Ciliary Morphology Research Articles

Role of Valosin-Containing Protein (VCP) in Ciliary Morphology and ADPKD: Exploring VCP as a Novel Genetic Interactor in Disease Progression

Role of Valosin-Containing Protein (VCP) in Ciliary Morphology and ADPKD: Exploring VCP as a Novel Genetic Interactor in Disease Progression

Comparison of rotational stability of the Implantable Collamer Lens after using a vertical or horizontal implanting orientation.

To compare rotational stability of the Implantable Collamer Lens (ICL) between horizontal and vertical implantation. Zhongshan Ophthalmic Center, China. Prospective 1:1 matched design. 94 cases (185 eyes with a vertical elliptical ciliary sulcus) were included with a 1:1 matched design based on ciliary sulcus morphology, preset deviation angle, and vault. Follow-ups at 4 days, 1 month, 3 months, and 6 months post-surgery measured rotational angles using slit-lamp photography. Latent class trajectory modeling was employed to investigate the postoperative rotational angle trajectories. Six months after surgery, both groups exhibited similar visual acuity and refractive outcomes. The horizontal group had a significantly greater rotation angle than the vertical group (F group = 13.638, P < 0.001). Additionally, a statistically significant difference (P = 0.004) in the average trajectories of rotational angles was observed. The vertical group displayed a greater presence in the low-stable trajectory subgroup while demonstrating a reduced presence in the moderate-increase and high-fluctuation trajectory subgroups compared to the horizontal group. The horizontal group had a 3.750 times higher risk of rotation angle ≥3° compared to the vertical group, which represented a statistically significant difference (95% CI: 1.346∼10.446). In both groups, a positive correlation between the preset deviation angle and the rotation angle was observed, with correlation coefficients of 0.320 (P = 0.030) and 0.371 (P = 0.011), respectively. Vertical ICL implantation showed better rotational stability than horizontal implantation in eyes with a vertical elliptical ciliary sulcus, offering guidance for ICL surgery.

Rotational Stability, Footplate Position, and Visual Outcomes of Toric Implantable Collamer Lenses in Eyes With Low Vault.

To evaluate the clinical outcomes, rotational stability, and footplate position of the toric Implantable Collamer Lens (TICL) (STAAR Surgical) in eyes with low vault and analyze factors related to rotational stability. This prospective observational study included 59 eyes of 59 patients with insufficient vault (< 250 µm). Postoperative rotation was defined as the difference between the achieved angle and the intraoperative fixation angle, and assessed with a digital anterior segment photograph after full mydriasis at 1 week and 1, 3, and 6 months postoperatively. Ultrasound biomicroscopy was used to determine the ciliary body morphology and position of the footplate. Correlation analysis was employed to identify the risk factors associated with TICL rotation at 6 months postoperatively. At 6 months postoperatively, the mean central vault was 137.4 ± 61.0 µm (range: 40 to 236 µm), and the mean efficacy and safety indices were 1.04 and 1.15, respectively. The mean manifest refractive astigmatism decreased from -1.67 ± 0.82 diopters (D) preoperatively to -0.43 ± 0.33 D postoperatively, and the mean absolute rotation was 4.50 ± 3.08 degrees (range: 0 to 12.50 degrees). The angle of rotation was correlated with the preoperative spherical power (r = -0.318, P = .014), the average value of TICL footplates position (r = 0.284, P = .029), and postoperative astigmatism (r = -.469, P⩽ .001). TICL implantation is predictable, safe, and effective in correcting myopic astigmatism in eyes with low vault. The rotational stability was acceptable and related to the malposition of the footplate and preoperative spherical power. [J Refract Surg. 2024;40(7):e460-e467.].

GDF15 controls primary cilia morphology and function thereby affecting progenitor proliferation.

We recently reported that growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous sonic hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, Gdf15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors.

