Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins

Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro and determining the localization and dynamics of Arl13b within the cilium. Previously, we showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study, we show the dynamics of Shh signaling component localization to the cilium are disrupted in the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant embryos may result from defects in protein localization and distribution within the cilium.     

Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation

Arl13b is an atypical Arf/Arl-family GTPase consisting of an extending large C-terminal region (C domain) and Arf-homologous GTP-binding motifs in the N terminus (N domain). Although Arl13b appears to be involved in cilia formation, its precise function and roles of the domains remain unknown. Here, we show the unique domain architecture of Arl13b by analyzing the relationship between its biochemical properties and cilia formation. Arl13b binds guanine nucleotides and specifically localizes to cilia. The ciliary localization of Arl13b requires both N and C domains but is independent of its guanine nucleotide-binding ability. Arl13b is capable of self-associating via N domain, and overexpression of N domain inhibits not only cilia formation but also the maintenance of pre-generated cilia. These findings suggest that N and C domains of Arl13b cooperatively regulate its ciliary localization and that N domain-dependent self-association of Arl13b may be important for its function in cilia biogenesis.     

Arl13b and the exocyst interact synergistically in ciliogenesis

Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.     

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogues in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in mouse embryonic fibroblasts (MEFs) results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 knockout (KO) in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other intraflagellar transport (IFT) proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi–cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi.     

Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b hnn stems from abnormal Shh signaling

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b(hnn), to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b(hnn) mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b(hnn) dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b(hnn) mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b(hnn) embryos indirectly causes dorsal defects.     

Neutrophil gelatinase-associated lipocalin (NGAL) is localised to the primary cilium in renal tubular epithelial cells - A novel source of urinary biomarkers of renal injury

BackgroundPrimary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated.MethodsHuman renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort.ResultsImmunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells.ConclusionThe accumulation of NGAL in supernatants in vitro and in the urine of CAN patients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury.     

Mon2 is a negative regulator of the monomeric G protein, Arl1

Using site-directed mutants of ARL1 predicted to alter nucleotide binding, we examined phenotypes associated with the loss of ARL1 , including effects on membrane traffic and K (+) homeostasis. The GTP-restricted allele, ARL[Q72L] , complemented the membrane traffic phenotype (CPY secretion), but not the K (+) homeostasis phenotypes (sensitivity to hygromycin B, steady-state levels of K (+) , and accumulation of (86) Rb (+) ), while the XTP-restricted mutant, ARL1[D130N] , complemented the ion phenotypes, but not the membrane traffic phenotype. A GDP-restricted allele, ARL1[T32N] , did not effectively complement either phenotype. These results are consistent with a model in which Arl1 has three different conformations in vivo. We also explored the relationship between ARL1 and MON2 using the synthetic lethal phenotype exhibited by these two genes and demonstrated that MON2 is a negative regulator of the GTP-restricted allele of ARL1 , ARL1[Q72L] . Finally, we constructed several new alleles predicted to alter binding of Arl1 to the sole GRIP domain containing protein in yeast, Imh1, and found that ARL1[F52G] and ARL1[Y82G] were unable to complement the loss of ARL1 with respect to either the membrane traffic or K (+) homeostasis phenotypes. Our study expands understanding of the roles of Arl1 in vivo.     

The ciliary GTPase Arl13b regulates cell migration and cell cycle progression

The GTPase ARL13B is localized to primary cilia; small cellular protrusions that act as antennae. Its defective ARL13B hennin (HNN) variant is linked causally with Joubert Syndrome, a developmental ciliopathy attributed to poor sensing of extracellular chemical gradients. We tested the hypothesis that impaired detection of extracellular voltage gradients also contributes to the HNN phenotype.

In vitro, extracellular electric fields stimulated migration of wild type (WT) and HNN fibroblasts toward the cathode but the field only increased the migration speed of WT cells. Cilia on WT cells did not align to the field vector. HNN cells divided more slowly than WT cells, arresting at the G2/M phase. Mechanistically, HNN cells had reduced phospho-ERK1/2 signaling and elevated levels of Suppressor of Fused protein. These suggest that cells may not be able to read extracellular chemical cues appropriately, resulting in deficits in cell migration and proliferation. Finally, an increase in tubulin stabilization (more detyrosinated tubulin) confirmed the general stagnation of HNN cells, which may further contribute to slower migration and cell cycle progression.

