Short Cilia Research Articles

The role of primary cilia in pulmonary hypertension

IntroductionVascular remodeling in pulmonary hypertension (PH) is characterized by endothelial dysfunction and smooth muscle cell proliferation and hypertrophy ultimately causing right heart failure and death. Up to now, the molecular mechanisms of PH pathogenesis have not been fully resolved, preventing the development of novel therapies against PH.Based on increased expression and activity levels in both patients and animal models with PH, respectively, PDGF‐BB, TGF‐β and mTOR signaling have been proposed to play critical roles as drivers of lung vascular remodeling. Among other effects, PDGF‐BB, TGF‐β and mTOR signaling cause shortening of the primary cilium – an antenna‐like organelle that functions as a flow‐sensor and signaling hub. Loss or shortening of cilia is characteristically associated with cell proliferation and promotes mTOR signaling, thus potentially establishing a positive feedback loop. Here, we hypothesized that PH is associated with a loss of primary cilia in pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs) which in turn drives cell proliferation and thus, remodeling in the pulmonary arterial wall.MethodsPulmonary artery tissue from PH‐patients and non‐PH donors was fixed and stained for acetylated α‐tubulin to determine the number of primary cilia. PAECs and PASMCs from PH patients and non‐PH donors were exposed in vitro to three different characteristic stimuli or mediators, respectively, of PH, namely PDGF‐BB, TGF‐β1, or hypoxia (1%). Cells were fixed and stained for measurement of primary cilia length. IFT88 ‐ an essential structural protein of the primary cilium ‐ was knocked down by siRNA in PAECs and PASMCs to assess the effect of cilium loss on migration and proliferation. Mice with an endothelial‐specific deletion of IFT88 (Cdh5‐CreERT2+, IFT88fl/fl) were housed for 5 weeks in normoxia or hypoxia (10% O2) to test for the role of endothelial primary cilia in the development of PH.ResultsIn pulmonary arteries of PH patients, the number of primary cilia per area was reduced by 80% as compared to healthy donor lungs. Analogously, PASMCs from PH patients had shorter cilia. Following IFT88 knock‐down, PAECs and PASMCs showed increased migration and proliferation as compared to control cells. In both PAECs and PASMCs, stimulation with PDGF‐BB, TGF‐β1, or hypoxia shortened primary cilia as compared to controls. Mice lacking primary cilia in endothelial cells showed a significant increase in right ventricular systolic pressure after 5 weeks of hypoxia as compared to control mice.ConclusionsHere, we demonstrate that PH is associated with a loss of cilia in vivo and reduced cilium length in vitro. Primary cilium loss promotes proliferation and migration of PAECs and PASMCs in vitro, as well as the development of PH in vivo. Cilium loss may as such be an important propagator of vascular remodeling in PH and may present a target for novel therapeutic interventions.

Research and Analysis on the Micro-Morphology of Hollow Alumina-Based Fibers Prepared by Using Reeds as Templates

Al2O3 fiber has high temperature resistance, chemical corrosion resistance, and low density. It is a good thermal insulation material. Reed is one of the most widely distributed angiosperms. The short cilia of the reed have a hollow structure, which can improve the thermal insulation performance of the fiber. Using reed fibers as a biological template, Al2O3 fibers with a hollow structure were prepared. After the reed fibers were immersed in the precursor solution, they were dried and sintered at a high temperature to obtain Al2O3 hollow fibers. The fibers replicated the hollow structure and the excellent continuity of the reed fibers.

Modeling Human Primary Microcephaly With hiPSC-Derived Brain Organoids Carrying CPAP-E1235V Disease-Associated Mutant Protein.

