Coordinated motion of cilia is a fascinating and vital aspect of very diverse forms of eukaryotic life, enabling swimming and propulsion of fluid across cellular epithelia. There are many questions ...still unresolved, and broadly they fall into two classes. (i) The mechanism of how cilia physically transmit forces onto each other. It is not known for many systems if the forces are mainly of hydrodynamical origin, or if elastic forces within the cytoskeleton are important. (ii) In those systems where we know that forces are purely hydrodynamical, we do not have a framework for linking our understanding of how each cilium behaves in isolation to the collective properties of two or more cilia. In this work, we take biological data of cilia dynamics from a variety of organisms as an input for an analytical and numerical study. We calculate the relative importance of
flows versus
cilia flows on cilia coupling. This study contributes to both the open questions outlined above: firstly, we show that it is, in general, incorrect to infer cilium-cilium coupling strength on the basis of experiments with external flows, and secondly, we show a framework to recapitulate the dynamics of single cilia (the waveform) showing classes that correspond to biological systems with the same physiological activity (swimming by propulsion, versus forming collective waves). This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.
Novel invertebrate‐killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic ...screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole‐organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here, we use high‐throughput imaging and quantitative phenotyping to measure Caenorhabditis elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover substructure that is not captured in broad mode‐of‐action labels. High‐throughput imaging and automated phenotyping could therefore accelerate mode‐of‐action discovery in invertebrate‐targeting compound development and help to refine mode‐of‐action categories.
Synopsis
A combination of imaging and machine learning is used to predict compound mode of action using the unique behavioural responses of the roundworm C. elegans to different pesticides and anthelmintics.
Insecticides affect phenotypes in multiple behavioural dimensions.
Compounds with the same mode of action have similar effects on behaviour.
Combining classifiers by voting enables mode of action prediction.
The approach allows mode of action deconvolution within classes.
A combination of imaging and machine learning is used to predict compound mode of action using the unique behavioural responses of the roundworm C. elegans to different pesticides and anthelmintics.
By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we ...investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC
mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.
Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. An imaging system with sufficient throughput ...and spatiotemporal resolution would be capable of imaging a large number of animals, estimating their pose, and quantifying detailed behavioural differences at a scale where hundreds of treatments could be tested simultaneously. Here we report an array of six 12-megapixel cameras that record all the wells of a 96-well plate with sufficient resolution to estimate the pose of C. elegans worms and to extract high-dimensional phenotypic fingerprints. We use the system to study behavioural variability across wild isolates, the sensitisation of worms to repeated blue light stimulation, the phenotypes of worm disease models, and worms' behavioural responses to drug treatment. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines.
The technique of differential dynamic microscopy is extended here, showing that it can provide a powerful and objective method of video analysis for optical microscopy videos of in vitro samples of ...live human bronchial epithelial ciliated cells. These cells are multiciliated, with motile cilia that play key physiological roles. It is shown that the ciliary beat frequency can be recovered to match conventional analysis, but in a fully automated fashion. Furthermore, it is shown that the properties of spatial and temporal coherence of cilia beat can be recovered and distinguished, and that if a collective traveling wave (the metachronal wave) is present, this has a distinct signature and its wavelength and direction can be measured.
•A protocol for hemispherical micro-structuring of PDMS surfaces is outlined.•Phase separated ternary lipid GUVs are brought to the patterned surface.•Liquid disordered domains are shown to ...co-localise to the bumps.•The domain caging behaviour is quantified, and its strength is estimated.•The effect is discussed in terms of possible applications.