We conclude that Arl13b dysfunction resulted in HNN cell stagnation due to poor growth factor signaling and impaired detection of extracellular electrical gradients, and that the role of Arl13b in cell proliferation may be understated.     

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet-Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.     

Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b stems from abnormal Shh signaling

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b^hnn, to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b^hnn mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b^hnn dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b^hnn mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b^hnn embryos indirectly causes dorsal defects.     

Molecular views of Arf-like small GTPases in cilia and ciliopathies

The primary cilia are microtubule-based organelles that protrude from most of the eukaryotic cells. Recognized as the cell's antenna, primary cilium functions as a signaling hub for many physiologically and developmentally important signaling cascades. Ciliary dysfunction causes a wide spectrum of syndromic human genetic diseases collectively termed “ciliopathies”. Mounting evidences have shown that various small GTPases have been implicated in the context of cilia as well as human ciliopathies. However, how these small GTPases affect cilia formation and function remains poorly understood. Here we review and discuss the ciliary role of three Arf-like small GTPases (Arls), Arl3, Arl6, and Arl13b.     

Review series: The cell biology of vision

The small ciliary G protein Arl13b is required for cilium biogenesis and sonic hedgehog signaling and is mutated in patients with Joubert syndrome (JS). In this study, using Caenorhabditis elegans and mammalian cell culture systems, we investigated the poorly understood ciliary and molecular basis of Arl13b function. First, we show that Arl13b/ARL-13 localization is frequently restricted to a proximal ciliary compartment, where it associates with ciliary membranes via palmitoylation modification motifs. Next, we find that loss-of-function C. elegans arl-13 mutants possess defects in cilium morphology and ultrastructure, as well as defects in ciliary protein localization and transport; ciliary transmembrane proteins abnormally accumulate, PKD-2 ciliary abundance is elevated, and anterograde intraflagellar transport (IFT) is destabilized. Finally, we show that arl-13 interacts genetically with other ciliogenic and ciliary transport–associated genes in maintaining cilium structure/morphology and anterograde IFT stability. Together, these data implicate a role for JS-associated Arl13b at ciliary membranes, where it regulates ciliary transmembrane protein localizations and anterograde IFT assembly stability.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus

The ARLs are a diverse family of GTPases that are related to ADP-ribosylation factors (ARFs), but whose function is poorly understood. There are at least ten ARLs in humans, two of which have homologs in the yeast Saccharomyces cerevisiae (ARL1/Arl1p and ARFRP1/Arl3p). The function of ARFRP1 is unknown, but mammalian ARL1 has recently been found to interact with a number of effectors including the GRIP domain that is present in a family of Golgi-localized long coiled-coil proteins. We find that in yeast, the intracellular targeting of Imh1p, the only yeast GRIP domain protein, is dependent on both Arl1p and Arl3p, but not on the ARF proteins. A recombinant form of the Imh1p GRIP domain binds to Arl1p in a GTP-dependent manner, but not to Arl3p. Yeast also contain a relative of SCOCO, a protein proposed to bind human ARL1, but this yeast protein, Slo1p, appears to bind Arl3p rather than Arl1p in vitro. However, Imh1p is not the sole effector of Arl1p since affinity chromatography of cytosol with immobilized Arl1p:GTP revealed an interaction with the GARP/VFT complex that is thought to act in the tethering of vesicles to the Golgi apparatus. Finally, we find that Arl3p is required in vivo for the targeting of Arl1p, explaining its requirement for the normal distribution of Imh1p.     