The centrosome is composed of a pair of centrioles and serves as the major microtubule-organizing center (MTOC) in cells. Centrosome dysfunction has been linked to autosomal recessive primary microcephaly (MCPH), which is a rare human neurodevelopmental disorder characterized by small brain size with intellectual disability. Recently, several mouse models carrying mutated genes encoding centrosomal proteins have been generated to address the genotype–phenotype relationships in MCPH. However, several human-specific features were not observed in the mouse models during brain development. Herein, we generated isogenic hiPSCs carrying the gene encoding centrosomal CPAP-E1235V mutant protein using the CRISPR-Cas9 genome editing system, and examined the phenotypic features of wild-type and mutant hiPSCs and their derived brain organoids. Our results showed that the CPAP-E1235V mutant perturbed the recruitment of several centriolar proteins involved in centriole elongation, including CEP120, CEP295, CENTROBIN, POC5, and POC1B, onto nascent centrioles, resulting in the production of short centrioles but long cilia. Importantly, our wild-type hiPSC-derived brain organoid recapitulated many cellular events seen in the developing human brain, including neuronal differentiation and cortical spatial lamination. Interestingly, hiPSC-CPAP-E1235V-derived brain organoids induced p53-dependent neuronal cell death, resulting in the production of smaller brain organoids that mimic the microcephaly phenotype. Furthermore, we observed that the CPAP-E1235V mutation altered the spindle orientation of neuronal progenitor cells and induced premature neuronal differentiation. In summary, we have shown that the hiPSC-derived brain organoid coupled with CRISPR/Cas9 gene editing technology can recapitulate the centrosome/centriole-associated MCPH pathological features. Possible mechanisms for MCPH with centriole/centrosome dysfunction are discussed.

Insights into the Regulation of Ciliary Disassembly.

The primary cilium, an antenna-like structure that protrudes out from the cell surface, is present in most cell types. It is a microtubule-based organelle that serves as a mega-signaling center and is important for sensing biochemical and mechanical signals to carry out various cellular processes such as proliferation, migration, differentiation, and many others. At any given time, cilia length is determined by a dynamic balance of cilia assembly and disassembly processes. Abnormally short or long cilia can cause a plethora of human diseases commonly referred to as ciliopathies, including, but not limited to, skeletal malformations, obesity, autosomal dominant polycystic kidney disease, retinal degeneration, and bardet-biedl syndrome. While the process of cilia assembly is studied extensively, the process of cilia disassembly and its biological role(s) are less well understood. This review discusses current knowledge on ciliary disassembly and how different cellular processes and molecular signals converge to carry out this process. This information will help us understand how the process of ciliary disassembly is regulated, identify the key steps that need further investigation, and possibly design therapeutic targets for a subset of ciliopathies that are causally linked to defective ciliary disassembly.

TRIP6 functions in brain ciliogenesis

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.

IFT88 deficiency in proximal tubular cells exaggerates cisplatin-induced injury by suppressing autophagy.

Primary cilia are widely regarded as specialized sensors in differentiated cells that have been implicated in the regulation of cell proliferation, differentiation, and viability. We have previously shown that shortening of primary cilia sensitizes cultured kidney tubular cells to cisplatin-induced apoptosis. Intraflagellar transport 88 (IFT88) is an essential component for ciliogenesis and maintenance. Here, we have further examined the effect of proximal tubule-specific IFT88 ablation on cisplatin-induced acute kidney injury (AKI). In this study, more severe AKI occurred in IFT88 knockout mice than age- and sex-matched wild-type mice. Mechanistically, cisplatin stimulated autophagy in kidney tubular cells as an intrinsic protective mechanism. However, renal autophagy was severely impaired in IFT88 knockout mice. In cultured HK-2 cells, cisplatin induced more apoptosis when IFT88 was knocked down. Tat-beclin 1 peptide, a specific autophagy activator, could partially prevent IFT88-associated cell death during cisplatin treatment, although cilium length was not improved significantly. Reexpression of IFT88 partially restored autophagy in IFT88 knockdown cells and suppressed apoptosis during cisplatin treatment. Taken together, these results indicate that defective autophagy in IFT88-deficient kidney cells and tissues contributes to the exaggerated AKI following cisplatin exposure.NEW & NOTEWORTHY Almost every cell has one hair-like, nonmotile antenna projecting from the cell surface, named the primary cilium. In kidney tubular cells, the primary cilium has a protective role, but the underlying mechanism is unclear. This study shows that a short cilium leads to the suppression of autophagy, which is responsible for the heightened injury sensitivity. These findings provide the clues of how to manipulate primary cilium and autophagy to save kidneys.

Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse.

Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1−/−, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species.

