Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3T31A and ARL3T31A/C118F mutations.

ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3T31A , ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, and TGF-ß signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.

RABL2 promotes the outward transition zone passage of signaling proteins in cilia via ARL3.

Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Chlamydomonas Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that Chlamydomonas RABL2 in a GTP-bound form (RABL2GTP) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2GDP for activating ARL3GDP as an ARL3 guanine nucleotide exchange factor. This confers ARL3GTP to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2GDP exits cilia by being bound to the ARL3GTP/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by RABL2 mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.

Non-syndromic Retinal Degeneration Caused by Pathogenic Variants in Joubert Syndrome Genes.

Inherited retinal degenerations (IRDs) are a group of genetic disorders characterized by progressive dysfunction and loss of photoreceptors. IRDs are classified as non-syndromic or syndromic, depending on whether retinal degeneration manifests alone or in combination with other associated symptoms. Joubert syndrome (JBTS) is a genetically and clinically heterogeneous disorder affecting the central nervous system and other organs and tissues, including the neuroretina. To date, 39 genes have been associated with JBTS, a majority of which encode structural or functional components of the primary cilium, a specialized sensory organelle present in most post-mitotic cells, including photoreceptors. The use of whole exome and IRD panel next-generation sequencing in routine diagnostics of non-syndromic IRD cases led to the discovery of pathogenic variants in JBTS genes that cause photoreceptor loss without other syndromic features. Here, we recapitulate these findings, describing the JBTS gene defects leading to non-syndromic IRDs.

Human Mutations in Arl3, a Small GTPase Involved in Lipidated Cargo Delivery to the Cilia, Cause Retinal Dystrophy.

Photoreceptors are highly polarized sensory neurons. Precise localization of signaling molecules within the ciliary outer segment is critical for photoreceptor function and viability. The small GTPase Arl3 plays a particularly important role in photoreceptors as it regulates outer segment enrichment of lipidated proteins essential for the visual response: transducin-α, transducin-γ, PDEα, PDE ß, and Grk1. Recently, mutations in Arl3 have been identified in human patients with nonsyndromic autosomal recessive and dominant inherited retinal degenerations as well as syndromic Joubert syndrome including retinal dystrophy.

Ciliary ARL13B inhibits developmental kidney cystogenesis in mouse.

ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.

Ciliary ARL13B inhibits developmental kidney cystogenesis in mouse.

ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.

Disrupting the ciliary gradient of active Arl3 affects rod photoreceptor nuclear migration.

The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in increased active G-protein-Arl3-D67V has constitutive activity and Arl3-Y90C is fast cycling-and their expression in mouse rods resulted in a displaced nuclear phenotype due to an aberrant Arl3-GTP gradient. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that an Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development.

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

ARL3 and ARL13B GTPases participate in distinct steps of INPP5E targeting to the ciliary membrane.

INPP5E, a phosphoinositide 5-phosphatase, localizes on the ciliary membrane via its C-terminal prenyl moiety, and maintains the distinct ciliary phosphoinositide composition. The ARL3 GTPase contributes to the ciliary membrane localization of INPP5E by stimulating the release of PDE6D bound to prenylated INPP5E. Another GTPase, ARL13B, which is localized on the ciliary membrane, contributes to the ciliary membrane retention of INPP5E by directly binding to its ciliary targeting sequence. However, as ARL13B was shown to act as a guanine nucleotide exchange factor (GEF) for ARL3, it is also possible that ARL13B indirectly mediates the ciliary INPP5E localization via activating ARL3. We here show that INPP5E is delocalized from cilia in both ARL3-knockout (KO) and ARL13B-KO cells. However, some of the abnormal phenotypes were different between these KO cells, while others were found to be common, indicating the parallel roles of ARL3 and ARL13B, at least concerning some cellular functions. For several variants of ARL13B, their ability to interact with INPP5E, rather than their ability as an ARL3-GEF, was associated with whether they could rescue the ciliary localization of INPP5E in ARL13B-KO cells. These observations together indicate that ARL13B determines the ciliary localization of INPP5E, mainly by its direct binding to INPP5E.

