BACKGROUND: Cilia loss and impaired motile ciliary functions are one of the typical pathological features of chronic rhinosinusitis with nasal polyps (CRSwNP). Interleukin-17A (IL-17A) and interleukin-22 (IL-22) are the canonical cytokines of type 3 inflammation, exhibiting similar functional effects on epithelial cells. In this study, we sought to examine the effects of IL-17A and IL-22 on ciliated cells and investigate the potential involvement of Hippo-Yes-associated protein (YAP) signaling in their influence on ciliogenesis.
METHODS: We assessed both the mRNA and protein expression levels of IL-17A and IL-22 in nasal tissues obtained from patients with CRSwNP and compared them to those from healthy controls. To further explore the impact of IL-17A and IL-22, we established a primary human nasal epithelial cell (hNEC) model using different concentrations (2 ng/mL, 10 ng/mL, 50 ng/mL) for a duration of 28 days in an air-liquid interface (ALI) culture. Additionally, we employed the inhibitor verteporfin (VP) to investigate whether IL-17A andIL-22 exert their effects on ciliated cells via Hippo-YAP pathway.
RESULTS: The mRNA and protein levels of IL-17A and IL-22 in CRSwNP were significantly higher than those in healthy controls, revealing a robust correlation between IL-17A and IL-22. YAP was highly expressed in the nucleus of ciliated cells in CRSwNP and displayed a positive correlation with clinical symptoms. Both IL-17A and IL-22 were found to reduce the number of ciliated cells. IL-17A, but not IL-22, suppressed ciliogenesis by disrupting the proper development and docking of the basal body of ciliated cells, resulting in motile ciliary dysfunctions. Furthermore, the expression of YAP within the nucleus of ciliated cells gradually declined as these cells reached the final stage of differentiation. However, this process was obstructed by IL-17A only. YAP inhibitors, such as Verteporfin, markedly reversed the effects of IL-17A by increasing the proportion of ciliated cells, suppressing nuclear YAP expression in these cells, and enhancing ciliary beating frequency.
CONCLUSIONS: Both IL-17A and IL-22 are overexpressed in nasal epithelium of CRSwNP, which is associated with the impairment of epithelial cell differentiation. Furthermore, IL-17A has been shown to exert a disruptive effect on morphogenesis of motile cilia via activation of YAP.

The unicellular green alga, Chlamydomonas reinhardtii, has played a central role in discovering much of what is currently known about the composition, assembly, and function of cilia and flagella. Chlamydomonas combines excellent genetics, such as the ability to grow cells as haploids or diploids and to perform tetrad analysis, with an unparalleled ability to detach and isolate flagella in a single step without cell lysis. The combination of genetics and biochemistry that is possible in Chlamydomonas has allowed many of the key components of the cilium to be identified by looking for proteins that are missing in a defined mutant. Few if any other model organisms allow such a seamless combination of genetic and biochemical approaches. Other major advantages of Chlamydomonas compared to other systems include the ability to induce flagella to regenerate in a highly synchronous manner, allowing the kinetics of flagellar growth to be measured, and the ability of Chlamydomonas flagella to adhere to glass coverslips allowing Intraflagellar Transport to be easily imaged inside the flagella of living cells, with quantitative precision and single-molecule resolution. These advantages continue to work in favor of Chlamydomonas as a model system going forward, and are now augmented by extensive genomic resources, a knockout strain collection, and efficient CRISPR gene editing. While Chlamydomonas has obvious limitations for studying ciliary functions related to animal development or organ physiology, when it comes to studying the fundamental biology of cilia and flagella, Chlamydomonas is simply unmatched in terms of speed, efficiency, cost, and the variety of approaches that can be brought to bear on a question.

Cilia are hair-like projections that assemble at the surface of cells in various tissues of multicellular organisms through a complex cell biological process called ciliogenesis. Cilia can assemble as single structures per cell (i.e. non-motile primary cilia), which act as cell signaling centers that dictate cell fate, or can be assembled in distinct cell types as many copies per cell (i.e. motile cilia) that beat to move fluids at the cell surface. The mechanisms that orchestrate formation and function of cilia, which are dysregulated in pathological settings such as ciliopathies, remain incompletely understood. Stem cell-derived organoids represent valuable models to study the mechanisms of ciliogenesis, ciliary signaling, and ciliary beating that collectively promote tissue development and homeostasis. Here, we present a comprehensive protocol for the growth of mammary organoids derived from mouse mammary stem cells and for immunofluorescence staining of primary cilia in these three-dimensional structures.

