UNASSIGNED: This study aimed to investigate whether the impaired ciliary length and aberrant ciliary ultrastructure marker, dynein axonemal intermediate chain 1 (DNAI1), are important pathological characteristics in nasal mucosa from patients with allergic rhinitis (AR).
UNASSIGNED: Biopsies were taken from the inferior turbinate (IT) of controls (n = 20) and patients with AR (n = 20). The ciliary length and the DNAI1 location patterns were assessed by using immunofluorescent staining. Three patterns of DNAI1 localization were defined using a semi-quantitative scoring system: normal (N), partial (P) and absence (A). Every individual section was assigned a score between 0 and 2 in each high-power field (5 fields per sample). The score of 0 = pattern N >70%; 1 = patterns N + P >70%; and 2 = pattern A ≥30%. The receiver operating characteristic (ROC) curve was used to evaluate the predicted value of DNAI1 score for AR.
UNASSIGNED: The ciliary length was reduced by 33.3% in patients with AR compared with controls (P < 0.0001). The higher DNAI1 score was found in the AR group, with a median (first and third quartile) of 0.9 (0.4 and 1.08), which was 0.1 (0 and 0.76) in the control group (P = 0.0071). The ROC of DNAI1 was calculated based on the area under the curve of 0.74 (P = 0.0094). The cutoff value of ROC was 0.5833, with a sensitivity and specificity of 70%.
UNASSIGNED: These results suggested that the shorter ciliary length and aberrant localization of DNAI1 are potentially important pathological characteristics of the allergic nasal mucosa. The aberrant localization of DNAI1 may provide a novel candidate target for clinical management of AR.

Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including osteoarthritis. Matrix (or substrate) stiffness plays a major role in cell spreading, adhesion, proliferation, and differentiation. However, how specific cells sense substrate stiffness still remains unclear. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. We hypothesized that the primary cilium dynamically alters its length and position to fine-tune cell mechanosignaling based on substrate stiffness alone. We used a hydrogel system of varying substrate stiffness to examine the role of stiffness on cilia frequency, length, and centriole position as well as cell and nuclei area over time. Contrary to other cell types, we show that chondrocyte primary cilia shorten on softer substrates, demonstrating tissue-specific mechanosensing that is aligned with the tissue stiffness the cells originate from. We further show that stiffness determines centriole positioning to either the basal or apical membrane during attachment and spreading, with centrioles positioned toward the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibers in a time-dependent manner. Finally, we show on stiff substrates that primary cilia are involved in tension-mediated cell spreading. We propose that substrate stiffness plays a role in cilia positioning, regulating cellular responses to external forces, and maybe a key driver of mechanosignaling-associated diseases.

The left-right (LR) field recognizes the importance of the mechanism involving the calcium permeable channel Polycystin-2. However, whether the early LR symmetry breaking mechanism is exclusively via Polycystin-2 has not been tested. For that purpose, we need to be able to isolate the effects of decreasing the levels of Pkd2 protein from any eventual effects on flow dynamics. Here we demonstrate that curly-up (cup) homozygous mutants have abnormal flow dynamics. In addition, we performed one cell stage Pkd2 knockdowns and LR organizer specific Pkd2 knockdowns and observed that both techniques resulted in shorter cilia length and abnormal flow dynamics. We conclude that Pkd2 reduction leads to LR defects that cannot be assigned exclusively to its putative role in mediating mechanosensation because indirectly, by modifying cell shape or decreasing cilia length, Pkd2 deficit affects LR flow dynamics.

Primary cilia are microtubule-based organelles that are involved in sensing micro-environmental cues and regulating cellular homeostasis via triggering signaling cascades. Hypoxia is one of the most common environmental stresses that organ and tissue cells may often encounter during embryogenesis, cell differentiation, infection, inflammation, injury, cerebral and cardiac ischemia, or tumorigenesis. Although hypoxia has been reported to promote or inhibit primary ciliogenesis in different tissues or cultured cell lines, the role of hypoxia in ciliogenesis is controversial and still unclear. Here we investigated the primary cilia change under cobalt chloride (CoCl2)-simulated hypoxia in immortalized human retina pigment epithelial-1 (hTERT RPE-1) cells. We found CoCl2 treatment elongated primary cilia in a time- and dose-dependent manner. The prolonged cilia recovered back to near normal length when CoCl2 was washed out from the cell culture medium. Under CoCl2-simulated hypoxia, the protein expression levels of HIF-1/2α and acetylated-α-tubulin (cilia marker) were increased, while the protein expression level of Rabaptin-5 is decreased during hypoxia. Taken together, our results suggest that hypoxia may elongate primary cilia by downregulating Rabaptin-5 involved endocytosis. The coordination between endocytosis and ciliogenesis may be utilized by cells to adapt to hypoxia.