Mechanism of Guanosine Triphosphate Hydrolysis by the Visual Proteins Arl3-RP2: Free Energy Reaction Profiles Computed with Ab Initio Type QM/MM Potentials.

We report the results of calculations of the Gibbs energy profiles of the guanosine triphosphate (GTP) hydrolysis by the Arl3-RP2 protein complex using molecular dynamics (MD) simulations with ab initio type QM/MM potentials. The chemical reaction of GTP hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) is catalyzed by GTPases, the enzymes, which are responsible for signal transduction in live cells. A small GTPase Arl3, catalyzing the GTP → GDP reaction in complex with the activating protein RP2, constitute an essential part of the human vision cycle. To simulate the reaction mechanism, a model system is constructed by motifs of the crystal structure of the Arl3-RP2 complexed with a substrate analog. After selection of reaction coordinates, energy profiles for elementary steps along the reaction pathway GTP + H2O → GDP + Pi are computed using the umbrella sampling and umbrella integration procedures. QM/MM MD calculations are carried out, interfacing the molecular dynamics program NAMD and the quantum chemistry program TeraChem. Ab initio type QM(DFT)/MM potentials are computed with atom-centered basis sets 6-31G** and two hybrid functionals (PBE0-D3 and ωB97x-D3) of the density functional theory, describing a large QM subsystem. Results of these simulations of the reaction mechanism are compared to those obtained with QM/MM calculations on the potential energy surface using a similar description of the QM part. We find that both approaches, QM/MM and QM/MM MD, support the mechanism of GTP hydrolysis by GTPases, according to which the catalytic glutamine side chain (Gln71, in this system) actively participates in the reaction. Both approaches distinguish two parts of the reaction: the cleavage of the phosphorus-oxygen bond in GTP coupled with the formation of Pi, and the enzyme regeneration. Newly performed QM/MM MD simulations confirmed the profile predicted in the QM/MM minimum energy calculations, called here the pathway-I, and corrected its relief at the first elementary step from the enzyme-substrate complex. The QM/MM MD simulations also revealed another mechanism at the part of enzyme regeneration leading to pathway-II. Pathway-II is more consistent with the experimental kinetic data of the wild-type complex Arl3-RP2, whereas pathway-I explains the role of the mutation Glu138Gly in RP2 slowing down the hydrolysis rate.

Autosomal Recessive Rod-Cone Dystrophy Associated With Compound Heterozygous Variants in ARL3 Gene.

Purpose: ARL3 (ADP-ribosylation factor-like 3) variants cause autosomal dominant retinitis pigmentosa (RP) or autosomal recessive Joubert syndrome. We found a family with rod-cone dystrophy (RCD) and verified it was associated with compound heterozygous variants in ARL3 gene. Methods: Ophthalmic examinations including optical coherence tomography and electroretinogram (ERG) were performed. Targeted next generation sequencing (NGS) was performed for the proband using a custom designed panel. Sanger sequencing and co-segregation were conducted in the family members. Changes of protein structure mediated by the variants were studied in vitro. ARL3 protein stability and its interaction with RP2 protein were assessed by cycloheximide chase assay and co-immunoprecipitation (Co-IP) assay. Results: Visual acuity of the 18-year-old male proband was 0.25 in the right and 0.20 in the left eye, while his non-consanguineous parents and sister was normal. The proband showed signs of RCD, including nyctalopia, peripheral field loss, bone-spicule deposits in the retina, and reduced ERG responses. The father, aged 50 years old, showed visual acuity of 1.0 in both eyes. Unlike the proband, he presented late onset and mild cone-rod dystrophy (CRD), including macular atrophy, central scotomata, moderate reduction in photopic ERG responses. None of all the family members had hearing abnormality, mental dysplasia or gait instability. We identified two novel compound heterozygous variants (c.91A>G, p.T31A; c.353G>T, p.C118F) in ARL3 in the proband, while his father only had variant c.91A>G. Bioinformatics analysis indicated amino acid positions of the two variants are highly conserved among species. The in silico tools predicted the variants to be harmful. Protein structure analysis showed the two variants had potential to alter the protein structure. Based on the ACMG guidelines, the two variants were likely pathogenic. In addition, the ARL3 mutations destabilized ARL3 protein, and the mutation c.353G>T disrupted the interaction between ARL3 and RP2 in HEK293T cells. Conclusions: We showed novel compound heterozygous variants in ARL3 were associated with early onset of autosomal recessive RCD, while c.91A>G along may be associated with a late onset of dominant CRD. The two variants in ARL3 could be causative by destabilizing ARL3 protein and impairing its interaction with RP2 protein.

