To prove this hypothesis, we performed numerical simulations by using a developed hydrodynamic model for swimming ciliates. The model revealed that the loss of cilia activity on the wall side physically induces a sliding motion, and the aspect ratio of the cell body and effective cilium area are critical functions for the sliding behavior on a wall. In addition, we investigated the stability of the sliding motion against an external flow. We found that ciliates slide upstream on a wall. Interestingly, the dynamics of this upstream sliding, called rheotaxis, were also explained by the identical physical conditions for no-flow sliding. Only two simple physical conditions are required to explain the dynamics of ciliate survival behavior. This review article is an extended version of the Japanese article, Fluid Dynamic Model Reveals a Mechano-sensing System Underlying the Behavior of Ciliates, published in SEIBUTSU BUTSURI Vol. 61, p. 16–19 (2021).

Фундаментальная и прикладная технологии диагностики нарушения двигательной активности реснитчатого эпителия верхних дыхательных путей

The modern development of computer and mathematical technologies makes it possible to improve the study of the movement of biological objects of extremely small sizes, including the cilia of epithelial cells of the upper respiratory tract. One of the directions of such research is the mathematical processing of video recordings that record the movement of the cilia of the epithelium of the respiratory tract. The methodology for conducting such studies is laborious and time-consuming. Such research is of a fundamental nature and is carried out primarily for scientific purposes. At the same time, clinical practice requires a fairly simple, objective method for diagnosing the disorder, which allows you to quickly obtain a result in inpatient and outpatient settings. The article presents the results of the development of algorithms for fundamental and applied research of the motor activity of the cilia of the epithelium of the upper respiratory tract using television microscopy and computer mathematical processing. The algorithms and integral criteria developed by the authors for an objective assessment of the motor activity of cilia make it possible to choose a research method depending on the goal. The use of an in-depth fundamental method in research work allows to reveal in detail the mechanism of impairment of motor activity of cilia beating. The use of the method of express diagnostics of cilia movements makes it possible to diagnose a violation, prescribe adequate treatment for correction and objectively evaluate its effectiveness.

Respiratory Cilia as a Therapeutic Target of Phosphodiesterase Inhibitors

Mucociliary clearance is an essential airway defense mechanism dependent predominantly on the proper ciliary function and mucus rheology. The crucial role of cilia is evident in `a variety of respiratory diseases, as the ciliary dysfunction is associated with a progressive decline in lung function over time. The activity of cilia is under supervision of multiple physiological regulators, including second messengers. Their role is to enable a movement in coordinated metachronal waves at certain beat frequency. Ciliary function can be modulated by various stimuli, including agents from the group of beta2 agonists, cholinergic drugs, and adenosine triphosphate (ATP). They trigger cilia to move faster in response to elevated cytoplasmic Ca2+ originated from intracellular sources or replenished from extracellular space. Well-known cilia-stimulatory effect of Ca2+ ions can be abolished or even reversed by modulating the phosphodiesterase (PDE)-mediated breakdown of cyclic adenosine monophosphate (cAMP) since the overall change in ciliary beating has been dependent on the balance between Ca2+ ions and cAMP. Moreover, in chronic respiratory diseases, high ATP levels may contribute to cAMP hydrolysis and thus to a decrease in the ciliary beat frequency (CBF). The role of PDE inhibitors in airway cilia-driven transport may help in prevention of progressive loss of pulmonary function often observed despite current therapy. Furthermore, administration of selective PDE inhibitors by inhalation lowers the risk of their systemic effects. Based on this review we may conclude that selective (PDE1, PDE4) or dual PDE inhibitors (PDE3/4) increase the intracellular level of cyclic nucleotides in airway epithelial cells and thus may be an important target in the development of new inhaled mucokinetic agents. Further research is required to provide evidence of their effectiveness and feasibility regarding their cilia-modulating properties.

Origin and role of the cerebrospinal fluid bidirectional flow in the central canal.

Circulation of the cerebrospinal fluid (CSF) contributes to body axis formation and brain development. Here, we investigated the unexplained origins of the CSF flow bidirectionality in the central canal of the spinal cord of 30 hpf zebrafish embryos and its impact on development. Experiments combined with modeling and simulations demonstrate that the CSF flow is generated locally by caudally-polarized motile cilia along the ventral wall of the central canal. The closed geometry of the canal imposes the average flow rate to be null, explaining the reported bidirectionality. We also demonstrate that at this early stage, motile cilia ensure the proper formation of the central canal. Furthermore, we demonstrate that the bidirectional flow accelerates the transport of particles in the CSF via a coupled convective-diffusive transport process. Our study demonstrates that cilia activity combined with muscle contractions sustain the long-range transport of extracellular lipidic particles, enabling embryonic growth.

Reorganization of complex ciliary flows around regenerating Stentor coeruleus.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such as Chlamydomonas. Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliate Stentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring in Stentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures.This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.