Relating ciliary propulsion morphology and flow to particle acquisition in marine planktonic ciliates I: the tintinnid ciliate Amphorides quadrilineata

Abstract The marine tintinnid ciliate Amphorides quadrilineata is a feeding-current feeder, creating flows for particle encounter, capture and rejection. Individual-level behaviors were observed using high-speed, high-magnification digital imaging. Cells beat their cilia backward to swim forward, simultaneously generating a feeding current that brings in particles. These particles are then individually captured through localized ciliary reversals. When swimming backward, cells beat their cilia forward (=ciliary reversals involving the entire ring of cilia), actively rejecting unwanted particles. Cells achieve path-averaged speeds averaging 3–4 total lengths per second. Both micro-particle image velocimetry and computational fluid dynamics were employed to characterize the cell-scale flows. Forward swimming generates a feeding current, a saddle flow vector field in front of the cell, whereas backward swimming creates an inverse saddle flow vector field behind the cell; these ciliary flows facilitate particle encounter, capture and rejection. The model-tintinnid with a full-length lorica achieves an encounter rate Q ~29% higher than that without a lorica, albeit at a ~142% increase in mechanical power and a decrease in quasi-propulsive efficiency (~0.24 vs. ~ 0.38). It is also suggested that Q can be approximated by π(W/2 + l)2U, where W, l and U represent the lorica oral diameter, ciliary length and swimming speed, respectively.

Engrailed 1 deficiency induces changes in ciliogenesis during human neuronal differentiation

A key pathological feature of Parkinson’s Disease (PD) is the progressive degeneration of dopaminergic neurons (DAns) in the substantia nigra pars compacta. Considering the major role of EN1 in the development and maintenance of these DAns and the implications from En1 mouse models, it is highly interesting to study the molecular and protective effect of EN1 also in a human cellular model. Therefore, we generated EN1 knock-out (ko) human induced pluripotent stem cell (hiPSCs) lines and analyzed these during neuronal differentiation. Although the EN1 ko didn’t interfere with neuronal differentiation and generation of tyrosine hydroxylase positive (TH+) neurons per se, the neurons exhibited shorter neurites. Furthermore, mitochondrial respiration, as well as mitochondrial complex I abundance was significantly reduced in fully differentiated neurons. To understand the implications of an EN1 ko during differentiation, we performed a transcriptome analysis of human neuronal precursor cells (hNPCs) which unveiled alterations in cilia-associated pathways. Further analysis of ciliary morphology revealed an elongation of primary cilia in EN1-deficient hNPCs. Besides, also Wnt signaling pathways were severely affected. Upon stimulating hNPCs with Wnt which drastically increased EN1 expression in WT lines, the phenotypes concerning mitochondrial function and cilia were exacerbated in EN1 ko hNPCs. They failed to enhance the expression of the complex I subunits NDUFS1 and 3, and now displayed a reduced mitochondrial respiration. Furthermore, Wnt stimulation decreased ciliogenesis in EN1 ko hNPCs but increased ciliary length even further. This further highlights the relevance of primary cilia next to mitochondria for the functionality and correct maintenance of human DAns and provides new possibilities to establish neuroprotective therapies for PD.

IFT27 regulates the long-term maintenance of photoreceptor outer segments in zebrafish

Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27′s integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.

Evaluation of Ciliary Body Morphology and Position of Implantable Collamer Lens in Low-Vault Eyes Using Ultrasound Biomicroscopy.

To investigate the ciliary body anatomy and position of the implantable collamer lens (ICL) in low-vault eyes and analyze factors related to insufficient vault. Zhongshan Ophthalmic Center, Guangzhou, China. Retrospective case-control observational study. In this study, 73 eyes of 73 patients with insufficient vault (＜250 µm) were matched with 73 eyes with an ideal vault (250 to 750 µm). Ultrasound biomicroscopy (UBM) was used to determine the ciliary body morphology and ICL position. The biometric parameters acquired by Scheimpflug tomography were compared. The correlation between the vault and these factors was analyzed, and the Least Absolute Shrinkage and Selection Operator (LASSO) method was used to screen the risk factors for low-vault. The low-vault group had a steeper corneal curvature, thicker lens thickness (LT), higher crystalline lens rise, and shorter axial length (AL) (All P <.005). The ciliary process length (CPL) and maximum ciliary body thickness (CBTmax) were significantly smaller, and the trabecular-ciliary angle (TCA), iris-ciliary angle (ICA), and ciliary sulus width (CSW) were significantly greater in the low-vault eyes. (All P <.005). The low-vault group had more ICL haptics below the ciliary process, and TCA, ICA, CPL, CBTmax, CSW, and haptic position were related to the postoperative vault (All P <.05). CPL, AL, and LT were identified as predictors of a low-vault. Malposition of ICL haptics behind the ciliary process is a risk factor for low-vault. A shorter CPL, thicker LT, and shorter AL are significant risk factors for postoperative low-vault.

