Search

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.

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.

Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes., Author Summary Ciliopathies, diseases arising from defects in the functions of primary cilia, have many different manifestations and vary dramatically in severity. How genetics influence ciliopathy phenotypes is poorly understood. Building off of our increasing knowledge of how different biochemical complexes contribute to ciliary function, we investigated how ciliopathy-associated genes interact to support ciliogenesis. Using a combination of nematode and mouse genetics, we found that genes encoding components of different biochemical complexes interact, whereas genes encoding different components within a single complex do not. These results revealed overlapping ciliary functions of biochemically distinct proteins complexes such as the BBSome, the transition zone MKS complex and the transition zone NPHP complex. This work indicates the genetic interactions that may alter the phenotypic consequences of human ciliopathy mutations.

Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT), consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. Tetratricopeptide repeat-containing (TTC) proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs) into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, ttc4, -9c, -36, and -39c, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of ttc4 and ttc25, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia.

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated., Author summary The BBSome is a protein complex that regulates ciliary trafficking in primary cilia, and mutations that impair BBSome function cause Bardet-Biedl Syndrome (BBS). BBS patients have retinal degeneration leading to blindness, but the disease pathophysiology has not been fully elucidated. In this study, we found that the BBSome is necessary for the structural organization of photoreceptor outer segments, and that the loss of different functional BBSome subunits causes outer segment malformation. Using a mouse model that allows the temporal inactivation of the BBSome, we inactivated BBSome function after the outer segment had formed normally. We found that the BBSome is required for both the initial formation and the continual maintenance of outer segment structures throughout life. In addition, using a mouse model that allows the temporal restoration of the BBSome, we restored BBSome function in immature photoreceptors and show that the malformed outer segment discs are displaced distally by normally formed outer segment structures. This finding indicates that when gene function is restored in immature retinas shortly after initial outer segment malformation, morphologically normal outer segments and a functionally normal retina can still result. This study has important implications for the timing of treatment of human retinal diseases.

The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure.

Primary cilia are important sensory organelles. They exist in a wide variety of lengths, which could reflect different cell-specific functions. How cilium length is regulated is unclear, but it probably involves intraflagellar transport (IFT), which transports protein complexes along the ciliary axoneme. Studies in various organisms have identified the small, conserved family of ros-cross hybridizing kinases (RCK) as regulators of cilium length. Here we show that Intestinal Cell Kinase (ICK) and MAPK/MAK/MRK overlapping kinase (MOK), two members of this family, localize to cilia of mouse renal epithelial (IMCD-3) cells and negatively regulate cilium length. To analyze the effects of ICK and MOK on the IFT machinery, we set up live imaging of five fluorescently tagged IFT proteins: KIF3B, a subunit of kinesin-II, the main anterograde IFT motor, complex A protein IFT43, complex B protein IFT20, BBSome protein BBS8 and homodimeric kinesin KIF17, whose function in mammalian cilia is unclear. Interestingly, all five proteins moved at ∼0.45 µm/s in anterograde and retrograde direction, suggesting they are all transported by the same machinery. Moreover, GFP tagged ICK and MOK moved at similar velocities as the IFT proteins, suggesting they are part of, or transported by the IFT machinery. Indeed, loss- or gain-of-function of ICK affected IFT speeds: knockdown increased anterograde velocities, whereas overexpression reduced retrograde speed. In contrast, MOK knockdown or overexpression did not affect IFT speeds. Finally, we found that the effects of ICK or MOK knockdown on cilium length and IFT are suppressed by rapamycin treatment, suggesting that these effects require the mTORC1 pathway. Our results confirm the importance of RCK kinases as regulators of cilium length and IFT. However, whereas some of our results suggest a direct correlation between cilium length and IFT speed, other results indicate that cilium length can be modulated independent of IFT speed.

Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity., Author Summary The brain plays an important role in maintaining energy homeostasis. The hormone leptin is a critical afferent signal in metabolic homeostasis through its action in the brain. Here we show that neuronal Bardet-Biedl syndrome (BBS) proteins, encoded by genes that cause obesity when mutated, govern energy homeostasis through the control of cell surface expression of the leptin receptor. Selective disruption of BBS proteins causes obesity in mice and impairs the transport of the leptin receptor to the plasma membrane leading to leptin resistance in a manner independent of obesity. These results establish BBS proteins as a fundamental mechanism underlying transport of the leptin receptor and explain why BBS patients develop obesity.

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals.

We analyzed the relatively poorly understood IFT-dynein (class DYNC2)-driven retrograde IFT pathway in C. elegans cilia, which yielded results that are surprising in the context of current models of IFT. Assays of C. elegans dynein gene expression and intraflagellar transport (IFT) suggest that conventional IFT-dynein contains essential heavy (CHE-3), light-intermediate (XBX-1), plus three light polypeptide chains that participate in IFT, but no “essential” intermediate chain. IFT assays of XBX-1::YFP suggest that IFT-dynein is transported as cargo to the distal tip of the cilium by kinesin-2 motors, but independent of the IFT-particle/BBSome complexes. Finally, we were surprised to find that the subset of cilia present on the OLQ (outer labial quadrant) neurons assemble independently of conventional “CHE-3” IFT-dynein, implying that there is a second IFT-dynein acting in these cilia. We have found a novel gene encoding a dynein heavy chain, DHC-3, and two light chains, in OLQ neurons, which could constitute an IFT-dynein complex in OLQ neuronal cilia. Our results underscore several surprising features of retrograde IFT that require clarification.