Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

Regulated trafficking of G protein–coupled receptors (GPCRs) controls cilium-based signaling pathways. β-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet–Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between β-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a β-arrestin–dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.     

Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling.     

Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity

Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt^T308 and P-Akt^S473, and previous work showed decreased P-Akt^S473 in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt^T308 and P-Akt^S473 in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt^T308. Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt^S473 or P-Akt^T308 upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA–dependent phosphorylation of Akt^S473 and Akt^T308 in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.     

Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity

Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.     

A role for the primary cilium in Notch signaling and epidermal differentiation during skin development

Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar transport proteins (IFTs) and conditional ablation of Ift-88 and Kif3a during embryogenesis. In both cultured keratinocytes and embryonic epidermis, all of these eliminated cilia, and many (not Kif3a) caused hyperproliferation. Surprisingly and independent of proliferation, ciliary mutants displayed defects in Notch signaling and commitment of?progenitors to differentiate. Notch receptors and Notch-processing enzymes colocalized with cilia in wild-type epidermal cells. Moreover, differentiation defects in ciliary mutants were cell autonomous and rescued by activated Notch (NICD). By contrast, Shh signaling was neither operative nor required for?epidermal ciliogenesis, Notch signaling, or differentiation. Rather, Shh signaling defects in ciliary mutants occurred later, arresting hair follicle morphogenesis in the skin. These findings unveil temporally and spatially distinct functions for primary cilia?at the nexus of signaling, proliferation, and differentiation.     

Mechanical Properties of a Primary Cilium As Measured by Resonant Oscillation

Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10⁻¹² N/rad and nonlinear spring constant α to be (−1 ± 0.34) × 10⁻¹⁰ N/rad²; and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia—anatomically similar structures with very different physiological functions.     

Primary cilia control telencephalic patterning and morphogenesis via Gli3 proteolytic processing

Primary cilia have essential functions in vertebrate development and signaling. However, little is known about cilia function in brain morphogenesis, a process that is severely affected in human ciliopathies. Here, we study telencephalic morphogenesis in a mouse mutant for the ciliopathy gene Ftm (Rpgrip1l). We show that the olfactory bulbs are present in an ectopic location in the telencephalon of Ftm(-/-) fetuses and do not display morphological outgrowth at the end of gestation. Investigating the developmental origin of this defect, we have established that E12.5 Ftm(-/-) telencephalic neuroepithelial cells lack primary cilia. Moreover, in the anterior telencephalon, the subpallium is expanded at the expense of the pallium, a phenotype reminiscent of Gli3 mutants. This phenotype indeed correlates with a decreased production of the short form of the Gli3 protein. Introduction of a Gli3 mutant allele encoding the short form of Gli3 into Ftm mutants rescues both telencephalic patterning and olfactory bulb morphogenesis, despite the persistence of cilia defects. Together, our results show that olfactory bulb morphogenesis depends on primary cilia and that the essential role of cilia in this process is to produce processed Gli3R required for developmental patterning. Our analysis thus provides the first in vivo demonstration that primary cilia control a developmental process via production of the short, repressor form of Gli3. Moreover, our findings shed light on the developmental origin of olfactory bulb agenesis and of other brain morphogenetic defects found in human diseases affecting the primary cilium.     

Smoothened determines β-arrestin–mediated removal of the G protein–coupled receptor Gpr161 from the primary cilium

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein–coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161–β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.     

Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import

Cilia sense and transduce extracellular signals via specific receptors. The intraflagellar transport (IFT) machinery mediates not only bidirectional protein trafficking within cilia but also the import/export of ciliary proteins across the ciliary gate. The IFT machinery is known to comprise two multisubunit complexes, namely, IFT-A and IFT-B; however, little is known about how the two complexes cooperate to mediate ciliary protein trafficking. We here show that IFT144–IFT122 from IFT-A and IFT88–IFT52 from IFT-B make major contributions to the interface between the two complexes. Exogenous expression of the IFT88(Δα) mutant, which has decreased binding to IFT-A, partially restores the ciliogenesis defect of IFT88-knockout (KO) cells. However, IFT88(Δα)-expressing IFT88-KO cells demonstrate a defect in IFT-A entry into cilia, aberrant accumulation of IFT-B proteins at the bulged ciliary tips, and impaired import of ciliary G protein–coupled receptors (GPCRs). Furthermore, overaccumulated IFT proteins at the bulged tips appeared to be released as extracellular vesicles. These phenotypes of IFT88(Δα)-expressing IFT88-KO cells resembled those of IFT144-KO cells. These observations together indicate that the IFT-A complex cooperates with the IFT-B complex to mediate the ciliary entry of GPCRs as well as retrograde trafficking of the IFT machinery from the ciliary tip.     

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.     

Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity,Density, and Beat

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.     

Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking

Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB’s capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP₂ binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.     

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-α in the primary cilium

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) α–mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-α and the Na⁺/H⁺ exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737^orpk MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-α ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5′-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA–mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737^orpk MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-α signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-α stimulation.     

Rer1p maintains ciliary length and signaling by regulating γ-secretase activity and Foxj1a levels

Cilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi–localized membrane protein involved in ciliogenesis. Rer1p, a protein quality control receptor, was highly expressed in zebrafish ciliated organs and regulated ciliary structure and function. Both in zebrafish and mammalian cells, loss of Rer1p resulted in the shortening of cilium and impairment of its motile or sensory function, which was reflected by hearing, vision, and left–right asymmetry defects as well as decreased Hedgehog signaling. We further demonstrate that Rer1p depletion reduced ciliary length and function by increasing γ-secretase complex assembly and activity and, consequently, enhancing Notch signaling as well as reducing Foxj1a expression.     

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.     

Sept6 Is Required for Ciliogenesis in Kupffer's Vesicle,the Pronephros,and the Neural Tube during Early Embryonic Development

Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer''s vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated α-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pronephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish.     

Torque Generation by Axonemal Outer-Arm Dynein

Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (αβγ) as well as proteolytically generated two-headed (βγ) and one-headed (α) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed αβγ and one-headed α exhibited significantly longer pitch. In contrast, the pitch driven by two-headed βγ did not display this sensitivity. In the assays on lawns containing mixtures of α and βγ at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of α in the mixture increased. Even small proportions of α-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (αβγ) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.     

Effector Contributions to Gβγ-mediated Signaling as Revealed by Muscarinic Potassium Channel Gating

Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the Gα subunit from Gβγ is a highly conserved signaling strategy used by numerous extracellular stimuli. Although Gβγ subunits regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403–406), few tools exist for probing instantaneous Gβγ-effector interactions, and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the atrial muscarinic K⁺ channel, which is activated by direct interactions with Gβγ subunits (Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321–326; Wickman, K., J.A. Iniguez-Liuhi, P.A. Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366: 654–663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133–1143), as a sensitive reporter of the dynamics of Gβγ-effector interactions. Muscarinic K⁺ channels exhibit bursting behavior upon G protein activation, shifting between three distinct functional modes, characterized by the frequency of channel openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated Gβγ) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a Gβγ effector with allosteric regulation and an intrinsic “off” switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in Gβγ concentration, and may be indicative of the factors regulating other Gβγ-modulated effectors.