IK is essentially involved in ciliogenesis as an upstream regulator of oral-facial-digital syndrome ciliopathy gene, ofd1

BackgroundThe cilia are microtubule-based organelles that protrude from the cell surface. Abnormalities in cilia result in various ciliopathies, including polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS), and oral-facial-digital syndrome type I (OFD1), which show genetic defects associated with cilia formation. Although an increasing number of human diseases is attributed to ciliary defects, the functions or regulatory mechanisms of several ciliopathy genes remain unclear. Because multi ciliated cells (MCCs) are especially deep in vivo, studying ciliogenesis is challenging. Here, we demonstrate that ik is essential for ciliogenesis in vivo.ResultsIn the absence of ik, zebrafish embryos showed various ciliopathy phenotypes, such as body curvature, abnormal otoliths, and cyst formation in the kidney. RNA sequencing analysis revealed that ik positively regulated ofd1 expression required for cilium assembly. In fact, depletion of ik resulted in the downregulation of ofd1 expression with ciliary defects, and these ciliary defects in ik mutants were rescued by restoring ofd1 expression. Interestingly, ik affected ciliogenesis particularly in the proximal tubule but not in the distal tubule in the kidney.ConclusionsThis study demonstrates the role of ik in ciliogenesis in vivo for the first time. Loss of ik in zebrafish embryos displays various ciliopathy phenotypes with abnormal ciliary morphology in ciliary tissues. Our findings on the ik–ofd1 axis provide new insights into the biological function of ik in clinical ciliopathy studies in humans.

Improvement in the ideal range of vault after implantable collamer lens implantation: a new vault prediction formula.

To derive and validate a novel vault prediction formula to improve the predictability and safety of implantable collamer lens (ICL) implantation. Thirty-five patients (61 eyes) with previous posterior chamber intraocular lens implantation were included. Various parameters, such as horizontal-visible iris diameter (HVID), photopic pupil diameter (PPD), axial length (AL), white-to-white (WTW), anterior chamber width (ACW), angle-to-angle (ATA), crystalline lens rise (CLR), anterior chamber depth (ACD), horizontal sulcus-to-sulcus (HSTS), and ciliary sulcus angle (CSA) were measured. Vault was measured at 3 months after surgery using CASIA2 anterior segment optical coherence tomography. The formula was derived using multiple linear regression analysis and named as WH formula. It was validated in 65 patients (118 eyes) to determine the percentage of the ideal postoperative vault range and to compare the differences between the WH formula and the NK, KS, and STAAR formulas. Final ICL size, ATA, CSA, and CLR were included in the prediction formula model (adjusted R2 = 0.67, p < 0.001). The achieved vault 1 month after the surgery was 556.19 μm ± 166.98 μm in the validation group, and the ideal vault range was 200-800 μm (92%). The difference between the achieved vault and that predicted using the WH formula was not statistically significant (p = 0.165), whereas the difference between the achieved vault and that predicted using the NK and KS formulas was statistically significant (p < 0.001 and p < 0.001, respectively). The 95% agreement limit range of the achieved vault and the vault predicted using the WH formula was narrower than those predicted using the NK and KS formulas (-295.20-258.82 μm). This study combined the results of optical coherence tomography and ultrasound biomicroscopy measurements of the anterior segment of the eye and incorporated ciliary sulcus morphology quantification into the prediction formula. The study derived a prediction formula for vault by combining ICL size, ATA, and CLR. The derived formula was found to be superior to the currently available formulas.

