bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2023–05–14
eightteen papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne
  1. An update on treatments for autosomal dominant polycystic kidney disease.
  2. Fixation methods and immunolabeling for cilia proteins in ciliary and extraciliary locations.
  3. Aortic, hepatic and renal artery aneurysms in autosomal dominant polycystic kidney disease.
  4. The safety and efficacy of tolvaptan in the treatment of patients with autosomal dominant polycystic kidney disease: A systematic review and meta-analysis.
  5. Sperm morphological abnormalities in autosomal dominant polycystic kidney disease are associated with the Hippo signaling pathway via PC1.
  6. Fatal acute portal vein thrombosis associated with hepatic cysts in a patient with autosomal dominant polycystic kidney disease.
  7. High-SPEED super-resolution SPEED microscopy to study primary cilium signaling in vivo.
  8. Energetic landscape of polycystin channel gating.
  9. Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.
  10. Haemoperitoneum in peritoneal dialysis and polycystic kidney disease: An interesting cause.
  11. Tolvaptan-induced isolated elevation of bilirubin in a patient with Gilbert syndrome.
  12. Human LUHMES and NES cells as models for studying primary cilia in neurons.
  13. Enhanced primary ciliogenesis via mitochondrial oxidative stress activates AKT to prevent neurotoxicity in HSPA9/mortalin-depleted SH-SY5Y cells.
  14. Modeling ciliopathies in patient-derived primary cells.
  15. Methods to study primary cilia and autophagy in the brain.
  16. Single-molecule imaging in the primary cilium.
  17. Methods to study motile ciliated cell types in the zebrafish brain.
  18. Analysis of motility and mucociliary function of tracheal epithelial cilia.
  1. JAAPA. 2023 May 10. Published Ahead of Print
    An update on treatments for autosomal dominant polycystic kidney disease.
    Suhani Janak Patel, Catherine K Sadowski.
       ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is less common than primary hypertension or diabetes but should be considered as a possible cause of end-stage renal disease, especially in young patients without comorbidities. Because of ADPKD's nonspecific symptoms, the diagnosis, treatment, and pertinent patient education may be delayed. This article describes ADPKD and its management, including tolvaptan, a new treatment with the potential to reduce or delay morbidity. However, only a subset of patients qualifies for this expensive treatment.
    DOI:  https://doi.org/10.1097/01.JAA.0000931420.46207.82
  2. Methods Cell Biol. 2023 ;pii: S0091-679X(23)00002-X. [Epub ahead of print]176 43-57
    Fixation methods and immunolabeling for cilia proteins in ciliary and extraciliary locations.
    Kiet Hua, Russell J Ferland.
      Primary cilia are complex organelles, usually singularly located on cell surfaces that are now known to be important for signaling and whose defect is implicated in a category of developmental diseases known as ciliopathies. They are composed of a microtubule axoneme and contain a cilia membrane that is unique and distinct from the plasma membrane. Primary cilia also have their own transport system termed the intraflagellar transport (IFT) system that allows for proteins to be trafficked along the microtubule axoneme in either an anterograde or retrograde manner. Proteins that localize to the primary cilium are referred to as ciliary proteins and have been implicated directly or indirectly in ciliogenesis or ciliary function. It is now recognized that cilia proteins can localize to different compartments of cilia, but can also localize to multiple sites outside of cilia (extraciliary sites). This complexity results in a need for a better understanding of ciliary protein fixation and immunolabeling protocols, as different methods are required to visualize different cilia proteins and reveal novel or unique localizations. Here, we detail a variety of fixation methods and their effects on ciliary protein immunolabeling.
    Keywords:  Axoneme; Basal body; Fixation; Immunolabel; Methods; Primary cilia
    DOI:  https://doi.org/10.1016/bs.mcb.2023.01.002
  3. Natl Med J India. 2023 Nov-Dec;35(6):pii: 10.25259/NMJI_35_6_372. [Epub ahead of print]35(6): 372-373
    Aortic, hepatic and renal artery aneurysms in autosomal dominant polycystic kidney disease.
    Deepak Kumar, Himanshu Mishra, Umakant Prasad.
      
    DOI:  https://doi.org/10.25259/NMJI_35_6_372
  4. Nefrologia (Engl Ed). 2023 May 05. pii: S2013-2514(23)00071-8. [Epub ahead of print]
    The safety and efficacy of tolvaptan in the treatment of patients with autosomal dominant polycystic kidney disease: A systematic review and meta-analysis.
