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Author(s): Bahl, Kriti; Yang, Shuang; Chou, Hui-Ting; Woodsmith, Jonathan; Stelzl, Ulrich; Walz, Thomas; Nachury, Maxence | Abstract: ABSTRACT The BBSome is a complex of eight Bardet-Biedl Syndrome (BBS) proteins that removes signaling receptors from cilia. The GTPase ARL6/BBS3 recruits the BBSome to the ciliary membrane where the BBSome–ARL6 GTP complex ferries G protein-coupled receptors (GPCRs) across the transition zone, a diffusion barrier at the base of cilia. Here, we find that the BBSome undergoes a conformational change upon recruitment to membranes by ARL6 GTP . Modeling the binding of the BBSome to membranes and to the GPCR Smoothened (SMO) reveals that the amphipathic helix 8 of SMO must be released from the membrane for SMO to be recognized by the BBSome. Underscoring the functional importance of amphipathic helix extraction in TZ crossing, we find that exchanging the amphipathic helix of ARL6 for one that embeds deeper into the membrane blocks BBSome-mediated exit of GPCRs from cilia. We propose that the rigid curvature and dense lipid packing of the transition zone reject asymmetric insertions in the inner leaflet and that the BBSome licenses transition zone crossing by extracting bulky amphipathic helices from the inner leaflet.

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the β-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.

Bardet-Biedl syndrome (BBS) is an incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a master regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the β-propeller domain of BBS1, the key subunit implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis predominantly results from disruption of autoinhibition and activation.

Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

Cilia, organelles that move to execute functions like fertilization and signal to execute functions like photoreception and embryonic patterning, are comprised of a core of nine-fold doublet microtubules overlain by a membrane. Distinct types of cilia display distinct membrane morphologies, ranging from simple domed cylinders to the invaginations and membrane disks of photoreceptor outer segments. Critical for the ability of cilia to signal, both the protein and the lipid compositions of ciliary membranes are differentiated from those of other cellular membranes. This specialization presents a unique challenge for the cell as, unlike other membrane-associated organelles, the ciliary membrane is contiguous with the surrounding plasma membrane. This distinct ciliary membrane is generated in concert with the multiple membrane remodeling events that comprise the process of ciliogenesis. Once the cilium is formed, control of ciliary membrane composition relies on discrete molecular machines, including a barrier to membrane proteins entering the cilium at a specialized region of the base of the cilium called the transition zone and a trafficking adaptor that controls G protein-coupled receptor (GPCR) localization to the cilium called the BBSome. The ciliary membrane can be further remodeled by the removal of membrane proteins by the release of ciliary extracellular vesicles that may function in intercellular communication, removal of unneeded proteins or ciliary disassembly. Here, we review the structures and transport mechanisms that control ciliary membrane composition, and discuss how membrane specialization enables the cilium to function as the antenna of the cell.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by developmental abnormalities and vision loss. To date, mutations in 21 genes have been linked to BBS. The products of eight of these BBS genes form a stable octameric complex termed the BBSome. Mutations in BBS8, a component of the BBSome, cause early vision loss, but the role of BBS8 in supporting vision is not known. To understand the mechanisms by which BBS8 supports rod and cone photoreceptor function, we generated animal models lacking BBS8. The loss of BBS8 protein led to concomitant decrease in the levels of BBSome subunits, BBS2 and BBS5 and increase in the levels of the BBS1 and BBS4 subunits. BBS8 ablation was associated with severe reduction of rod and cone photoreceptor function and progressive degeneration of each photoreceptor subtype. We observed disorganized and shortened photoreceptor outer segments (OS) at post-natal day 10 as the OS elaborates. Interestingly, loss of BBS8 led to changes in the distribution of photoreceptor axonemal proteins and hyper-acetylation of ciliary microtubules. In contrast to properly localized phototransduction machinery, we observed OS accumulation of syntaxin3, a protein normally found in the cytoplasm and the synaptic termini. In conclusion, our studies demonstrate the requirement for BBS8 in early development and elaboration of ciliated photoreceptor OS, explaining the need for BBS8 in normal vision. The findings from our study also imply that early targeting of both rods and cones in BBS8 patients is crucial for successful restoration of vision.

