Primary cilia frequency and length are key metrics in studies of ciliogenesis and ciliopathies. Typically, quantitative cilia analysis is done manually, which is very time-consuming. While some open-source and commercial image analysis software applications can segment input data, they still require the user to optimize many parameters, suffer from user bias, and often lack rigorous performance quality assessment (e.g., false positives and false negatives). Further, optimal parameter combinations vary in detection accuracy depending on cilia reporter, cell type, and imaging modality. A good automated solution would analyze images quickly, robustly, and adaptably—across different experimental data sets—without significantly compromising the accuracy of manual analysis. To solve this problem, we developed a new software for automated cilia detection in cells (ACDC). The software operates through four main steps: image importation, pre-processing, detection auto-optimization, and analysis. From a data set, a representative image with manually selected cilia (i.e., Ground Truth) is used for detection auto-optimization based on four parameters: signal-to-noise ratio, length, directional score, and intensity standard deviation. Millions of parameter combinations are automatically evaluated and optimized according to an accuracy ‘F1’ score, based on the amount of false positives and false negatives. Afterwards, the optimized parameter combination is used for automated detection and analysis of the entire data set. The ACDC software accurately and adaptably detected nuclei and primary cilia across different cell types (NIH3T3, RPE1), cilia reporters (AcTub, Smo-GFP, Arl13b), and image magnifications (60×, 40×). We found that false-positive and false-negative rates for Arl13b-stained cilia were 1–6%, yielding high F1 scores of 0.96–0.97 (max. = 1.00). The software detected significant differences in mean cilia length between control and cytochalasin D-treated cell populations and could monitor dynamic changes in cilia length from movie recordings. Automated analysis offered up to a 96-fold speed enhancement compared to manual analysis, requiring around 5 s/image, or nearly 18,000 cilia analyzed/hour. The ACDC software is a solution for robust automated analysis of microscopic images of ciliated cells. The software is extremely adaptable, accurate, and offers immense time-savings compared to traditional manual analysis.
Cilia emanate from basal bodies just underneath the cell membrane. Basal bodies must withstand torque from the ciliary beat and be appropriately spaced for cilia to beat in metachronal waves. Basal body rootlets provide stability for motile cilia. Paramecium has three. Our focus is on the largest one, the striated rootlet (SR). Paramecium basal bodies align in straight rows. Previously we found a potential role for the SR in this alignment. Here we present a phylogeny of the Paramecium homologs of the SF-Assemblin gene of the SR of Chlamydomonas, and the organization of these genes. We describe the phenotypes from RNA interference (RNAi) silencing of genes and gene groups. Phenotypes of the RNAi depletions were characterized by immunofluorescence (IF), electron microscopy, and mass spectrometry. We found 30 genes for Paramecium SF-Assemblin homologs (SFA) organized into 13 Paralog Groups (further categorized in five Structural Groups). Representatives of Paralog Groups were found in the SRs. Silencing the transcripts of any of the Structural Groups correlates with misaligned rows of basal bodies, SRs, and cortical units. The silencing of Structural Groups was key and gave us the ability to systematically disrupt SR structures and cell surface organization. Silencing of SFA genes and Paralog Groups shows no effects on the SR or the cell surface organization. Silencing of the larger Structural Groups has an enormous impact on rows of basal bodies, SRs and cortical units, and SR striations, and length. Misaligned basal bodies have cilia causing the cells to swim in abnormal paths.