    Xuanwei Li, Wenlai Li, Yue Li, Chuanjiang Dong, Ping Zhu.
       BACKGROUND: The irreversible progression of autosomal dominant polycystic kidney disease (ADPKD) to end-stage renal disease (ESRD) is delayed by tolvaptan. Therefore, we aim to systematically estimate and evaluate the efficacy and safety of tolvaptan in the treatment of ADPKD.
    METHODS: Two reviewers independently searched all published randomized controlled trials studies in PubMed, EMBASE, Web of Science and Cochrane databases, extracted data, assessed bias risk and rated the quality of evidence. Data were analyzed by the RevMan software.
    RESULTS: We identified 8 trials including 2135 patients. Both of the decline of estimated glomerular filtration rate (eGFR) [MD=1.89, 95% CI (0.74, 3.04), P=0.001] and total kidney volume (TKV) [MD=-3.32, 95% CI (-4.57, -2.07), P<0.001] were delayed in tolvaptan group compared with placebo group in ADPKD patients. The use of tolvaptan delayed TKV progression in the different-month subgroups [MD=-69.99, 95% CI (-91.05, -48.94), P<0.001]. Tolvaptan reduced renal pain [RR=0.66, 95% CI (0.54, 0.81), P<0.001] and hematuria events [RR=0.55, 95% CI (0.41, 0.74), P<0.001] in ADPKD patients. However, the prevalence of thirst [RR=2.75, 95% CI (2.34, 3.24), P<0.001] and nocturia events [RR=3.01, 95% CI (1.27, 7.11), P=0.01] were increased in tolvaptan group. There is no significant difference of hypertension events [RR=0.92, 95% CI (0.82, 1.03), P=0.13] in tolvaptan group compared placebo group.
    CONCLUSIONS: This meta-analysis suggests that tolvaptan may improve clinical progression in patients with ADPKD without significantly increasing the risk of adverse reactions.
    Keywords:  ADPKD (autosomal dominant polycystic kidney disease); ERPAD (enfermedad renal poliquística autosómica dominante); TKV (total kidney volume); Tolvaptan; VRT (volumen renal total); eGFR (estimated glomerular filtration rate); eGFR (tasa de filtración glomerular estimada)
    DOI:  https://doi.org/10.1016/j.nefroe.2023.04.002
  5. Front Endocrinol (Lausanne). 2023 ;14 1130536
    Sperm morphological abnormalities in autosomal dominant polycystic kidney disease are associated with the Hippo signaling pathway via PC1.
    Wei-Hui Shi, Zhi-Yang Zhou, Mu-Jin Ye, Ning-Xin Qin, Zi-Ru Jiang, Xuan-You Zhou, Nai-Xin Xu, Xian-Lin Cao, Song-Chang Chen, He-Feng Huang, Chen-Ming Xu.
       Background: Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary kidney disorder mostly caused by mutations in PKD1 or PKD2 genes. Here, we report thirteen ADPKD males with infertility and investigated the sperm morphological defects associated with PC1 disruption.
    Methods: Targeted next-generation sequencing was performed to detect PKD1 variants in patients. Sperm morphology was observed by immunostaining and transmission electron microscopy, and the sperm motility was assessed using the computer-assisted sperm analysis system. The Hippo signaling pathway was analyzed with by quantitative reverse transcription polymerase chain reaction (qPCR) and western blotting in vitro.
    Results: The ADPKD patients were infertile and their sperm tails showed morphological abnormalities, including coiled flagella, absent central microtubules, and irregular peripheral doublets. In addition, the length of sperm flagella was shorter in patients than in controls of in in. In vitro, ciliogenesis was impaired in Pkd1-depleted mouse kidney tubule cells. The absence of PC1 resulted in a reduction of MST1 and LATS1, leading to nuclear accumulation of YAP/TAZ and consequently increased transcription of Aurka. which might promote HDAC6-mediated ciliary disassembly.
    Conclusion: Our results suggest the dysregulated Hippo signaling significantly contributes to ciliary abnormalities in and may be associated with flagellar defects in spermatozoa from ADPKD patients.
    Keywords:  PKD1; autosomal dominant polycystic kidney disease; male infertility; sperm flagella; the Hippo pathway
    DOI:  https://doi.org/10.3389/fendo.2023.1130536
  6. CEN Case Rep. 2023 May 10.
    Fatal acute portal vein thrombosis associated with hepatic cysts in a patient with autosomal dominant polycystic kidney disease.