The function of the primary cilia, which is assembled in most vertebrate cells, is achieved by transport in and out of kinds of signaling receptors. The BBSome protein complex could recognize and target membrane proteins to the cilia, but how the BBSome itself is transported into the cilia is poorly understood. Here we demonstrate that the centrosome protein Dzip1 mediates the assembly of the BBSome-Dzip1-PCM1 complex in the centriolar satellites (CS) at the G0 phase for ciliary translocation of the BBSome. Phosphorylation of Dzip1 at Ser-210 by Plk1 (polo-like kinase 1) during the G2 phase promotes disassembly of this complex, resulting in removal of Dzip1 and the BBSome from the CS. Inhibiting the kinase activity of Plk1 maintains the CS localization of the BBSome and Dzip1 at the G2 phase. Collectively, our findings reveal the cell cycle-dependent regulation of BBSome transport to the CS and highlight a potential mechanism that the BBSome-mediated signaling pathways are accordingly regulated during the cell cycle.

Cilia and flagella on eukaryotic cells are slender microtubule-based projections surrounded by a membrane with a unique lipid and protein composition. It is now appreciated that cilia in addition to their established roles in motility also constitute hubs for cellular signaling by sensing external environmental cues necessary for organ development and maintenance of human health. Pathways reported to rely on the cilium organelle include Hedgehog, TGF-β, Wnt, PDGFRα, integrin and DNA damage repair signaling. An emerging theme in ciliary signaling is the requirement for active transport of signaling components into and out of the cilium proper. Here, we review the current state-of-the-art regarding the importance of intraflagellar transport and BBSome multi-subunit complexes in ciliary signaling.

Almost every single cell in the human body possesses primary cilium. The primary cilium function as a sensor of blood flow and sheer stress and contribute to both calcium and nitric oxide signaling in the endothelium. The BBS1 protein is an important member of the BBSome complex which mediates protein trafficking to the cilia membrane and other cellular compartments. To investigate the role of the BBSome in endothelial cells, we generated mice lacking the Bbs1 gene specifically in endothelial cells by crossing the Bbs1 fl/fl mice with the endothelial-specific Tie2 cre mice. The Bbs1 fl/fl /Tie2 Cre mice developed normally compared to littermate controls. We assessed vascular function ex vivo using aortic rings and resistance-sized mesenteric arteries. Interestingly, loss of the Bbs1 gene in endothelial cells caused endothelial dysfunction with a more severe effect in female mice as indicated by the impaired acetylcholine (ACh)-induced relaxation (Max relax of mesenteric artery: males: 43.6±7.6% vs 56.4±6.2% in controls, Pfl/fl /Tie2 Cre mice, indicative of endothelial but not smooth muscle dysfunction in the endothelium-specific Bbs1 null mice. Blood pressure tended to be elevated in Bbs1 fl/fl /Tie2 Cre mice particularly females. Strikingly, endothelial Bbs1 gene deletion caused hepatosteatosis as indicated by the significant lipid deposition in the liver of Bbs1 fl/fl /Tie2 Cre mice by oil-red-O staining (females: 7.75±2.34 vs 0.59±0.16 % area in controls, Pfl/fl /Tie2 Cre mice. Consistent with this, Bbs1 fl/fl /Tie2 Cre mice exhibited 80-90% reduction (P