Cilia are evolutionary conserved structures protruding from the surface of almost every cell type. Initially motile cilia got all the attentions, while primary cilia were presumed to be vestigial organs. In the last few decades, it has come to light that these solitary, non-motile, microtubule rich structures are indispensable for development and sensory functions. Perturbation in the structure or function of cilia cause an array of related diseases, commonly termed ciliopathies. The most common ciliopathy phenotype is retinal degeneration, which is largely attributed to defects of the retinal photoreceptor, which contains a highly modified primary cilium. This chapter discusses the role of cilia in the development, function, and maintenance of the eye and how cilia defects affect visual function. Research into the sensory role of the photoreceptor outer segment, a modified cilium, has exploded over the past decades. The biogenesis of retinal primary cilium, ciliary gene mutations causing retinal degenerations and functional insights from respective animal models have been extensively discussed. This chapter also discusses the role of primary cilia and ciliary signaling in other ocular cells, and highlights how these findings brings about insights into disease mechanisms and theraupeutics.
Basal body and ciliary dysfunction can give rise to a multitude of different developmental and degenerative disorders that are referred to as ciliopathies. Although cilia are virtually ubiquitously present in vertebrates and mammals, the first human diseases linked to ciliary dysfunction mainly affect the kidney- leading to end stage renal disease. This has led early research efforts towards understanding the role of ciliary function in the kidney. As many of the syndromic ciliopathies such as Bardet Biedl Syndrome (BBS), Joubert Syndrome or Senior-Loken Syndrome present with symptoms in several organs, more recent studies have addressed the role of ciliary function in other organs. This chapter summarizes the information gained from spontaneous and targeted ciliary mutants such as the Oregon Ridge Polycystic Kidney (orpk) mouse or pancreas specific deletion of core ciliary genes. In addition, it discusses the evidence of signalling pathways compromised by impaired ciliary function in the pancreas. Although ciliary function has been implicated in many pathways, the majority of these links have been observed in tissues other than the pancreas or in cultured cells. This overview focuses on the phenotypes caused by ciliary impairment in the pancreas and attempts to reach conclusions about the role of cilia in this context. Emphasis will be placed on metabolic disease that at least partially arises from ciliary dysfunction in the pancreas.
Cilia are specialized, hair-like structures that project from the cell bodies of eukaryotic cells. With increased understanding of the distribution and functions of various types of cilia, interest in these organelles is accelerating. To effectively use this great expansion in knowledge, this information must be made digitally accessible and available for large-scale analytical and computational investigation. Capture and integration of knowledge about cilia into existing knowledge bases, thus providing the ability to improve comparative genomic data analysis, is the objective of this work.
Cilia and ciliary proteins play a crucial role in development and the dysfunction of cilia and ciliary proteins often results in complex syndromal disorders. One site of frequent manifestation of ciliary disorders is the skeleton and the resulting ciliary chondrodysplasias represent a heterogeneous group of rare, nearly exclusivelyautosomal recessive inherited conditions. Frequent manifestations include shortened limbs and ribs, polydactyly as well as craniofacial abnormalities. In addition, extraskeletal disease affecting the kidneys, liver, heart, eyes and other organs and tissues is observed inconsistently. Nevertheless, perinatal and childhood lethality due to cardiorespiratory failure resulting from congenital heart disease and/or thoracic constriction as well as from renal and hepatic insufficiency is significantly observed. Brain malformation, developmental delay and loss of eyesight are additional disabling phenotypes observed. While the exact pathomechanism in these disorders remains to be understood, the skeletal phenotype has been largely attributed to imbalances in the hedgehog signalling pathway, for which chondrocyte cilia play a crucial role. Although phenotypes have been historically distinguished based on clinical features into short rib-polydactyly syndrome (SRPS), Jeune Asphyxiating Thoracic Dystrophy (JATD), Mainzer-Saldino Syndrome (MZDS), Sensenbrenner Syndrome (cranioectodermal dysplasia, CED), Oral-Facial-Digital syndrome (OFD), Axial Spondylometaphyseal Dysplasia (axial SMD) and Ellis-van Creveld syndrome (EVC), recent research suggests that there is significant genetic as well as phenotypic overlap between the conditions. This chapter summarizes phenotypic hallmarks as well as the molecular basis of ciliary chondrodysplasias and gives a short outlook towards clinical management and potential therapeutic approaches.