Ciliary rootlet morphology shows distinct pattern in each region of mouse brain

Ciliary rootlet morphology shows distinct pattern in each region of mouse brain

Evaluation of ciliary-GPCR dynamics using a validated organotypic brain slice culture method.

The primary cilium is a structural organelle present in most mammalian cells. Primary cilia are enriched with a unique protein repertoire distinct from that of the cytosol and the plasma membrane. Such a highly organized microenvironment creates effective machinery for translating extracellular cues into intracellular signals. G protein-coupled receptors (GPCRs) are key receptors in sensing environmental stimuli transmitted via a second messenger into a cellular response. Recent data has demonstrated that a limited number of non-olfactory GPCRs, including melanin-concentrating hormone receptor 1 (MCHR1), are preferentially localized to ciliary membranes of several mammalian cell types, including neuronal cells. Evidence was accumulated to support the functional importance of ciliary-GPCR signaling accompanying ciliary structural changes using cilia-specific cell and molecular biology techniques. Thus, cilia are now considered to function as a unique sensory platform for the integration of GPCR signaling and various cytoplasmic domains. Dissociated neurons expressing ciliary-GPCRs can be a useful tool for examining ciliary dynamics. However, losing preexisting neuronal connectivity may alter neuronal ciliary morphology, such as abnormal elongation. Brain slices prepared under ex vitro conditions are a powerful approach that maintains the cytoarchitecture, enabling researchers to have accurate control over experimental conditions and to study individual cells from subregions of the brain. Here, we present a detailed description of our novel modified method for organotypic culture of rat brain slice and a validated immunostaining protocol to characterize ciliary-GPCR dynamics in coupling with neuropeptides or aminergic activation.

A WDR47 homolog facilitates ciliogenesis by modulating intraflagellar transport.

Cilia are conserved organelles found in many cell types in eukaryotes, and their dysfunction causes defects in environmental sensing and signaling transduction; such defects are termed ciliopathies. Distinct cilia have cell-specific morphologies and exert distinct functions. However, the underlying mechanisms of cell-specific ciliogenesis and regulation are unclear. Here, we identified a WD40-repeat (WDR) protein, NMTN-1 (the homolog of mammalian WDR47), and show that it is specifically required for ciliogenesis of AWB chemosensory neurons in C. elegans. NMTN-1 is expressed in the AWB chemosensory neuron pair, and is enriched at the basal body (BB) of the AWB cilia. Knockout of nmtn-1 causes abnormal AWB neuron cilia morphology, structural integrity, and induces aberrant AWB-mediated aversive behaviors. We further demonstrate that nmtn-1 deletion affects movement of intraflagellar transport (IFT) particles and their cargo delivery in AWB neurons. Our results indicate that NMTN-1 is essential for AWB neuron ciliary morphology and function, which reveal a novel mechanism for cell-specific ciliogenesis. Given that WDR47/NMTN-1 is conserved in mammals, our findings may help understanding of the process of cell-specific ciliogenesis and provide insights for treating ciliopathies.

Performance of the Deep Neural Network Ciloctunet, Integrated with Open-Source Software for Ciliary Muscle Segmentation in Anterior Segment OCT Images, Is on Par with Experienced Examiners.

Anterior segment optical coherence tomography (AS-OCT), being non-invasive and well-tolerated, is the method of choice for an in vivo investigation of ciliary muscle morphology and function. The analysis requires the segmentation of the ciliary muscle, which is, when performed manually, both time-consuming and prone to examiner bias. Here, we present a convolutional neural network trained for the automatic segmentation of the ciliary muscle in AS-OCT images. Ciloctunet is based on the Freiburg U-net and was trained and validated using 1244 manually segmented OCT images from two previous studies. An accuracy of 97.5% for the validation dataset was achieved. Ciloctunet's performance was evaluated by replicating the findings of a third study with 180 images as the test data. The replication demonstrated that Ciloctunet performed on par with two experienced examiners. The intersection-over-union index (0.84) of the ciliary muscle thickness profiles between Ciloctunet and an experienced examiner was the same as between the two examiners. The mean absolute error between the ciliary muscle thickness profiles of Ciloctunet and the two examiners (35.16 µm and 45.86 µm) was comparable to the one between the examiners (34.99 µm). A statistically significant effect of the segmentation type on the derived biometric parameters was found for the ciliary muscle area but not for the selective thickness reading ("perpendicular axis"). Both the inter-rater and the intra-rater reliability of Ciloctunet were good to excellent. Ciloctunet avoids time-consuming manual segmentation, thus enabling the analysis of large numbers of images of ample study cohorts while avoiding possible examiner biases. Ciloctunet is available as open-source.