The role of the primary cilium in pulmonary arterial hypertension

Introduction Vascular remodeling in pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction and smooth muscle cell proliferation and hypertrophy ultimately causing right heart failure and death. Up to now, the molecular mechanisms of PAH pathogenesis have not been adequately resolved, preventing the development of novel therapies specifically targeting vascular remodeling in PAH. Based on increased expression levels of PDGF-BB and TGF-β in both patients and animal models with PAH, these signaling pathways have been proposed to play a critical role as drivers of lung vascular remodeling. Among multiple other effects, PDGF-BB and TGF-β cause shortening of the primary cilium – an antenna-like organelle functioning as a flow-sensor and signaling hub on most eukaryotic cells. Loss or shortening of cilia is characteristically associated with cell proliferation and promotes PDGF signaling, thus potentially establishing a positive feedback loop. We thus hypothesized that PAH is associated with a loss or shortening of primary cilia in pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs) which in turn drives cell proliferation and thus, remodeling in the pulmonary arterial wall. Methods Pulmonary artery tissue from PH-patients and non-PH donors was fixed and stained for acetylated α-tubulin to determine the number of primary cilia. PAECs and PASMCs from PAH patients and non-PAH donors were exposed in vitro to three different characteristic stimuli or mediators, respectively, of PAH, namely PDGF-BB, TGF-β1, or hypoxia (1%). Cells were fixed and stained for measurement of primary cilia length. In parallel, donor and PAH-SMCs were compared functionally in migration and proliferation assays. IFT88 - an essential structural protein of the primary cilium - was knocked down by siRNA in PAECs and PASMCs to assess the effect of cilium loss on migration and proliferation. Results In pulmonary arteries of PAH patients, the number of primary cilia per area was reduced by 80% as compared to donor lungs. PAH-SMCs were found to have shorter cilia and similar to IFT88 knock-down ECs and SMCs showed increased migration and proliferation as compared to healthy control cells. Additionally, primary cilia of PAECs, PASMCs, and PAH-SMCs stimulated with PDGF-BB, TGF-β1, or hypoxia were shorter as compared to controls. Lithium, which has been shown to elongate cilia in other cell types, was able to rescue cilia length and reduced migration and proliferation in PAH-SMCs. Conclusions Here, we demonstrate that PAH is associated with a loss of cilia in vivo and reduced cilium length in vitro. Primary cilium loss or shortening may promote progression of vascular remodeling as indicated by the fact that cilium loss causes proliferation/migration in PAH-SMCs, and their parallel reversal by lithium. Cilium loss may as such be an important propagator of vascular remodeling in PAH and may present a target for novel therapeutic interventions such as lithium.

POS-075 Decreased IFT88 causes cilia shortening and mitochondrial dysfunction in cisplatin-induced tubular injury

Renal primary cilia are associated with the pathogenesis of various diseases, including acute kidney injury (AKI). The length of the primary cilia dynamically change during the progression of diseases: tubular primary cilia shortened in cisplatin-induced AKI mouse model. However, its relevance in kidney disease and the underlying mechanism are largely unknown. Tubular damage in AKI closely links to mitochondrial dysfunction. Thus, we investigated the interaction between primary cilia and mitochondria in cisplatin-induced tubular injury. C57BL6 mice with cisplatin-induced AKI were euthanized at 72 h after disease induction to collect blood and kidney samples. In in vitro experiments, we used RPTEC/TERT1 cells, which are human proximal tubular epithelial cells that maintain the cilia length at high cell densities, and knocked down IFT88 (IFT88-KD), a cilia maintenance protein, by siRNA. Cisplatin-treated or IFT88-KD cells were assessed for the cellular phenotypic changes or mitochondrial metabolic function using the Flux Analyzer. We found that the expression of protein IFT88 was decreased in damaged tubules of cisplatin-induced AKI mice. These data were consistent with that IFT88 expression decreased in the cilia of cisplatin-treated RPTEC/TERT1 at mRNA and protein levels in association with shortening of the primary cilia, suggesting the pathogenic link between tubular damage and IFT88-mediated cilia alteration. Interestingly, IFT88-KD cells significantly exhibited shorter cilia as compared to control siRNA-transfected cells and showed downregulation of mitochondrial oxidative phosphorylation capacity and decreased ATP production, indicating the contribution of IFT88 to mitochondrial homeostasis. In addition, the knockdown of IFT88 decreases fatty acid oxidation (FAO) in the same direction as cisplatin treatment. Our findings suggest that tubular mitochondrial dysfunction in cisplatin-induced AKI is mediated by a decreased IFT88 with shortening cilia, at least in part. Tubular mitochondrial damage followed by tubular injury in AKI may occur by the alteration of IFT88 expression, and subsequent cilia shortening in the tubular epithelial cells.