The auditory system is one of the most remarkable organs in vertebrate organisms. Not only is the auditory system required for our perception of sound, but also for our perception of spatial orientation. Although much is known about how this system develops and functions, little is known about the role of cilia during these processes, which is unfortunate considering that auditory dysfunction is one of the phenotypes of several human ciliopathy syndromes. In this chapter we summarize what is currently known about cilia function in the auditory system with a particular focus on the mammalian cochlea and the kinocilium found on the hair cells of the Organ of Corti. We describe the different regions of the cochlea in which primary cilia have been identified and discuss signaling pathways that are thought to be regulated by cilia function in the inner ear. We examine the auditory phenotype of various ciliopathy animal models and conclude by discussing human diseases relevant to ciliary defects in the auditory system.
The lungs are the site of gaseous exchange, enabling delivery of oxygen to the blood and removal of carbon dioxide from it. To efficiently perform this function, the lungs are connected to the outside environment via the trachea, mouth and nose. Because of this direct connection to the external environment, mechanisms are needed to help remove inhaled particles such as toxins and pathogens. One such mechanism is the trapping of particles within the protective mucus layer that sits above the airway epithelium. The co-ordinated beating of multiple motile cilia in the airways enables directed flow of mucus away from the lower regions of the lungs and up towards the mouth and nose, from where it can be safely removed. In addition to the important role played by motile cilia, cells in the lungs also contain non-motile, primary cilia. Currently, much less is known about the role of primary cilia in the lungs, though evidence is beginning to emerge. Here we describe the cellular structure of the lungs with an emphasis on the development of primary (non-motile) and motile cilia. We also consider the relationships between cilia and lung disease, when cilia structure/function is directly affected for example as occurs in primary ciliary dyskinesia and also when cilia are indirectly impaired for example in asthma or cystic fibrosis.
The kidney is a uniquely shaped organ that our body requires to maintain fluid homeostasis. Its formation begins with a mesenchymal to epithelial transition, where epithelial branching will give rise to the distinct kidney sub-structures. During kidney development, the primary cilium, a sensory organelle serving as the cell's antenna contributes to correct renal formation and function. One of the signalling pathways that are transduced via the primary cilium in the kidney is Wnt signalling. Wnt signalling consists of three branches: canonical Wnt signalling through catenin, non-canonical Wnt/Planar Cell Polarity (PCP) signalling and non-canonical Wnt/Ca+2 mediated signalling. Defects in either of the three pathways have been shown to affect kidney morphogenesis and function and have been associated with ciliary abnormalities that could also result in chronic, inherited kidney disease. This chapter examines the role of primary cilia in kidney development and function, dissects the significance of Wnt signalling in the context of the kidney and relates ciliary and Wnt signalling perturbations with renal development and disease.
Mainzer–Saldino syndrome (MZSDS) is a skeletal ciliopathy and part of the short-rib thoracic dysplasia (SRTD) group of ciliary disorders. The main characteristics of MZSDS are short limbs, mild narrow thorax, blindness, and renal failure. Thus far, variants in two genes are associated with MZSDS: IFT140, and IFT172. In this study, we describe a 1-year-old girl presenting with mild skeletal abnormalities, Leber congenital amaurosis, and bilateral hearing difficulties. For establishing an accurate diagnosis, we combined clinical, molecular, and functional analyses.
The mechanisms by which primary cilia affect glioma pathogenesis are unclear. Depending on the glioma cell line, primary cilia can promote or inhibit tumor development. Here, we used piggyBac-mediated transgenesis to generate patient-derived glioblastoma (GBM) cell lines that stably express Arl13b:GFP in their cilia. This allowed us to visualize and analyze the behavior of cilia and ciliated cells during live GBM cell proliferation.