    Yohei Tsuchida, Yutaka Tsubata, Ryosuke Nozawa, Shuntaro Maruyama, Kouzo Ikarashi, Noriko Saito, Tetsuo Morioka, Takeshi Kamura, Hisaki Shimada, Ichiei Narita.
      Autosomal dominant polycystic kidney disease (ADPKD) often involves polycystic liver disease (PLD). In severe cases, PLD can develop various complications. However, fatal acute portal vein thrombosis (APVT) associated with PLD has not been reported. A 64-year-old male reported mild consciousness disorder. He had been under maintenance hemodialysis for end-stage renal disease due to ADPKD with PLD. Because of recurring hepatic cyst infections, he had sustained high levels of C-reactive protein. Regarding the mild consciousness disorder, a diagnosis of hepatic encephalopathy was made based on an elevation of serum ammonia without any other abnormal liver function tests. Several days after his admission, hepatobiliary enzymes elevated, and acute liver failure progressed. Enhanced abdominal computed tomography suggested the possibility of complete occlusion of the portal vein by a thrombus. Based on an absence of obvious portosystemic collaterals, a diagnosis of APVT was made. The patient died 19 days after admission. Patients with PLD with repeated cystic infections have been seen to develop liver failure, and APVT formation may be one cause of the rapid progression of fatal liver failure. In conclusion, this is the first paper to report on the involvement of APVT in patients with PLD.
    Keywords:  Acute liver failure; Acute portal vein thrombosis; Autosomal dominant polycystic kidney disease; Hemodialysis; Hepatic encephalopathy; Polycystic liver disease
    DOI:  https://doi.org/10.1007/s13730-023-00795-6
  7. Methods Cell Biol. 2023 ;pii: S0091-679X(22)00204-7. [Epub ahead of print]176 181-197
    High-SPEED super-resolution SPEED microscopy to study primary cilium signaling in vivo.
    Mark Tingey, Andrew Ruba, Weidong Yang.
      The primary cilium is a surface exposed organelle found in eukaryotic cells that functions to decode a variety of intracellular signals with significant implications in human developmental disorders and diseases. It is therefore highly desirable to obtain in vivo information regarding the dynamic processes occurring within the primary cilium. However, current techniques are limited by either the physical limitations of light microscopy or the static nature of electron microscopy. To overcome these limitations, single-point edge-excitation sub-diffraction (SPEED) microscopy was developed to obtain dynamic in vivo information in subcellular organelles such as cilia and nuclear pore complexes using single-molecule super-resolution light microscopy with a spatiotemporal resolution of 10-20nm and 0.4-2ms. Three-dimensional (3D) structural and dynamic information in these organelles can be further obtained through a post-processing 2D-to-3D transformation algorithm. Here we present a modular step-by-step protocol for studying primary cilium signaling dynamics, including Intraflagellar transport (IFT) via IFT20 and somatostatin g-protein-coupled receptor activity via SSTR3.
    Keywords:  Primary cilia; Super-resolution light microscopy; Transformation algorithm
    DOI:  https://doi.org/10.1016/bs.mcb.2022.12.016
  8. EMBO Rep. 2023 May 09. e56783
    Energetic landscape of polycystin channel gating.
    Leo Ct Ng, Brandon J Harris, Megan Larmore, My C Ta, Thuy N Vien, Valerie L Tokars, Vladimir Yarov-Yarovoy, Paul G DeCaen.
      Members of the polycystin family (PKD2 and PKD2L1) of transient receptor potential (TRP) channels conduct Ca2+ and depolarizing monovalent cations. Variants in PKD2 cause autosomal dominant polycystic kidney disease (ADPKD) in humans, whereas loss of PKD2L1 expression causes seizure susceptibility in mice. Understanding structural and functional regulation of these channels will provide the basis for interpreting their molecular dysregulation in disease states. However, the complete structures of polycystins are unresolved, as are the conformational changes regulating their conductive states. To provide a holistic understanding of the polycystin gating cycle, we use computational prediction tools to model missing PKD2L1 structural motifs and evaluate more than 150 mutations in an unbiased mutagenic functional screen of the entire pore module. Our results provide an energetic landscape of the polycystin pore, which enumerates gating sensitive sites and interactions required for opening, inactivation, and subsequent desensitization. These findings identify the external pore helices and specific cross-domain interactions as critical structural regulators controlling the polycystin ion channel conductive and nonconductive states.