Primary cilia (PC) are sensory organelles that function as cellular antennas, transmitting signals between the extracellular and intracellular spaces in many vertebrate tissues. The cell generates and assembles PC through a highly regulated process called ciliogenesis. This complex process is involved in several physiological functions, including embryonic development, locomotion, cell cycle regulation or energetic homeostasis control. In general, when a cell finishes its cell division, the oldest centriole usually migrates to the plasma membrane and becomes a basal body that gives rise to the formation of a cilium. For this reason, the presence of cilia is incompatible with cell division, so when a cell is going to divide, the cilium and the basal body disappear. Ciliogenesis is triggered by various stimuli, all of them related to cell cycle blockade. This cell cycle, and ciliogenesis induction, can be observed by: (1) the influence of growth factors (lack of serum and consequent inability to promote cell cycle exit and increase the proportion of cells in G0); (2) pharmacological cell cycle inhibitors (staurosporine or etoposide); or (3) physiological cell cycle inhibition (excessive contact between neighboring cells). Evaluation of ciliogenesis induction is vitally important for the study of diseases related to ciliary dysfunction, called ciliopathies. That is why the use of correct protocols for inducing cilia formation and an accurate posterior visualization of the cilia after performing said protocols are essential parts in the study of these diseases. To facilitate this task, here we described detailed protocols to induce ciliogenesis in vitro and visualize PC by immunofluorescence microscopy in cultured cells.

UNASSIGNED: Primary ciliary dyskinesia (PCD) is characterised by an imbalance in mucociliary clearance leading to chronic respiratory infections. Cilia length is considered to be a contributing factor in cilia movement. Recently, IFT46 protein has been related to cilia length. Therefore, this work aims to study IFT46 expression in a PCD patients cohort and analyse its relationship with cilia length and function, as it was not previously described.
UNASSIGNED: The expression of one intraflagellar transport (IFT46) and two regulating ciliary architecture (FOXJ1 and DNAI2) genes, as well as cilia length of 27 PCD patients, were measured. PCD patients were diagnosed based on clinical data, and cilia function and ultrastructure. Gene expression was estimated by real-time RT-PCR and cilia length by electron microscopy in nasal epithelium biopsies.Results and conclusions: While IFT46 expression was only diminished in patients with short cilia, FOXJ1, and DNAI2 expression were reduced in all PCD patient groups compared to controls levels. Among the PCD patients, cilia were short in 44% (5.9 ± 0.70 µm); nine of these (33% from the total) patients\' cilia also had an abnormal ultrastructure. Cilia length was normal in 33% of patients (6.4 ± 0.39 µm), and only three patients\' biopsies indicated decreased expression of dynein.

The primary cilium is a microtubule-based organelle that is considered to be a cellular antennae, because proteins related to multiple signaling pathways such as Wnt, PDGFRα, Hh, and mechanosignaling are localized to the membrane of the primary cilium. In the kidney, primary cilia extend from the cell membrane to the lumen of renal tubules to respond to fluidic stress. Recent studies have indicated that the disruption of ciliary proteins including polycystin-1 (PC1), polycystin-2 (PC2), and members of the intraflagellar transport (IFT) family induce the development of polycystic kidney disease (PKD), suggesting that the malformation or absence of primary cilia is a driving force of the onset of PKD. Therefore, in this chapter, the renal cystogenesis mechanism induced by cilia defects and pathogenic ciliary proteins associated with PKD development will be described.

BACKGROUND: Nearly all cells have a primary cilia on their surface, which functions as a cellular antennae. Primary cilia assembly begins intracellularly and eventually emerges extracellularly. However, current ciliogenesis assays, which detect cilia length and number, do not monitor ciliary stages.
METHODS: We developed a new assay that detects antibody access to a fluorescently tagged ciliary transmembrane protein, which revealed three ciliary states: classified as \'inside,\' \'outside,\' or \'partial\' cilia.
RESULTS: Strikingly, most cilia in RPE cells only partially emerged and many others were long and intracellular, which would be indistinguishable by conventional assays. Importantly, these states switch with starvation-induced ciliogenesis and the cilia can emerge both on the dorsal and ventral surface of the cell. Our assay further allows new molecular and functional studies of the \'ciliary pocket,\' a deep plasma membrane invagination whose function is unclear. Molecularly, we show colocalization of EHD1, Septin 9 and glutamylated tubulin with the ciliary pocket.
CONCLUSIONS: Together, the IN/OUT assay is not only a new tool for easy and quantifiable visualization of different ciliary stages, but also allows molecular characterization of intermediate ciliary states.