BACKGROUND:Primary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons. These small organelles serve as sensory structures sampling the extracellular environment and reprogramming the transcriptional machinery in response to environmental change. Primary cilia are decorated with a variety of receptor proteins and are necessary for specific signaling cascades such as the Sonic hedgehog (Shh) pathway. Disrupting cilia structure or function results in a spectrum of diseases collectively referred to as ciliopathies. Common to human ciliopathies is cognitive impairment, a symptom also observed in Alzheimer's disease (AD). One hallmark of AD is accumulation of senile plaques composed of neurotoxic Amyloid-β (Aβ) peptide. The Aβ peptide is generated by the proteolytic cleavage of the amyloid precursor protein (APP). We set out to determine if Aβ affects primary cilia structure and the Shh signaling cascade.METHODS:We utilized in vitro cell-based assays in combination with fluorescent confocal microscopy to address our study goals. Shh signaling and cilia structure was studied using two different cell lines, mouse NIH3T3 and human HeLa cells. To investigate how Aβ levels affect Shh signaling and cilia structure in these cells, we utilized naturally secreted Aβ as well as synthetic Aβ. Effects on Shh signaling were assessed by luciferase activity while cilia structure was analyzed by fluorescent microscopy.RESULTS:Here, we report that APP localizes to primary cilia and Aβ treatment results in distorted primary cilia structure. In addition, we demonstrate that Aβ treatment interrupts canonical Shh signal transduction.CONCLUSIONS:Overall, our study illustrates that Aβ can alter primary cilia structure suggesting that elevated Aβ levels, like those observed in AD patients, could have similar effects on neuronal primary cilia in the brain. Additionally, our study suggests that Aβ impairs the Shh signaling pathway. Together our findings shed light on two novel targets for future AD therapeutics.
BACKGROUND:A transient increase in cytosolic Ca2+ (the "Ca2+ transient") determines the degree and duration of myocyte force development in the heart. However, we have previously observed that, under the same experimental conditions, the Ca2+ transients from isolated cardiac myocytes are reduced in amplitude in comparison to those from multicellular cardiac preparations. We therefore questioned whether the enzymatic cell isolation procedure might remove structures that modulate intracellular Ca2+ in some way. Primary cilia are found in a diverse range of cell types, and have an abundance of Ca2+-permeable membrane channels that result in Ca2+ influx when activated. Although primary cilia are reportedly ubiquitous, their presence and function in the heart remain controversial. If present, we hypothesized they might provide an additional Ca2+ entry pathway in multicellular cardiac tissue that was lost during cell isolation. The aim of our study was to look for evidence of primary cilia in isolated myocytes and ventricular tissue from rat hearts.METHODS:Immunohistochemical techniques were used to identify primary cilia-specific proteins in isolated myocytes from adult rat hearts, and in tissue sections from embryonic, neonatal, young, and adult rat hearts. Either mouse anti-acetylated α-tubulin or rabbit polyclonal ARL13B antibodies were used, counterstained with Hoechst dye. Selected sections were also labelled with markers for other cell types found in the heart and for myocyte F-actin.RESULTS:No evidence of primary cilia was found in either tissue sections or isolated myocytes from adult rat ventricles. However, primary cilia were present in tissue sections from embryonic, neonatal (P2) and young (P21 and P28) rat hearts.CONCLUSION:The lack of primary cilia in adult rat hearts rules out their contribution to myocyte Ca2+ homoeostasis by providing a Ca2+ entry pathway. However, evidence of primary cilia in tissue from embryonic and very young rat hearts suggests they have a role during development.