A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice

Primary cilia are ubiquitous cellular antennae whose dysfunction collectively causes various disorders, including vision and hearing impairment, as well as renal, skeletal, and central nervous system anomalies. One ciliopathy, Alström syndrome, is closely related to Bardet–Biedl syndrome (BBS), sharing amongst other phenotypic features morbid obesity. As the cellular and molecular links between weight regulation and cilia are poorly understood, we used the obese mouse strain foz/foz, bearing a truncating mutation in the Alström syndrome protein (Alms1), to help elucidate why it develops hyperphagia, leading to early onset obesity and metabolic anomalies. Our in vivo studies reveal that Alms1 localizes at the base of cilia in hypothalamic neurons, which are implicated in the control of satiety. Alms1 is lost from this location in foz/foz mice, coinciding with a strong postnatal reduction (～70%) in neurons displaying cilia marked with adenylyl cyclase 3 (AC3), a signaling protein implicated in obesity. Notably, the reduction in AC3‐bearing cilia parallels the decrease in cilia containing two appetite‐regulating proteins, Mchr1 and Sstr3, as well as another established Arl13b ciliary marker, consistent with progressive loss of cilia during development. Together, our results suggest that Alms1 maintains the function of neuronal cilia implicated in weight regulation by influencing the maintenance and/or stability of the organelle. Given that Mchr1 and Sstr3 localization to remaining cilia is maintained in foz/foz animals but known to be lost from BBS knockout mice, our findings suggest different molecular etiologies for the satiety defects associated with the Alström syndrome and BBS ciliopathies. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.     

A transgenic zebrafish for in vivo visualization of cilia

Cilia are organelles for cellular signalling and motility. Mutations affecting ciliary function are also associated with cilia-related disorders (ciliopathies). The identification of cilia markers is critical for studying their function at the cellular level. Due to the lack of a conserved, short ciliary localization motif, the full-length ARL13b or 5HT₆ proteins are normally used for cilia labelling. Overexpression of these genes, however, can affect the function of cilia, leading to artefacts in cilia studies. Here, we show that Nephrocystin-3 (Nphp3) is highly conserved among vertebrates and demonstrate that the N-terminal truncated peptide of zebrafish Nphp3 can be used as a gratuitous cilia-specific marker. To visualize the dynamics of cilia in vivo, we generated a stable transgenic zebrafish Tg (β-actin: nphp3N-mCherry)^sx1001. The cilia in multiple cell types are efficiently labelled by the encoded fusion protein from embryonic stages to adulthood, without any developmental and physiological defects. We show that the line allows live imaging of ciliary dynamics and trafficking of cilia proteins, such as Kif7 and Smo, key regulators of the Hedgehog signalling pathway. Thus, we have generated an effective new tool for in vivo cilia studies that will help shed further light on the roles of these important organelles.     

Rabs and other small GTPases in ciliary transport

The non-motile primary cilium is a single, microtubule-based hair-like projection that emanates from most, if not all, non-dividing mammalian cells. Enriched in a variety of signalling receptors and accessories, the cilium mediates crucial sensory and regulatory functions during development and postnatal tissue homoeostasis. Maintenance of ciliary morphology and function requires continuous IFT (intraflagellar transport), and recent findings have shed light on some molecular details of how ciliogenesis is dependent on targeted exocytic membrane trafficking from the Golgi. The ARL [Arf (ADP ribosylation factor)-related] small GTPase Arf4 functions in TGN (trans-Golgi network) sorting of cilia-targeted rhodopsin into carrier vesicles, while Arl6 (Arf-like 6) and Arl13b regulate aspects of ciliary transport and IFT. Ciliogenesis and ciliary functions are also regulated by small Rabs. Rab8a, in conjunction with Rab11a, and via its interaction with a multitude of proteins associated with the ciliary basal body and axoneme/membrane, appears to be critical for ciliogenesis. Rab8's close homologue Rab10 may also play a ciliogenic role in some cells. Rab23, the depletion or inactivation of which affects cilia formation, may regulate specific ciliary protein targeting and turnover, particularly those involved in Shh (Sonic hedgehog) signalling. Recent findings have also implicated Ran, a small GTPase better known for nuclear import, in ciliary targeting of the KIF17 motor protein. We highlight and discuss recent findings on how Rabs and other small GTPases mediate ciliogenesis and ciliary traffic.