The role of lipid composition as a regulator or mediator of processes that take place in biological membranes is a very topical question, and important insights can be gained by studying in vitro model lipid mixture systems. A particular question is the coupling of local curvature to the local phases in membranes of mixed composition. Working with an experimental system of giant unilamellar vesicles of ternary composition, the curvature is imposed by approaching the membrane to a topographically (on the micron scale) patterned surface. Performing experiments, we show that domains of the more disordered phase localise preferentially to regions of higher curvature. We characterise and discuss the strength of this “caging” behaviour. In future, the setup we discuss here could prove useful as a platform to localise domains rich in membrane proteins, or to promote the onset of biochemical processes at specific locations. Finally, we note that the methods developed here could have also applications in bio-sensing, as a similar but metal coated topography can sustain plasmonic resonances.
Recent advances in molecular genotyping have helped establish the association between genetic defects and aberrant cilia ultrastructure in PCD, as detected by transmission electron microscopy. Even ...in cases where ultrastructural defects do exist, they can be difficult to detect using standard transmission electron microscopy techniques, and therefore patients who harbor such subtle defects are at risk of being misdiagnosed. Principal component analysis was applied on 85 cilia from 11 patients (three healthy, three HYDIN, and five DNAH11), choosing five specific parameters to build “barcodes” for our samples: the maximum amplitude of the beat; the curvature of the cilium, measured at midlength and averaged for the time cycle; the SD of the curvature over time measured at midlength; the autocorrelation of the curvature; and the mean force exerted on the fluid. Principal component analysis combined a set of five different measurements: 1) maximum amplitude of the beat, measured at 5 μm from the cell surface (healthy: 8.7 ± 1.6 μm, HYDIN: 2.5 ± 0.8 μm, DNAH11: 1.3 ± 0.6 μm); 2) curvature of the cilium, measured at midlength and averaged for the time cycle (healthy: 0.19 ± 0.03 μm−1, HYDIN: 0.082 ± 0.006 μm−1, DNAH11: 0.16 ± 0.04 μm−1), 3) SD of the curvature over time measured at midlength (healthy: 0.13 ± 0.03 μm−1, HYDIN: 0.060 ± 0.008 μm−1, DNAH11: 0.044 ± 0.005 μm−1), 4) the autocorrelation of the curvature measured using couples of points separated by 3/20 of the cilium length (healthy: 0.59 ± 0.03, HYDIN: 0.52 ± 0.02, DNAH11: 0.52 ± 0.02) and 5) the mean force per unit length exerted on the fluid, measured at 9/10 of the cilium length (healthy: 1.17 ± 0.14 pN/μm, HYDIN: 0.38 ± 0.17 pN/μm, DNAH11: 0.40 ± 0.08 pN/μm).
The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize ...IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.
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•IL-13 alters secretory expression states in all airway epithelial cell types•Chronic IL-13 weakens innate defense but spurs interferon signaling in ciliated cells•IL-13 reprogramming leads to pathologic mucus that slows ciliary beat frequency•IL-13-induced metaplasia recapitulates reprograming in type 2 inflamed children
Using airway epithelial cell cultures, Jackson et al. show that IL-13, a driver of type 2-high asthma, induces emergent mucus secretory expression states for each cell type. This program universally diminishes innate airway defense, produces a pathologic mucus secretome that arrests mucociliary movement, and is recapitulated in type 2 inflamed children.
Data collected for the eLife OpenAccess paper: Systematic creation and phenotyping of Mendelian disease models in C. elegans: towards large-scale drug repurposing. (doi: 10.7554/eLife.92491.1)
...Contains: extracted features, calculated stats, normalised z-scores and timerseries data of all the disease model mutants generated. In addition, there is a static .html file that allows for mousing over the clustermaps to easily view differences in strains compared to the N2 wild-type. Dataset also contains, metadata and feature summary/file name information of FDA-library drug screen and the confirmation screen of the hit from this (i.e., all data collected in published in the associated paper).
