Using Paramecium as a Model for Ciliopathies

Paramecium has served as a model organism for the studies of many aspects of genetics and cell biology: non-Mendelian inheritance, genome duplication, genome rearrangements, and exocytosis, to name a few. However, the large number and patterning of cilia that cover its surface have inspired extraordinary ultrastructural work. Its swimming patterns inspired exquisite electrophysiological studies that led to a description of the bioelectric control of ciliary motion. A genetic dissection of swimming behavior moved the field toward the genes and gene products underlying ciliary function. With the advent of molecular technologies, it became clear that there was not only great conservation of ciliary structure but also of the genes coding for ciliary structure and function. It is this conservation and the legacy of past research that allow us to use Paramecium as a model for cilia and ciliary diseases called ciliopathies. However, there would be no compelling reason to study Paramecium as this model if there were no new insights into cilia and ciliopathies to be gained. In this review, we present studies that we believe will do this. For example, while the literature continues to state that immotile cilia are sensory and motile cilia are not, we will provide evidence that Paramecium cilia are clearly sensory. Other examples show that while a Paramecium protein is highly conserved it takes a different interacting partner or conducts a different ion than expected. Perhaps these exceptions will provoke new ideas about mammalian systems.

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left–right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

Primary ciliary dyskinesia: state of the problem and prospects

This review article provides an up-to-date understanding of primary ciliary dyskinesia (immotile-cilia syndrome) and its particular variant, Cartagener syndrome, a genetically determined pathology leading to chronic inflammatory lesions of the respiratory tract, hearing organs, and impaired fertile function. This orphan disease is not well known to the general medical community. Primary ciliary dyskinesia is a rare hereditary disease of the group of ciliopathies that is based on a genetically determined defect in the ultrastructure of the cilia of the respiratory tract epithelium and similar structures, leading to impaired motor function. Various step-by-step algorithms have been proposed to verify the diagnosis, the obligatory components of which are assessment of the motor ability of the cilia of the atopic epithelium, nasal nitric oxide (nNO) level, electron microscopic examination of a bronchial mucosal biopsy specimen, and genetic examination. There is no gold standard for diagnosis of primary ciliary dyskinesia. Diagnostic search in patients should be complex and consist of certain stages. Currently, therapeutic strategies for primary ciliary dyskinesia are based on approved clinical guidelines. In many countries, the therapy of patients with primary ciliary dyskinesia is based on treatment protocols for patients with cystic fibrosis, despite the obvious differences in these diseases. The main goal of therapy is adequate airway clearance, control and prevention of infectious diseases, and elimination of potential airway exposure to various types of pollutants, including tobacco smoke. The article describes the clinic, characteristic symptoms of the disease, its prevalence and genetic aspects, discusses the problems of diagnosis, treatment, prognosis and monitoring of these children, as well as the need for a national register of patients with this pathology.

Central Apparatus, the Molecular Kickstarter of Ciliary and Flagellar Nanomachines

Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.

Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.

Homozygous truncating NEK10 mutation, associated with primary ciliary dyskinesia: a case report

Background Primary Ciliary Dyskinesia (PCD) is also known as immotile-cilia syndrome, an autosomal recessive disorder of ciliary function, leading to mucus retention in the respiratory system in childhood. Our knowledge in the pathophysiological aspect of this devastating disorder is increasing with the advancement of genetic and molecular testing. Case presentation Here in, we report two siblings with a classical clinical and radiological presentation of PCD. Using whole exome sequencing we identified a homozygous truncating variant (c.3402 T > A); p.(Tyr1134*) in the NEK10 gene. Western bolt analysis revealed a decrease in the expression of NEK10 protein in the patient cells. Conclusions NEK10 plays a central role in the post-mitotic process of cilia assembly, regulating ciliary length and functions during physiological and pathological status. This study highlights the challenges of identifying disease-causing variants for a highly heterogeneous disorder and reports on the identification of a novel variant in NEK10 which recently associated with PCD.

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex

SUMMARYMolecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the 3’ untranslated region (3’-UTR) of Dand5 mRNA is necessary and sufficient for the left-sided decay and is responsive to the flow direction, loss of the cation channel Pkd2, and Ca2+. The 200-nucleotide proximal-most portion of the 3’-UTR, which is conserved among mammals, is essential for the asymmetric mRNA decay and binds Bicc1, an RNA binding protein specifically expressed at the node. Bicc1 preferentially recognizes GAC and GACR sequences in RNA, and these motifs are enriched in the 200-nucleotide region of the Dand5 3’-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. Our results thus suggest that leftward fluid flow induces binding of Bicc1 to the 3’-UTR of Dand5 mRNA in crown cells on the left side of the node, and that consequent recruitment of Ccr4-Not mediates mRNA degradation.