    Keywords:  TRP channels; calcium; polycystic kidney disease; polycystins; structural biology
    DOI:  https://doi.org/10.15252/embr.202356783
  9. Methods Cell Biol. 2023 ;pii: S0091-679X(23)00045-6. [Epub ahead of print]176 235-250
    Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.
    Quanlong Lu, Christopher J Westlake.
      The ciliary membrane is continuous with the plasma membrane but has distinct lipid and protein composition, which is key to defining the function of the primary cilium. Ciliary membranes dynamically assemble and disassemble in association with the cell cycle and directly transmit signals and molecules through budding membranes. Various imaging approaches have greatly advanced the understanding of the ciliary membrane function. In particular, fluorescence live-cell imaging has revealed important insights into the dynamics of ciliary membrane assembly by monitoring the changes of fluorescent-tagged ciliary proteins. Protein dynamics can be tracked simultaneously using multi-color live cell imaging by coupling ciliary-associated factors with different colored fluorescent tags. Ciliary membrane and membrane associated-proteins such as Smoothened, 5-HTr6, SSTR3, Rab8a, and Arl13b have been used to track ciliary membranes and centriole proteins like Centrin1/2, CEP164, and CEP83 are often used to mark the ciliary basal body. Here, we describe a method for studying ciliogenesis membrane dynamics using spinning disk confocal live-cell imaging.
    Keywords:  Ciliary dynamics; Ciliary proteins; Ciliogenesis; Live-cell fluorescence imaging; Membrane trafficking; Primary cilia
    DOI:  https://doi.org/10.1016/bs.mcb.2023.02.001
  10. Perit Dial Int. 2023 May 07. 8968608231169834
    Haemoperitoneum in peritoneal dialysis and polycystic kidney disease: An interesting cause.
    Zheng-Zhe Wu, Chih-Hen Yu, Junne-Ming Sung.
      
    Keywords:  Cyst rupture; haemoperitoneum; peritoneal dialysis; polycystic kidney disease; retroperitoneal haematoma
    DOI:  https://doi.org/10.1177/08968608231169834
  11. SAGE Open Med Case Rep. 2023 ;11 2050313X231169841
    Tolvaptan-induced isolated elevation of bilirubin in a patient with Gilbert syndrome.
    Matthew Nguyen, Beshoy T Yanny, Tai Ld Truong, Hongyu Zhao, Ramy Hanna.
      Tolvaptan is the current standard of treatment for autosomal dominant polycystic kidney disease. It operates by acting on V2 receptors and blocks vasopressin interactions, causing a reduction in the rate of renal cyst growth and preserving kidney function. The current known risks of tolvaptan involve a serious liver injury characterized by an elevation in total bilirubin and alanine transaminase and aspartate transaminase levels. In this report, we document a unique liver injury characterized by an elevated bilirubin with normal alanine transaminase and aspartate transaminase levels in a patient who is homozygous for the UGT1A1 consistent with Gilbert syndrome.
    Keywords:  Gilbert’s syndrome; Tolvaptan; autosomal dominant polycystic kidney disease
    DOI:  https://doi.org/10.1177/2050313X231169841
  12. Methods Cell Biol. 2023 ;pii: S0091-679X(22)00200-X. [Epub ahead of print]176 27-41
    Human LUHMES and NES cells as models for studying primary cilia in neurons.
    Andrea Coschiera, Michelle Evelyn Watts, Juha Kere, Kristiina Tammimies, Peter Swoboda.
      Primary cilia are antenna-like organelles emanating from the cell surface. They are involved in cell-to-cell communication and bidirectional signal transduction to/from the extracellular environment. During brain formation, cilia critically aid in neurogenesis and maturation of neuronal structures such as axons, dendrites and synapses. Aberrations in cilia function can induce neuron differentiation defects and pathological consequences of varying severity, resulting in ciliopathies and likely a number of neurodevelopmental disorders. Despite the documented relevance of cilia for proper brain development, human neuronal models to recognize and study cilia biology are still scarce. We have established two types of cell models, Lund Human Mesencephalic (LUHMES) cells and neuroepithelial stem (NES) cells derived from induced pluripotent stem cells (iPSC), to investigate cilia biology in both proliferating neuronal progenitors/precursors and during the entire neuron differentiation and maturation process. We employ improved immunocytochemistry assays able to specifically detect cilia by confocal and super-resolution microscopy. We provide straightforward and robust methods to easily maintain cells in culture, for immunostaining and characterization of cilia orientation, anatomy and shape in human neurons across all stages of differentiation.