Fluorescence imaging of beta cell primary cilia.

Primary cilia are slender cell-surface organelles that project into the intercellular space. In pancreatic beta cells, primary cilia coordinate a variety of cell responses including GPCR signaling, calcium influx, and insulin secretion, along with likely many underappreciated roles in islet development and differentiation. To study cilia function in islet biology, direct visualization of primary cilia by microscopic methods is often a necessary first step. Ciliary abundance, distribution, and morphology are heterogeneous among islet cells and are best visualized by fluorescence microscopy, the tools for which are readily accessible to most researchers. Here we present a collection of fluorescence imaging methods that we have adopted and optimized for the observation of primary cilia in mouse and human islets. These include conventional confocal microscopy using fixed islets and pancreas sections, live-cell imaging with cilia-targeted biosensors and probes, cilia motion recordings, and quantitative analysis of primary cilia waveform in the ex vivo environment. We discuss practical considerations and limitations of our approaches as well as new tools on the horizon to facilitate the observation of primary cilia in pancreatic islets.

Mechanical analysis of the impact of the morphology of the iris and ciliary body on the central vault after posterior chamber phakic intraocular lens implantation

Objective: To investigate the impact of the iris and ciliary body morphology on the central vault after phakic posterior chamber intraocular lens (pIOL) implantation. Methods: This research was based on the retrospective analysis of 123 patients (123 eyes) who underwent pIOL implantation in the Department of Ophthalmology, Affiliated Hospital of Qingdao University between June 2018 and June 2020. The anterior segment structure was observed with an ultrasound biomicroscope before surgery, and all morphological parameters of the iris and ciliary body were measured manually using the ImageJ software, including iris span (IS), iris convexity (IC), iris-ciliary body contact distance (ICCD) and iris-lens contact distance (ILCD). The ICCD was divided into Q1, Q2 and Q3 groups according to the equidistant distance of 0.36 mm. The lens thickness was measured with the IOLMaster. The horizontal corneal diameter and anterior chamber depth were measured using the Pentacam. The central vaults were measured by optical coherence tomography at 1 week, 3 months and 1 year after surgery. The relationships between vault values and preoperative parameters of the anterior segment were evaluated using the Pearson correlation analysis, Spearman correlation analysis, and multiple linear regression. The repeated measures ANOVA was applied to identify changes of vault values over time. Results: The mean values of the vaults at 1 week, 3 months and 1 year after surgery were (723±265) μm, (642±255) μm and (613±280) μm, respectively. The difference among them was statistically significant (F=50.143, P<0.001). The vaults continued to decline within 1 year after pIOL implantation, and the total decline was (122±86) μm. The vaults declined by (69±98) μm from postoperative 1 week to 3 months and by (52±54) μm from postoperative 3 months to 1 year. The regression formula showed that the pIOL size and ILCD were positively related with the vault, while the LT, IS and IC were negatively related with the vault at 1 week postoperatively (adjusted R²=0.404, P<0.001). The pIOL size and ILCD were positively related with the vault, while the IS and IC were negatively related with the vault at 3 months postoperatively (adjusted R²=0.342, P<0.001). The pIOL size was positively related with the vault, while the IS and IC were negatively related with the vault at 1 year postoperatively (adjusted R²=0.661, P<0.001). The vault values were higher in group Q3 compared to group Q1 at every timepoint, and the vault value was higher in group Q2 compared to Q1 at 1 year postoperatively. Conclusions: In the early postoperative period, eyes with a larger pIOL, shorter iris span, longer contact distance between the iris and ciliary body, and longer contact distance between the iris and lens were associated with a higher rate of excessive vaults. Meanwhile, eyes with a thicker lens and larger iris reverse convexity were more likely to obtain insufficient vaults. Within one year after surgery, the pIOL size, IS, IC and ICCD continued to impact on the vault. The ICCD, ILCD and IC can reflect the posterior chamber volume and change the haptic location and force, thus affecting the vault.