The BBSome, a protein complex of 8 Bardet-Biedl Syndrome (BBS) protein including BBS1, has emerged as an important regulator of metabolic and cardiovascular function. We previously demonstrated that conditional disruption of the BBSome, by deleting the Bbs1 gene, throughout the nervous system leads to obesity in mice. The ventromedial hypothalamus (VMH) play a key role in energy balance, but the contribution of the BBSome in the VMH to the control of energy homeostasis is not known. To address this, we specifically ablated the Bbs1 gene in the VMH by crossing the Bbs1 fl/fl mice with mice expressing Cre recombinase selectively in the VMH, driven by the steroidogenic factor 1 promoter (SF1 Cre ). VMH-selective Cre-recombination was verified using td-Tomato reporter mice. Importantly, SF1 Cre /Bbs1 fl/fl mice develop obesity as indicated by the increased (PCre /Bbs1 fl/wt mice displayed an intermediate obesity phenotype (body weight: males: 39±2g and females: 30±1g). Daily food intake was not different in SF1 Cre /Bbs1 fl/fl mice relative to controls. However, energy expenditure (EE) was lower in SF1 Cre /Bbs1 fl/fl mice (ANCOVA corrected night EE: 0.27±0.06 vs 0.30±0.06 kcal/h in controls, PCre /Bbs1 fl/fl mice exhibited a blunted cooling-induced increase in sympathetic nerve activity (SNA) subserving thermogenic brown adipose tissue (BAT), although basal BAT SNA activity was not different. Next, we assessed the consequence of SF1 neuron-specific Bbs1 gene deletion on SNA subserving cardiovascular beds. SF1 Cre /Bbs1 fl/fl mice displayed a modest, but significant increase in renal SNA (49±24%, P=0.03) and splanchnic SNA (85±18%, PCre /Bbs1 fl/fl mice relative to controls (P=0.2). Radiotelemetry measurement of blood pressure in SF1 Cre /Bbs1 fl/fl and control mice is under way. These findings highlight the importance of the VMH BBSome for the regulation of energy homeostasis and sympathetic nerve outflow.

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that cells shed to influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis and environment release are almost entirely unknown. Here, we show that the ciliary G-protein RAB28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans. We also find that sequential targeting of lipidated RAB28 to periciliary and ciliary membranes is highly dependent on the BBSome and PDE6D, respectively, and that BBSome loss causes excessive and ectopic EV production. Our data indicate that RAB28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane. Our findings also suggest that EVs control sensory organ homeostasis by mediating communication between ciliated neurons and glia, and that defects in ciliary EV biogenesis may contribute to human ciliopathies.

In humans, dysfunctional primary cilia result in Bardet-Biedl syndrome (BBS), which presents with clinical features including intellectual disabilities, obesity, and retinal degeneration, and, in mouse models, the added feature of hydrocephalus. We observed increased Glial Fibrillary Acidic Protein (GFAP) immunoreactivity in BBS mouse brains. Increased GFAP expression is a hallmark of astrocyte reactivity that is associated with microglia activation and neuro-inflammation. To gain a better understanding of reactive astrocytes observed in BBS mice, we used two mouse models of BBS8, a BBSome protein, to characterize the reactive astrocyte phenotype. The finding of reactive astrocytes in young BBS mouse brains led us to hypothesize that loss of BBSome function leads to reactive astrocytes prior to hydrocephalus and obesity. By using two mouse models of BBS8, a congenital BBS8 knockout with hydrocephalus, and a tamoxifen-inducible BBS8 knockout without hydrocephalus, we were able to molecularly phenotype the reactive astrocytes. Molecular phenotype of reactive astrocytes shows differential regulation of inducers of Pan, A1 neurotoxic, and A2 neuroprotective astrocytes that are significantly altered in brains of both congenital and induced knockouts of BBS8, but without microglia activation. We find evidence for neuroinflammation in the brains of congenital knockout mice, but not in induced knockout mice. Protein levels of GFAP, SERPINA3N and post-synaptic density 95 (PSD95) are significantly increased in congenital knockout mice, but remain unchanged in induced knockout mice. Thus, despite the reactive astrocyte phenotype being present in both models, the molecular signature of reactive astrocytes in BBS8 mice models are distinct. Together, these findings suggest that BBS8, and by extension the BBSome, plays a role in neuro-astrocyte functions independent of hydrocephalus, and its dysregulation is associated with astrocyte reactivity without microglia activation. (Total word count 278).