ARL3, a small GTPase with a functionally conserved role in primary cilia and immune synapses.

The primary cilium and the immunological synapse are both specialized functional plasma membrane domains that share several similarities. Signalling output of membrane domains is regulated, spatially and temporally, by segregating and focusing lipids and proteins. ARL3, a small GTPase, plays a major role in concentrating lipid-modified proteins in both the immunological synapse and the primary cilia. Here in this review we will introduce the role of ARL3 in health and disease and its role in polarizing signalling at the primary cilia and immunological synapses.

Expression patterns of ciliopathy genes ARL3 and CEP120 reveal roles in multisystem development.

BACKGROUND: Joubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genes ARL3 and CEP120. METHODS: We sought to explore the developmental expression patterns of ARL3 and CEP120 in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characterise ARL3 and CEP120 expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource. RESULTS: Both ARL3 and CEP120 are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes. CONCLUSIONS: This study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development.

Flagellar targeting of an arginine kinase requires a conserved lipidated protein intraflagellar transport (LIFT) pathway in Trypanosoma brucei.

Both intraflagellar transport (IFT) and lipidated protein intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility, and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies have revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei, the causative agent of African sleeping sickness. TbArl13 catalyzes two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 has been identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting to and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacted with TbUnc119 in a GTP-dependent manner, suggesting functional specialization of Arl3-GTPases in T. brucei These results establish the function of TbUnc119 as a myristoylated cargo carrier and support the presence of a conserved LIFT pathway in T. brucei.

Diffuse or hitch a ride: how photoreceptor lipidated proteins get from here to there.

Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment (OS) where vision begins, an inner segment (IS) where protein synthesis occurs and a synaptic terminal for signal transmission to second-order neurons. The OS is a large, modified primary cilium attached to the IS by a slender connecting cilium (CC), the equivalent of the transition zone (TZ). Daily renewal of ~10% of the OS requires massive protein biosynthesis in the IS with reliable transport and targeting pathways. Transport of lipidated ('sticky') proteins depends on solubilization factors, phosphodiesterase δ (PDEδ) and uncoordinated protein-119 (UNC119), and the cargo dispensation factor (CDF), Arf-like protein 3-guanosine triphosphate (ARL3-GTP). As PDE6 and transducin still reside prominently in the OS of PDEδ and UNC119 germline knockout mice, respectively, we propose the existence of an alternate trafficking pathway, whereby lipidated proteins migrate in rhodopsin-containing vesicles of the secretory pathway.

The Dynamic and Complex Role of the Joubert Syndrome-Associated Ciliary Protein, ADP-Ribosylation Factor-Like GTPase 13B (ARL13B) in Photoreceptor Development and Maintenance.

Photoreceptor neurons are modified primary cilia with an extended ciliary compartment known as the outer segment (OS). The mechanism behind the elaboration of photoreceptor cilia and OS morphogenesis remains poorly understood. In this review, we discuss the role of ADP-ribosylation factor-like GTPase 13B (ARL13B), a small GTPase in OS morphogenesis and its impact on photoreceptor health and biology.