CCP1, a Tubulin Deglutamylase, Increases Survival of Rodent Spinal Cord Neurons following Glutamate-Induced Excitotoxicity.

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Posttranslational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates posttranslational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knock-down of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in Caenorhabditis elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knock-down of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries.

Characterization of Primary Cilia in Osteoblasts Isolated From Patients With ADPKD and CKD.

ABSTRACTAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of chronic kidney disease (CKD) and leads to a specific type of bone disease. The primary cilium is a major cellular organelle implicated in the pathophysiology of ADPKD caused by mutations in polycystin‐1 (PKD1) and polycystin‐2 (PKD2). In this study, for the first time, cilia were characterized in primary preosteoblasts isolated from patients with ADPKD. All patients with ADPKD had low bone turnover and primary osteoblasts were also obtained from patients with non‐ADPKD CKD with low bone turnover. Image‐based immunofluorescence assays analyzed cilia using standard markers, pericentrin, and acetylated‐α‐tubulin, where cilia induction and elongation were chosen as relevant endpoints for these initial investigations. Osteoblastic activity was examined by measuring alkaline phosphatase levels and mineralized matrix deposition rates. It was found that primary cilia can be visualized in patient‐derived osteoblasts and respond to elongation treatments. Compared with control cells, ADPKD osteoblasts displayed abnormal cilia elongation that was significantly more responsive in cells with PKD2 nontruncating mutations and PKD1 mutations. In contrast, non‐ADPKD CKD osteoblasts were unresponsive and had shorter cilia. Finally, ADPKD osteoblasts showed increased rates of mineralized matrix deposition compared with non‐ADPKD CKD. This work represents the first study of cilia in primary human‐derived osteoblasts from patients with CKD and patients with ADPKD who have normal kidney function, offering new insights as bone disease phenotypes are not well recapitulated in animal models. These data support a model whereby altered cilia occurs in PKD‐mutated osteoblasts, and that ADPKD‐related defects in bone cell activity and mineralization are distinct from adynamic bone disease from patients with non‐ADPKD CKD. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

IFT46 Expression in the Nasal Mucosa of Primary Ciliary Dyskinesia Patients: Preliminary Study.

BackgroundPrimary 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.Materials and methodsThe 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.

RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP).

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

Photoreceptor progenitor dynamics in the zebrafish embryo retina and its modulation by primary cilia and N-cadherin.

Photoreceptor cells of the vertebrate neural retina originate in the neuroepithelium, and like other neurons, must undergo cell body translocation and polarity transitions to acquire their final functional morphology, which includes features of neuronal and epithelial cells. We analyzed this process in detail in zebrafish embryos using in vivo confocal microscopy and electron microscopy. Photoreceptor progenitors were labeled by the transgenic expression of enhanced green fluorescent protein under the regulation of the photoreceptor-specific promoter crx, and structures of interest were disrupted using morpholino oligomers to knock-down specific genes. Photoreceptor progenitors detached from the basal retina at pre-mitotic stages, rapidly retracting a short basal process as the cell body translocated apically. They remained at an apical position indefinitely to form the outer nuclear layer (ONL), initially extending and retracting highly dynamic neurite-like processes, tangential to the apical surface. Many photoreceptor progenitors presented a short apical primary cilium. The number and length of these cilia was gradually reduced until nearly disappearing around 60 hpf. Their disruption by knocking-down ift88 and elipsa caused a notorious defect on basal process retraction. To assess the role of cell adhesion in the organization of photoreceptor progenitors, we knocked-down cdh2/N-cadherin and observed the cell behavior by time-lapse microscopy. The ectopic photoreceptor progenitors initially migrated in an apparent random manner, profusely extending cell processes, until they encountered other cells to establish cell rosettes in which they stayed, acquiring photoreceptor-like polarity. Altogether, our observations indicate a complex regulation of photoreceptor progenitor dynamics to form the retinal ONL, previous to the post-mitotic maturation stages.

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome.

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.

RNAseq in fat pads from Alstrom syndrome 1 (ALMS1) Knockout rats support a role for ALMS1 in leptin release and fat metabolism in adipocytes.