Bardet-Biedl syndrome (BBS) is an archetypical ciliopathy caused by defective ciliary trafficking and consequent function. Insights gained from BBS mouse models are applicable to other syndromic and nonsyndromic retinal diseases. This progressive characterization of the visual phenotype in three BBS mouse models sets a baseline for testing therapeutic interventions. Longitudinal acquisition of electroretinograms, optical coherence tomography scans, and visual acuity using the optomotor reflex in Bbs6/Mkks, Bbs8/Ttc8, and Bbs5 knockout mice. Gene and protein expression analysis in vivo and in vitro. Complete loss of BBS5, BBS6, or BBS8 leads to different rates of retinal degeneration and visual function over time. BBS8-deficient mice showed the fastest rate of degeneration, and BBS8 seems to be required for cone photoreceptors to reach functional maturity. In contrast, the loss of BBS5 (a further BBSome component) showed very little degeneration. Loss of BBS8 versus BBS5 resulted in different physiologic responses both in vivo and in vitro. BBS6-deficient mice show a slower rate of degeneration with both rod and cone function reducing at a similar rate. The mouse models analyzed show distinct and diverging courses of degeneration upon loss of BBS5, BBS6, or BBS8, which can be used as a benchmark to test therapeutic interventions. Close consideration of the different phenotypes reveal subtle but important differences relating to their function. Because we also see differences in terms of phenotype depending on the type of visual assessment used, our data highlight the importance of using a combinatorial approach for assessment of visual function.

The intraflagellar transport (IFT) machinery, which includes the IFT-A and IFT-B complexes, mediates bidirectional trafficking of ciliary proteins. In addition to these complexes, the BBSome, which is composed of eight subunits that are encoded by the causative genes of Bardet-Biedl syndrome (BBS), has been proposed to connect the IFT machinery to ciliary membrane proteins, such as G protein-coupled receptors, to mediate their export from cilia. However, little is known about the connection between the IFT machinery and the BBSome. Using the visible immunoprecipitation assay, we here identified the interaction between IFT38 from the IFT-B complex and BBS1, BBS2 and BBS9 from the BBSome. Furthermore, by analyzing phenotypes of IFT38-knockout cells exogenously expressing wild-type IFT38 or its mutant lacking the ability to interact with BBS1+BBS2+BBS9, we showed that knockout cells expressing the IFT38 mutant have restored ciliogenesis; however, similar to BBS1-knockout cells, they demonstrated significant accumulation of GPR161 within cilia upon stimulation of Hedgehog signaling. These results indicate that the IFT-B–BBSome interaction is required for the export of GPR161 across the ciliary gate., Summary: We here determine an interface between the IFT-B complex and the BBSome involving IFT38 and BBS1+BBS2+BBS9, and show that the interaction is crucial for GPR161 export from cilia.

Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar ‘membrane+matrix’ was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140. This article has an associated First Person interview with the first author of the paper.

© 2019 Ludlam et al., Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its α-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation.

Bardet–Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require intraflagellar transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1−/−-defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex.

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet–Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP.

© 2016, National Academy of Sciences. All rights reserved. Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms.

Most motile and all non-motile (also known as primary) eukaryotic cilia possess microtubule-based axonemes that are assembled at the cell surface to form hair-like or more elaborate compartments endowed with motility and/or signaling functions. Such compartmentalized ciliogenesis depends on the core intraflagellar transport (IFT) machinery and the associated Bardet-Biedl syndrome complex (BBSome) for dynamic delivery of ciliary components. The transition zone (TZ), an ultrastructurally complex barrier or 'gate' at the base of cilia, also contributes to the formation of compartmentalized cilia. Yet, some ciliated protists do not have IFT components and, like some metazoan spermatozoa, use IFT-independent mechanisms to build axonemes exposed to the cytosol. Moreover, various ciliated protists lack TZ components, whereas Drosophila sperm surprisingly requires the activity of dynamically localized TZ proteins for cytosolic ciliogenesis. Here, we discuss the various ways eukaryotes use IFT and/or TZ proteins to generate the wide assortment of compartmentalized and cytosolic cilia observed in nature. Consideration of the different ciliogenesis pathways allows us to propose how three types of cytosol-exposed cilia (primary, secondary and tertiary), including cilia found in the human sperm proximal segment, are likely generated by evolutionary derivations of compartmentalized ciliogenesis.