    Keywords:  Confocal microscopy; Immunocytochemistry; Neuron differentiation; Neuronal cilia; Primary cilia
    DOI:  https://doi.org/10.1016/bs.mcb.2022.12.012
  13. Mol Brain. 2023 May 11. 16(1): 41
    Enhanced primary ciliogenesis via mitochondrial oxidative stress activates AKT to prevent neurotoxicity in HSPA9/mortalin-depleted SH-SY5Y cells.
    Ji-Eun Bae, Soyoung Jang, Joon Bum Kim, Hyejin Hyung, Na Yeon Park, Yong Hwan Kim, So Hyun Kim, Seong Hyun Kim, Jin Min Ha, Gyeong Seok Oh, Kyuhee Park, Kwiwan Jeong, Jae Seon Jang, Doo Sin Jo, Pansoo Kim, Hyun-Shik Lee, Zae Young Ryoo, Dong-Hyung Cho.
      The primary cilium, an antenna-like structure on the cell surface, acts as a mechanical and chemical sensory organelle. Primary cilia play critical roles in sensing the extracellular environment to coordinate various developmental and homeostatic signaling pathways. Here, we showed that the depletion of heat shock protein family A member 9 (HSPA9)/mortalin stimulates primary ciliogenesis in SH-SY5Y cells. The downregulation of HSPA9 enhances mitochondrial stress by increasing mitochondrial fragmentation and mitochondrial reactive oxygen species (mtROS) generation. Notably, the inhibition of either mtROS production or mitochondrial fission significantly suppressed the increase in primary ciliogenesis in HSPA9-depleted cells. In addition, enhanced primary ciliogenesis contributed to cell survival by activating AKT in SH-SY5Y cells. The abrogation of ciliogenesis through the depletion of IFT88 potentiated neurotoxicity in HSPA9-knockdown cells. Furthermore, both caspase-3 activation and cell death were increased by MK-2206, an AKT inhibitor, in HSPA9-depleted cells. Taken together, our results suggest that enhanced primary ciliogenesis plays an important role in preventing neurotoxicity caused by the loss of HSPA9 in SH-SY5Y cells.
    Keywords:  HSPA9/mortalin; Mitochondrial stress; Neurotoxicity; Primary cilia; SH-SY5Y cells
    DOI:  https://doi.org/10.1186/s13041-023-01029-7
  14. Methods Cell Biol. 2023 ;pii: S0091-679X(23)00060-2. [Epub ahead of print]176 139-158
    Modeling ciliopathies in patient-derived primary cells.
    G Yealland, M Jevtic, K-U Eckardt, M Schueler.
      Ciliopathies comprise a group of inherited diseases caused by mutations in genes encoding proteins that localize to cilia or centrosomes. They afflict multiple organs and are one of the most frequent monogenic causes of kidney failure in adults, adolescents and children. Primary cilia play diverse roles in cell signaling, cell cycle regulation, planar cell polarity and mechanosensing. The use of patient-derived cells possessing endogenous disease causing mutations enables the study of these processes and their dysregulation in disease. Here we describe methods to cultivate patient-derived dermal fibroblast and renal epithelial cells isolated from urine. Fibroblasts are highly robust, long-lived, and easy to culture cells in which ciliary assembly can be easily induced. Similarly, the ability to acquire and culture ciliated renal epithelial cells without patient-invasive-intervention holds great potential to further our understanding of ciliopathies. In addition to monolayer cultures, we also detail the formation of three-dimensional renal-epithelial organoids-so-called tubuloids-that demonstrate epithelial-polarization and transepithelial transport activities like those seen in vivo renal-tubules. These in vitro models are powerful tools to investigate the underlying disease mechanisms of human ciliopathies that can be employed without the need for heavy-handed genetic or molecular manipulations.
    Keywords:  3D culture; Ciliopathy; Fibroblast; Organotypic; Patient derived; Personalized medicine; Renal epithelial cell; Tubuloid; Urine derived
    DOI:  https://doi.org/10.1016/bs.mcb.2023.02.016
  15. Methods Cell Biol. 2023 ;pii: S0091-679X(23)00010-9. [Epub ahead of print]176 217-234
    Methods to study primary cilia and autophagy in the brain.
    Laura de Las Heras-García, Irati Zabalegui, Olatz Pampliega.