Prominin 1 and Notch regulate ciliary length and dynamics in multiciliated cells of the airway epithelium

SummaryDifferences in ciliary morphology and dynamics among multiciliated cells of the respiratory tract contribute to efficient mucociliary clearance. Nevertheless, little is known about how these phenotypic differences are established. We show that Prominin 1 (Prom1), a transmembrane protein widely used as stem cell marker, is crucial to this process. During airway differentiation, Prom1 becomes restricted to multiciliated cells, where it is expressed at distinct levels along the proximal-distal axis of the airways. Prom1 is induced by Notch in multiciliated cells, and Notch inactivation abolishes this gradient of expression. Prom1 was not required for multicilia formation, but when inactivated resulted in longer cilia that beat at a lower frequency. Disruption of Notch resulted in opposite effects and suggested that Notch fine-tunes Prom1 levels to regulate the multiciliated cell phenotype and generate diversity among these cells. This mechanism could contribute to the innate defense of the lung and help prevent pulmonary disease.

Assessment of in vitro kinetics and biological impact of nebulized trehalose on human bronchial epithelium

Trehalose is added in drug formulations to act as fillers or improve aerosolization performance. Its characteristics as a carrier molecule have been explored; however, the fate of trehalose in human airway tissues has not been thoroughly investigated. Here, we investigated the fate of nebulized trehalose using in vitro human air-liquid bronchial epithelial cultures. First, a tracing experiment was conducted using 13C12-trehalose; we measured trehalose distribution in different culture compartments (apical surface liquid, epithelial culture, and basal side medium) at various time points following acute exposure to 13C12-labeled trehalose. We found that 13C12-trehalose was metabolized into 13C6-glucose. The data was then used to model the kinetics of trehalose disappearance from the apical surface of bronchial cultures. Secondly, we evaluated the potential adverse effects of nebulized trehalose on the bronchial cultures after they were acutely exposed to nebulized trehalose up to a level just below its solubility limit (50 g/100 g water). We assessed the ciliary beating frequency and histological characteristics. We found that nebulized trehalose did not lead to marked alteration in ciliary beating frequency and morphology of the epithelial cultures. The in vitro testing approach used here may enable the early selection of excipients for future development of inhalation products.

Roles for ELMOD2 and Rootletin in ciliogenesis.

ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway supports a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

CiliaQ: a simple, open-source software for automated quantification of ciliary morphology and fluorescence in 2D, 3D, and 4D images

Cilia are hair-like membrane protrusions that emanate from the surface of most vertebrate cells and are classified into motile and primary cilia. Motile cilia move fluid flow or propel cells, while also fulfill sensory functions. Primary cilia are immotile and act as a cellular antenna, translating environmental cues into cellular responses. Ciliary dysfunction leads to severe diseases, commonly termed ciliopathies. The molecular details underlying ciliopathies and ciliary function are, however, not well understood. Since cilia are small subcellular compartments, imaging-based approaches have been used to study them. However, tools to comprehensively analyze images are lacking. Automatic analysis approaches require commercial software and are limited to 2D analysis and only a few parameters. The widely used manual analysis approaches are time consuming, user-biased, and difficult to compare. Here, we present CiliaQ, a package of open-source, freely available, and easy-to-use ImageJ plugins. CiliaQ allows high-throughput analysis of 2D and 3D, static or time-lapse images from fluorescence microscopy of cilia in cell culture or tissues, and outputs a comprehensive list of parameters for ciliary morphology, length, bending, orientation, and fluorescence intensity, making it broadly applicable. We envision CiliaQ as a resource and platform for reproducible and comprehensive analysis of ciliary function in health and disease.Graphic abstract