Victor Hernandez-Hernandez, Dagan JenkinsGenetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UKAbstract: Bardet–Biedl syndrome (BBS) is an autosomal recessive condition characterized by important clinical features, including obesity, blindness, renal cystic disease, and intellectual disability. BBS is caused by mutations in >20 genes, a subset of which form the so-called BBSome. The BBSome is a complex that coats intracellular vesicles and interacts with key proteins, such as small GTPases, that regulate the trafficking of these vesicles to the base of cilia. Cilia are microtubular protusions present on the surface of most cells that are defective in a key group of disorders known as ciliopathies, of which BBS is one. BBSome components particularly localize to the basal body of cilia, and also centrosomes, where they interact with pericentriolar material proteins that regulate their function. The BBSome also facilitates the transport of key cargo within cilia by acting as an adaptor protein for intraflagellar transport complexes, and as such BBS mutations lead to a variety of functional defects in cilia in a tissue- and cell-type-specific manner. This might include defects in photoreceptor trafficking linked to the connecting cilium, abnormal hedgehog signaling within bone, and aberrant calcium signaling in response to fluid flow along renal tubules, although the precise mechanisms are still not completely understood. Taken together, the BBSome is an important complex that may be targeted for treatment of a variety of common and important disorders, and understanding the precise function of the BBSome will be essential to capitalize on this translationally.Keywords: retinitis pigmentosa GTPase regulator, polycystin, disrupted in schizophrenia 1, Hedgehog signaling, calcium signaling, photoreceptors

Purpose The microRNA cluster miR-183C, which includes miR-183 and two other genes, is critical for multiple sensory systems. In mouse retina, removal of this cluster results in photoreceptor defects in polarization, phototransduction, and outer segment elongation. However, the individual roles of the three components of this cluster are not clearly known. We studied the separate role of mouse miR-183 in in vivo. Methods miR-183 knockout mice were generated using the CRISPR/Cas9 genome-editing system. Electroretinography were carried out to investigate the changes of retinal structures and function. miR-183 was overexpressed by subretinal adeno-associated virus (AAV) injection in vivo. Rnf217, a target of miR-183 was overexpressed by cell transfection of the photoreceptor-derived cell line 661W in vitro. RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to compare the gene expression changes in AAV-injected mice and transfected cells. Results The miR-183 knockout mice showed progressively attenuated electroretinogram responses. Over- or under-expression of Rnf217, a direct target of miR-183, misregulated expression of cilia-related BBSome genes. Rnf217 overexpression also led to compromised electroretinography responses in WT mice, indicating that it may contribute to functional abnormalities in miR-183 knockout mice. Conclusions miR-183 is essential for mouse retinal function mediated directly and indirectly through Rnf217 and cilia-related genes. Our findings provide valuable insights into the explanation and analysis of the regulatory role of the individual miR-183 in miR-183C.