BACKGROUND:Primary cilia mediate signal transduction by acting as an organizing scaffold for receptors, signalling proteins and ion channels. Ciliated olfactory sensory neurons (OSNs) organize olfactory receptors and ion channels on cilia and generate a calcium influx as a primary signal in odourant detection. In the zebrafish olfactory placode, ciliated OSNs and microvillus OSNs constitute the major OSN cell types with distinct odourant sensitivity.METHODS:Using transgenic expression of the calcium biosensor GCaMP5 in OSNs, we analysed sensory cilia-dependent odour responses in live zebrafish, at individual cell resolution. oval/ift88 mutant and ift172 knockdown zebrafish were compared with wild-type siblings to establish ciliated OSN sensitivity to different classes of odourants.RESULTS:oval/ift88 mutant and ift172 knockdown zebrafish showed fewer and severely shortened OSN cilia without a reduction in OSN number. The fraction of responding OSNs and response amplitudes to bile acids and food odour, both sensed by ciliated OSNs, were significantly reduced in ift88 mutants and ift172-deficient embryos, while the amino acids responses were not significantly changed.CONCLUSIONS:Our approach presents a quantitative model for studying sensory cilia signalling using zebrafish OSNs. Our results also implicate ift172-deficiency as a novel cause of hyposmia, a reduced sense of smell, highlighting the value of directly assaying sensory cilia signalling in vivo and supporting the idea that hyposmia can be used as a diagnostic indicator of ciliopathies.
BACKGROUND:Recent research into ciliary structure and function provides important insights into inherited diseases termed ciliopathies and other cilia-related disorders. This wealth of knowledge needs to be translated into a computational representation to be fully exploitable by the research community. To this end, members of the Gene Ontology (GO) and SYSCILIA Consortia have worked together to improve representation of ciliary substructures and processes in GO.METHODS:Members of the SYSCILIA and Gene Ontology Consortia suggested additions and changes to GO, to reflect new knowledge in the field. The project initially aimed to improve coverage of ciliary parts, and was then broadened to cilia-related biological processes. Discussions were documented in a public tracker. We engaged the broader cilia community via direct consultation and by referring to the literature. Ontology updates were implemented via ontology editing tools.RESULTS:So far, we have created or modified 127 GO terms representing parts and processes related to eukaryotic cilia/flagella or prokaryotic flagella. A growing number of biological pathways are known to involve cilia, and we continue to incorporate this knowledge in GO. The resulting expansion in GO allows more precise representation of experimentally derived knowledge, and SYSCILIA and GO biocurators have created 199 annotations to 50 human ciliary proteins. The revised ontology was also used to curate mouse proteins in a collaborative project. The revised GO and annotations, used in comparative 'before and after' analyses of representative ciliary datasets, improve enrichment results significantly.CONCLUSIONS:Our work has resulted in a broader and deeper coverage of ciliary composition and function. These improvements in ontology and protein annotation will benefit all users of GO enrichment analysis tools, as well as the ciliary research community, in areas ranging from microscopy image annotation to interpretation of high-throughput studies. We welcome feedback to further enhance the representation of cilia biology in GO.
Primary cilia are immotile, microtubule-based organelles present on most cells. Defects in primary cilia presence/function result in a category of developmental diseases referred to as ciliopathies. As the cilia field progresses, there is a need to consider both the ciliary and extraciliary roles of cilia proteins. However, traditional fixation methods are not always suitable for examining the full range of localizations of cilia proteins. Here, we tested a variety of fixation methods with commonly used cilia markers to determine the most appropriate fixation method for different cilia proteins.
This is a history of cilia research before and after the discovery of intraflagellar transport (IFT) and the link between primary cilia ciliogenesis and polycystic kidney disease (PKD). Before IFT, ca. the beginning of the new millennium, although sensory and primary cilia were well described, research was largely focused on motile cilia, their structure, movement, and biogenesis. After IFT and the link to PKD, although work on motile cilia has continued to progress, research on primary cilia has exploded, leading to new insights into the role of cilia in cell signaling and development. Genomics, proteomics, and new imaging techniques have unified the field and pointed out the critical role of cilia as a restricted cell organellar compartment, functionally integrated with other cell organelles including the autophagosome and the nucleus.
BACKGROUND:The discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome.METHODS:We integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student's and Fisher's exact t tests.RESULTS:Glycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband's fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1.CONCLUSIONS:The rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.