Inactivating mutations in the ALMS1 gene in humans cause early onset obesity and metabolic syndrome. Deletion of ALMS1 in rodents induces early onset obesity and insulin resistance on normal chow. The mechanisms by which ALMS1 cause obesity are not clear but likely involve the development of leptin resistance since adult ALMS1 KO rats are hyperphagic and hyperleptinemic. It is not known whether ALMS1 controls leptin release and expression in adipocytes prior to development of obesity. ALMS1 has been associated to ciliary function which may play a role in adipogenesis. We hypothesized that deletion of ALMS1 would alter gene pathways involved in fat metabolism and cilia formation in young ALMS1 KO rats, which should contribute to hyperleptinemia. To study this, we obtained abdominal fat pads from young ALMS1 KO rats shortly after weaning (7 weeks) and prior to changes in body weight. We found that plasma leptin levels were higher in young ALMS1 KO rats (ALMS1: 3530±300 vs. WT: 172±16 pg/ml, p<0.05) despite no differences in body weight or baseline glucose. Leptin release (normalized to fat weight) was 120% higher (wild type: 30±8, ALMS1‐KO: 56± 15 pg/mg tissue/2h) in abdominal fat pads from ALMS1 KO rats. To study the genes involved in higher leptin release we performed RNAseq in freshly isolated abdominal fat pads. 12,860 genes were identified and quantified. In fat pads from ALMS1 KO there were 163 upregulated and 237 significantly downregulated genes. Leptin mRNA was significantly increased (1.68 Log2 fold, p<0.001, n=4) as were several genes involved in lipogenesis (Elovl6, Scd1, Scd2, Mogat2, Acly, Lpl, Dgat2, Fasn). Ingenuity pathway analysis indicated that the top canonical pathways impacted were: oleate biosynthesis, glycolysis I, hepatic fibrosis, and clathrin‐mediated endocytosis signaling. Importantly, at least 10 ciliary genes were significantly decreased (Traf3ip1, Pard6g, Cngb1, Ak7, Ttll1, Mlf1, Ccdc162, Dnah6, Ccdc187, Foxj1) suggesting a role for ALMS1 in ciliary function. To examine this, we immunolabeled cilia in abdominal adipocytes with gamma acetylated tubulin antibodies, and examined sections by confocal microscopy. Short cilia (1–2 μm) were observed in both WT and ALMS1 KO adipocytes. We concluded that ALMS1 is likely involved in leptin release from adipocytes. It is not clear whether increased leptin expression and release is secondary to neuroendocrine alterations or alterations in cilia‐induced signaling. Our data also point to an important role for ALMS1 in regulating adipocyte function and leptin physiology.Support or Funding InformationHenry Ford Hospital Research Fund

ARL13B regulates Sonic hedgehog signaling from outside primary cilia.

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B's role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

Diverged morphology changes of astrocytic and neuronal primary cilia under reactive insults

Primary cilia are centriole-derived sensory organelles that are present in most mammalian cells, including astrocytes and neurons. Evidence is emerging that astrocyte and neuronal primary cilia demonstrate a dichotomy in the mature mouse brain. However, it is unknown how astrocytic and neuronal primary cilia change their morphology and ciliary proteins when exposed to reactive insults including epilepsy and traumatic brain injury. We used a double transgenic mouse strain (Arl13b-mCherry; Centrin2-GFP), in which we found spontaneous seizures, and a cortical injury model to examine the morphological changes of astrocytic and neuronal primary cilia under reactive conditions. Transgenic overexpression of Arl13b drastically increases the length of astrocytic and neuronal primary cilia in the hippocampus, as well as the cilia lengths of cultured astrocytes and neurons. Spontaneous seizures shorten Arl13b-positive astrocytic cilia and AC3-positive neuronal cilia in the hippocampus. In a cortical injury model, Arl13b is not detectable in primary cilia, but Arl13b protein relocates to the cell body and has robust expression in the proximity of injured tissues. In contrast, the number of AC3-positive cilia near injured tissues remains unchanged, but their lengths become shorter. These results on astrocytic cilia implicate Arl13b in regulating astrocyte proliferation and tissue regeneration, while the shortening of AC3-positive cilia suggests adaptive changes of neuronal primary cilia under excitotoxicity.

Reorganization of complex ciliary flows around regenerating Stentor coeruleus.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such as Chlamydomonas. Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliate Stentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring in Stentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures.This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.

Primary cilia and the exocyst are linked to urinary extracellular vesicle production and content

The recently proposed idea of "urocrine signaling" hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.