ARL3 is downregulated and acts as a prognostic biomarker in glioma.

BACKGROUND: Glioma is the most common primary malignant brain tumor in adults with a poor prognosis. ARL3 is a member of the ARF family, and plays a key role in ciliary function and lipid-modified protein trafficking. ARL3 has been reported to be involved in ciliary diseases, in which it affects kidney and photoreceptor development. However, the functional role of ARL3 in cancer remains unknown. In this study, we aimed to explore ARL3 expression and its roles in glioma prognosis. METHODS: RT-PCR and immunohistochemistry were performed to examine the expression level of ARL3 in glioma samples. Data from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA) and Repository for Molecular Brain Neoplasia Data (REMBRANDT) databases were employed to investigate ARL3 expression and its roles in glioma prognosis. A nomogram for predicting 3- or 5-year survival was established using Cox proportional hazards regression. Finally, gene ontology (GO) analysis, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were performed to explore the biological function. RESULTS: ARL3 expression was downregulated in glioma, and associated with poor prognosis in glioma patients. The C-indexes, areas under the ROC curve and calibration plots of the nomogram indicated an effective predictive performance for glioma patients. In addition, GO and pathway analyses suggested the involvement of ARL3 in angiogenesis and immune cell infiltration in the microenvironment. CONCLUSIONS: Low ARL3 expression predicted poor prognosis and contributed to antiangiogenesis and the proportion of infiltrating immune cells in the GBM microenvironment. Thus, ARL3 may be a prognostic marker and therapeutic target for glioma.

ARL3 Mutations Cause Joubert Syndrome by Disrupting Ciliary Protein Composition.

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

The Ciliary Machinery Is Repurposed for T Cell Immune Synapse Trafficking of LCK.

Upon engagement of the T cell receptor with an antigen-presenting cell, LCK initiates TCR signaling by phosphorylating its activation motifs. However, the mechanism of LCK activation specifically at the immune synapse is a major question. We show that phosphorylation of the LCK activating Y394, despite modestly increasing its catalytic rate, dramatically focuses LCK localization to the immune synapse. We describe a trafficking mechanism whereby UNC119A extracts membrane-bound LCK by sequestering the hydrophobic myristoyl group, followed by release at the target membrane under the control of the ciliary ARL3/ARL13B. The UNC119A N terminus acts as a "regulatory arm" by binding the LCK kinase domain, an interaction inhibited by LCK Y394 phosphorylation, thus together with the ARL3/ARL13B machinery ensuring immune synapse focusing of active LCK. We propose that the ciliary machinery has been repurposed by T cells to generate and maintain polarized segregation of signals such as activated LCK at the immune synapse.

ARL3 subcellular localization and its suspected role in autophagy.

ADP-ribosylation factor-like3 (ARL3) is a member of the ADP-ribosylation factor family of GTP-binding proteins that plays important role in regulating Ciliary trafficking. It ubiquitously expressed in normal tissues and tumor cell lines. However, the location and function of ARL3 in organelles are rarely known. In this study, we explored ARL3 subcellular localization in an all-round way in HEK293T, Neuro-2A and U251 cells by density gradient centrifugation and immunofluorescence. The results showed that ARL3 is expressed in most of organelles, and an iodixonal step gradient was further confirmed that ARL3 is mainly localized to the mitochondria, endosomes, lysosomes, and proteasome. By molecular functional analysis, we observed that ARL3 promotes the aggregation of GFP-LC3, up-regulation of LC3-II/LC3-I and down-regulation of SQSMT1/BECN1, and knocking down of ARL3 inbibits autophagy, which suggested that ARL3 is necessary for autophagy. this study presents a comprehensive evaluation of the subcellular localization for ARL3 and provides important on understanding the functions of ARL3.