      Autophagy is an intracellular catabolic pathway that allows proteins, organelles, and pathogens to be recycled. Thus, it is crucial to maintain cell homeostasis, especially important in post-mitotic cells as neurons that cannot dilute cellular damage through mitosis. In the last decade, autophagy has been connected to the primary cilium (PC), a small organelle that acts as a sensory hub and is present in most cell types, including astrocytes and neurons. In this chapter, we briefly describe the state-of-the-art of the interplay between autophagy, PC, and its implications for the brain, in healthy and pathophysiological conditions. Deregulations in autophagy can be monitored by numerous assays, both in vivo and in vitro, and so do changes in PC length/number. Here, we relate a practical and user-friendly description of immunofluorescence methods to study autophagy and PC changes in brain slices, including the tissue preparation, confocal microscopy, image analysis, and deconvolution process.
    Keywords:  Astrocyte; Autophagy; Immunohistochemistry; Neuron; Primary cilium
    DOI:  https://doi.org/10.1016/bs.mcb.2023.01.010
  16. Methods Cell Biol. 2023 ;pii: S0091-679X(23)00003-1. [Epub ahead of print]176 59-83
    Single-molecule imaging in the primary cilium.
    Lucien E Weiss, Julia F Love, Joshua Yoon, Colin J Comerci, Ljiljana Milenkovic, Tomoharu Kanie, Peter K Jackson, Tim Stearns, Anna-Karin Gustavsson.
      The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium. Specifically, we describe in detail how to capture and quantify the 2D dynamics of individual transmembrane proteins PTCH1 and SMO and how to map the 3D nanoscale distributions of the inversin compartment proteins INVS, ANKS6, and NPHP3. This protocol can, with minor modifications, be adapted for studies of other proteins and cell lines to further elucidate the structure and function of the primary cilium at the molecular level.
    Keywords:  3D imaging; Hedgehog signaling; Inversin compartment; Point-spread-function engineering; Primary cilium; Single-molecule tracking; Super-resolution microscopy
    DOI:  https://doi.org/10.1016/bs.mcb.2023.01.003
  17. Methods Cell Biol. 2023 ;pii: S0091-679X(23)00043-2. [Epub ahead of print]176 103-123
    Methods to study motile ciliated cell types in the zebrafish brain.
    Percival P D'Gama, Nathalie Jurisch-Yaksi.
      Cilia are well conserved hair-like structures that have diverse sensory and motile functions. In the brain, motile ciliated cells, known as ependymal cells, line the cerebrospinal fluid (CSF) filled ventricles, where their beating contribute to fluid movement. Ependymal cells have gathered increasing interest since they are associated with hydrocephalus, a neurological condition with ventricular enlargement. In this article, we highlight methods to identify and characterize motile ciliated ependymal lineage in the brain of zebrafish using histological staining and transgenic reporter lines.
    Keywords:  Brain; Cilia; Ependymal cell; Glutamylated tubulin; Motile cilia; Multiciliation; Zebrafish
    DOI:  https://doi.org/10.1016/bs.mcb.2023.01.020
  18. Methods Cell Biol. 2023 ;pii: S0091-679X(22)00203-5. [Epub ahead of print]176 159-180
    Analysis of motility and mucociliary function of tracheal epithelial cilia.
    Tomoyuki Fujisawa, Yuko Tanaka, Koji Ikegami.
      The airway epithelium contains numerous multiciliated cells. The apical surface of multiciliated cells is covered with cilia that move at 15-25Hz. Ciliary movement is not a simple reciprocal movement and distinctly has forward and reverse movements called effective and recovery strokes, respectively. These "asymmetric" ciliary strokes push away the mucus covering the mucosa of the airway epithelium. Mucus flow created by ciliary stroke is important for capturing and expelling dust, pollen, PM2.5, pathogens, and other particles that enter the airways from outside the body. This mechanism for protecting the airways produced by ciliary movement is called mucociliary function. Defects in ciliary motility lead to impairment of mucociliary function, resulting in recurrent airway infections such as bronchitis and pneumonia, and consequently, bronchiectasis. While the analysis of ciliary beat frequency is relatively easy, the analyses of the amplitude, velocities of strokes, and the asymmetric level require specific techniques and tips. In this chapter, we present methods for the analysis of ciliary movements of a group of cilia on the luminal surface of the trachea ex vivo and individually isolated and ATP-reactivated cilia in vitro. In addition, a method for the analysis of mucociliary function is also presented.
    Keywords:  Asymmetric beating; Ciliary axoneme; Effective stroke; High-speed camera; Motile cilia; Mucociliary function; Mucociliary transport; Recovery stroke; Trachea
    DOI:  https://doi.org/10.1016/bs.mcb.2022.12.015
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