Eukaryotic organisms rely on the coordinated beating of motile cilia for a multitude of fundamental reasons. In smaller organisms, such as Paramecium and the single cell alga Chlamydomonas ...reinhardtii, it is a matter of propulsion, to swim towards a higher concentration of nutrients or away from damaging environments. Larger organisms use instead the coordinated motion of cilia to push fluid along an epithelium: examples common to mammals are the circulation of cerebrospinal fluid in the brain, the transport of ovules in the fallopian tubes, and breaking the left/right symmetry in the embryo. Another notable example, and one that is central to this thesis, is mucociliary clearance in human airways: A carpet of motile cilia helps keeping the cell surface free from pathogens and foreign particles by constantly evacuating from lungs, bronchi, and trachea a barrier of mucus. The question of how motile cilia interact with one another to beat in a coordinated fashion is an open and pressing one, with immediate implications for the medical community. In order for the fluid propulsion to be effective, the motion of cilia needs to be phase-locked across significant distances, in the form of travelling waves (``metachronal waves''). It is still not known how this long-range coordination emerges from local rules, as there is no central node regulating the coordination among cilia. In the first part of this thesis I will focus on studying the coordination in carpets of cilia with a top-down approach, by proposing, implementing, and applying a new method of analysing microscope videos of ciliated epithelia. Chapter 1 provides the reader with an introduction on motile cilia and flagella, treating their structure and motion and reporting the different open questions currently tackled by the scientific community, with particular interest in the coordination mechanisms of cilia and the mucociliary clearance apparatus. Chapter 2 introduces Differential Dynamic Microscopy (DDM), a powerful and versatile image analysis tool that bridges the gap between spectroscopy and microscopy by allowing to perform scattering experiments on a microscope. The most interesting aspects of DDM for this work are that it can be applied to microscope videos where it is not possible to resolve individual objects in the field of view, and it requires no user input. These two characteristics make DDM a perfect candidate for analysing several hundred microscope videos of weakly scattering filaments such as cilia. In Chapter 3 I will present how it is possible to employ DDM to extract a wealth of often-overlooked information from videos of ciliated epithelia: DDM can successfully probe the ciliary beat frequency (CBF) in a sample, measure the direction of beating of the cilia, and detect metachronal waves and read their direction and wavelength. In vitro ciliated epithelia however often do not show perfect coordination or alignment among cilia. For the analysis of these samples, where the metachronal coordination might not be evident, we developed a new approach, called multiscale DDM (multiDDM), to measure a coordination length scale, a characteristic length of the system over which the coordination between cilia is lost. The new technique of multiDDM is employed in Chapter 4 to study how the coordination among cilia changes as a response to changes in the rheology of the mucous layer. In particular, we show that cilia beating under a thick, gel-like mucus layer show a larger coordination length scale, as if the mucus acted as an elastic raft effectively coupling cilia over long distances. This is corroborated by the coordination length scale being larger in samples from patients affected by Cystic Fibrosis than in healthy samples, and much shorter when the mucus layer is washed and cilia therefore beat in a near-Newtonian fluid. We then show how it is possible to employ multiDDM to measure the effectiveness of drugs in recovering, in CF samples, a coordination length scale typical of a healthy phenotype. In the second part I will focus instead on the single cilium scale, showing how we can attempt to link the beating pattern of cilia to numerical simulations studying synchronisation in a model system. In particular in Chapter 5 I will describe our approach to quantitatively describe the beating pattern of single cilia obtained from human airway cells of either healthy individuals or patients affected by Primary Ciliary Dyskinesia. Our description of the beating pattern, and the selection of a few meaningful, summary parameters, are then shown to be accurate enough to discriminate between different mutations within Primary Ciliary Dyskinesia. In Chapter 6 instead I report the results obtained by coarse-graining the ciliary beat pattern into a model system consisting of two ``rotors''. The rotors are simulated colloidal particles driven along closed trajectories while leaving their phase free. In my study, the trajectories followed by the rotors are analytical fits of experimental trajectories of the centre of drag of real cilia. The rotors, that are coupled only via hydrodynamics interactions, are seen to phase-lock, and the shape of the trajectory they are driven along is seen to influence the steady state of the system.
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