The BBSome, a complex containing 8 Bardet-Biedl syndrome (BBS) proteins including BBS1, has merged as an important regulator of various cellular processes including trafficking of receptors to plasma membrane. We hypothesized that the BBSome in endothelial cells contribute to the control of vascular function. We generated mice lacking the BBSome specifically in endothelial cells by crossing Bbs1 fl/fl mice with the endothelial-specific Tie2 cre mice. The Bbs1 fl/fl /Tie2 Cre mice developed normally compared to littermate control mice with no difference in body weight (females: 24.3±1.3 vs 22.7±1.0 g in control; males: 31.8±1.7 vs 30.0±0.6 g at end of 16 weeks of age), fat mass or food intake. Circulating glucose, triglycerides and cholesterol were also unaltered in Bbs1 fl/fl /Tie2 Cre mice. Next, we assessed vascular function ex vivo using aortic rings and resistance-sized mesenteric arteries. Strikingly, endothelial cell-specific BBSome deficiency caused endothelial dysfunction in both male and female mice, with more severe changes observed in females. In aortic rings, relaxation responses to acetylcholine were decreased in female Bbs1 fl/fl /Tie2 Cre mice compared to controls (Max. relaxation: 37.8±15.2 vs 62.0±10.3%). The male Bbs1 fl/fl /Tie2 Cre mice displayed a slightly decreased response that was not statistically different (Max relaxation: 38.2±11.7 vs 53.7±12.0%). In the mesenteric arteries, both male and female Bbs1 fl/fl /Tie2 Cre mice showed significantly decreased relaxation response evoked by acetylcholine (Max. relaxation: male: 43.6±7.6 vs 56.4±6.2% in controls, Pfl/fl /Tie2 Cre mice compared to controls. Thus, endothelium-specific BBSome deficiency leads to endothelial, but not smooth muscle dysfunction. Notably, female Bbs1 fl/fl /Tie2 Cre mice displayed about 25 mmHg elevation in mean arterial pressure relative to controls. Thus, the BBSome in endothelial cells contribute to vascular endothelial function and blood pressure regulation.

The BBSome is a protein complex composed of 8 Bardet-Biedl syndrome (BBS) proteins, including Bbs1 , involved in the regulation of various cellular processes including trafficking of receptors to cilia and the plasma membrane. This protein complex has emerged as a key regulator of cardiovascular function and blood pressure. We tested the hypothesis that the BBSome in vascular smooth muscle contributes to the regulation of vascular function. We selectively disrupted the BBSome in smooth muscle cells by deleting the Bbs1 gene. We crossed mice harboring floxed alleles of the Bbs1 gene (Bbs1 F/F ) with mice expressing tamoxifen-inducible Cre in smooth muscle cells (smMHC CreERT2 ). Validation of Bbs1 gene deletion in smooth muscle was confirmed via decreased Bbs1 mRNA levels in the aorta (1.0±0.2 vs 0.4±0.1 AU). We used wire myography to assess isolated aortic and mesenteric arterial ring function. Bbs1 gene deletion from smooth muscle resulted in a significant decrease in the relaxation responses evoked by acetylcholine (Max relaxation: 55±7 vs 25±12%) and sodium nitroprusside (Max relaxation: 85±3 vs 61±8%) in aortic rings indicating dysfunction in both the endothelium and smooth muscle. Interestingly, aortic rings from SM Cre /Bbs1 F/F mice exhibited enhanced contractility to endothelin-1 compared to controls (Max contraction: 0.1±0.01 vs 0.06±0.01g). Additionally, resistance mesenteric arterial rings of SM Cre /Bbs1 F/F mice displayed enhanced contractile responses evoked by U-46619, phenylephrine, endothelin-1 and KCl-induced depolarization relative to controls (pCre /Bbs1 F/F mice compared to controls. Radiotelemetric mean arterial pressure was not different in SM Cre /Bbs1 F/F mice at 4 weeks post-tamoxifen (96±4 mmHg) relative to controls (99±1 mmHg). Finally, arterial stiffness, measured using pulse wave velocity 4 weeks post-tamoxifen, showed a trend toward increased aortic stiffness in SM Cre /Bbs1 F/F mice (2.6±0.1 m/s) compared to controls (2.3±0.1 m/s, p=0.06). These data point to the vascular smooth muscle BBSome as a novel regulator of vascular function in a manner independent of blood pressure.

Cilia are surface-exposed organelles that dynamically concentrate signaling molecules to organize sensory, developmental and homeostatic pathways. Entry and exit of signaling receptors is germane to the processing of signals and the molecular machines for entry and exit have strated to emerge. The IFT-A complex and its membrane recruitment factor Tulp3 complex promotes the entry of signaling receptors into cilia while the BBSome and its membrane recruitment factor Arl6(GTP) ferry activated signaling receptors out of cilia. Ciliary exit is a surprisingly complex process entailing passage through a first diffusion barrier at the transition zone, diffusion inside an intermediate compartment and crossing of a periciliary diffusion barrier. The two barriers may organize a privileged compartment where activated signaling receptors transiently reside.

Protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery composed of large protein complexes. The BBSome consists of eight BBS proteins encoded by causative genes of Bardet-Biedl syndrome (BBS), and has been implicated in the trafficking of ciliary membrane proteins, including G protein-coupled receptors (GPCRs), by connecting the IFT machinery to cargo GPCRs. Membrane recruitment of the BBSome to promote cargo trafficking has been proposed to be regulated by the Arf-like small GTPase ARL6/BBS3, through its interaction with the BBS1 subunit of the BBSome. We here investigated how the BBSome core subcomplex composed of BBS1, BBS2, BBS7, and BBS9 assembles and interacts with ARL6, and found that the ARL6–BBS1 interaction is reinforced by BBS9. BBS1-knockout (KO) cells showed defects in the ciliary entry of other BBSome subunits and ARL6, and in ciliary retrograde trafficking and the export of the GPCRs, Smoothened and GPR161. The trafficking defect of these GPCRs was rescued by the exogenous expression of wild-type BBS1, but not by its mutant lacking BBS9-binding ability. Our data thus indicate that the intact BBSome is required for retrograde trafficking of GPCRs out of cilia.

Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms including blindness. The detailed structures of these organelles and the functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We have combined cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of mouse retinas. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening space. Within this framework we report the spatial distributions of intraflagellar transport proteins and proteins of the BBSome complex in WT and Bardet-Biedl syndrome model mice.

BBSome is a protein complex crucial for trafficking of specific cargoes to the primary cilium. Although primary cilia are typically not present in cells of haematopoietic origin, such as T cells, recent research has revealed striking parallels between the primary cilium and the immunological synapse. Amongst other similarities, both structures are supposed to use the same transport machinery involving Rab8 and IFT20, the close interaction partners of BBSome. The first goal of this thesis was to investigate the role of BBSome in the biology of T cells. Using RT-qPCR, we have shown that BBSome subunits are expressed in lymphoid tissues and T cells. Studies of localization of BBSome subunits in Jurkat cell line and primary OT-I T cells revealed that the subunits have distinct localization patterns with BBS4 localizing to the centrosome and BBS1, BBS5, and BBip10 having dispersed localization. After the contact with an antigen presenting cell, BBS4 re-localizes to the immunological synapse. Mutations in BBSome encoding genes cause Bardet-Biedl syndrome (BBS), a rare ciliopathy presenting with multiorganic symptoms. The second goal of this thesis was to examine the associations between BBS and the immune system. Examination of medical records of more than 450 BBS patients revealed that autoimmune...

Nonsyndromic craniosynostosis (NCS) is the premature ossification of skull sutures, without associated clinical features. Mutations in several genes account for a small number of NCS patients; thus, the molecular etiopathogenesis of NCS remains largely unclear. Our study aimed at characterizing the molecular signaling implicated in the aberrant ossification of sutures in NCS patients. Comparative gene expression profiling of NCS patient sutures identified a fused suture-specific signature, including 17 genes involved in primary cilium signaling and assembly. Cells from fused sutures displayed a reduced potential to form primary cilia compared to cells from control patent sutures of the same patient. We identified specific upregulated splice variants of the Bardet Biedl syndrome-associated gene 9 (BBS9), which encodes a structural component of the ciliary BBSome complex. BBS9 expression increased during in vitro osteogenic differentiation of suture-derived mesenchymal cells of NCS patients. Also, Bbs9 expression increased during in vivo ossification of rat sutures. BBS9 functional knockdown affected the expression of primary cilia on patient suture cells and their osteogenic potential. Computational modeling of the upregulated protein isoforms (observed in patients) predicted that their binding affinity within the BBSome may be affected, providing a possible explanation for the aberrant suture ossification in NCS.