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 solitary organelle that organizes a sensitive signaling hub in a highly ordered microenvironment. Cilia are plastic structures, changing their length in response to bioactive substances, and ciliary length may be regulated to ensure efficient signaling capacity. Mammalian brain neurons possess primary cilia that are enriched in a set of G protein-coupled receptors (GPCRs), including the feeding-related melanin-concentrating hormone (MCH) receptor 1 (MCHR1). We previously demonstrated a novel biological phenomenon, ciliary MCHR1-mediated cilia length shortening through Gi/o and Akt signaling, using a simple cell culture model of human retinal pigmented epithelial RPE1 cells exogenously expressing MCHR1. In the present study, we characterized the properties of endogenous MCHR1-expressing primary cilia in hippocampal neurons in rodents. Using cultured dissociated rat hippocampal neurons in vitro, we showed that MCH triggered cilia length reduction involved in MCHR1-Gi/o and -Akt signaling. In rat hippocampal slice cultures with preservation of the cytoarchitecture and cell populations, ciliary MCHR1 was abundantly located in the CA1 and CA3 regions, but not in the dentate gyrus. Notably, treatment of slice cultures with MCH induced Gi/o- and Akt-dependent cilia shortening in the CA1 region without influencing cilia length in the CA3 region. Regarding the in vivo mouse brain, we observed higher levels of ciliary MCHR1 in the CA1 and CA3 regions as well as in slice cultures. In the starved state mice, a marked increase in MCH mRNA expression was detected in the lateral hypothalamus. Furthermore, MCHR1-positive cilia length in the hippocampal CA1 region was significantly shortened in fasted mice compared with fed mice. The present findings focused on the hippocampus provide a potential approach to investigate how MCHR1-driven cilia shortening regulates neuronal activity and physiological function toward feeding and memory tasks.

G-protein-coupled receptors (GPCRs) comprise the largest and most diverse cell surface receptor family, with more than 800 known GPCRs identified in the human genome. Binding of an extracellular cue to a GPCR results in intracellular G protein activation, after which a sequence of events, can be amplified and optimized by selective binding partners and downstream effectors in spatially discrete cellular environments. Because GPCRs are widely expressed in the body, they help to regulate an incredible range of physiological processes from sensation to growth to hormone responses. Indeed, it is estimated that ∼ 30% of all clinically approved drugs act by binding to GPCRs. The primary cilium is a sensory organelle composed of a microtubule axoneme that extends from the basal body. The ciliary membrane is highly enriched in specific signaling components, allowing the primary cilium to efficiently convey signaling cascades in a highly ordered microenvironment. Recent data demonstrated that a limited number of non-olfactory GPCRs, including somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1), are selectively localized to cilia on several mammalian cell types including neuronal cells. Utilizing cilia-specific cell biological and molecular biological approaches, evidence has accumulated to support the biological importance of ciliary GPCR signaling followed by cilia structural changes. Thus, cilia are now considered a unique sensory platform for integration of GPCR signaling toward juxtaposed cytoplasmic structures. Herein, we review ciliary GPCRs and focus on a novel role of MCHR1 in ciliary length control that will impact ciliary signaling capacity and neuronal function.

Primary cilia are singular, sensory organelles that extend from the plasma membrane of most quiescent mammalian cells. These slender, microtubule-based organelles receive and transduce extracellular cues and regulate signaling pathways. Primary cilia are critical to the development and function of many tissue types, and mutation of ciliary genes causes multi-system disorders, termed ciliopathies. Notably, renal cystic disease is one of the most common clinical features of ciliopathies, highlighting a central role for primary cilia in the kidney. Additionally, acute kidney injury and chronic kidney disease are associated with altered primary cilia lengths on renal epithelial cells, suggesting ciliary dynamics and renal physiology are linked. Here we describe methods to examine primary cilia in kidney tissue and in cultured renal cells. We include immunofluorescence and scanning electron microscopy to determine ciliary localization of proteins and cilia structure. Further, we detail cellular assays to measure cilia assembly and disassembly, which regulate cilia length.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK\'s kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

Previously, macroautophagy/autophagy was demonstrated to be regulated inter alia by the primary cilium. Mutations in RPGRIP1L cause ciliary dysfunctions resulting in severe human diseases summarized as ciliopathies. Recently, we showed that RPGRIP1L deficiency leads to a decreased proteasomal activity at the ciliary base in mice. Importantly, the drug-induced restoration of proteasomal activity does not rescue ciliary length alterations in the absence of RPGRIP1L indicating that RPGRIP1L affects ciliary function also via other mechanisms. Based on this knowledge, we analyzed autophagy in Rpgrip1l-negative mouse embryos. In these embryos, autophagic activity was decreased due to an increased activation of the MTOR complex 1 (MTORC1). Application of the MTORC1 inhibitor rapamycin rescued dysregulated MTORC1, autophagic activity and cilia length but not proteasomal activity in Rpgrip1l-deficient mouse embryonic fibroblasts demonstrating that RPGRIP1L seems to regulate autophagic and proteasomal activity independently from each other.