The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies

Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.     

The BBSome controls IFT assembly and turnaround in cilia

The bidirectional movement of intraflagellar transport (IFT) particles, which are composed of motors, IFT-A and IFT-B subcomplexes, and cargoes, is required for the biogenesis and signalling of cilia. A successful IFT cycle depends on the proper assembly of the massive IFT particle at the ciliary base and its turnaround from anterograde to retrograde transport at the ciliary tip. However, how IFT assembly and turnaround are regulated in vivo remains elusive. From a whole-genome mutagenesis screen in Caenorhabditis?elegans, we identified two hypomorphic mutations in dyf-2 and bbs-1 as the only mutants showing normal anterograde IFT transport but defective IFT turnaround at the ciliary tip. Further analyses revealed that the BBSome (refs?, ), a group of conserved proteins affected in human Bardet-Biedl syndrome (BBS), assembles IFT complexes at the ciliary base, then binds to the anterograde IFT particle in a DYF-2- (an orthologue of human WDR19) and BBS-1-dependent manner, and lastly reaches the ciliary tip to regulate proper IFT recycling. Our results identify the BBSome as the key player regulating IFT assembly and turnaround in cilia.     

ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.     

Architecture of the IFT ciliary trafficking machinery and interplay between its components

Abstract

Cilia and flagella serve as cellular antennae and propellers in various eukaryotic cells, and contain specific receptors and ion channels as well as components of axonemal microtubules and molecular motors to achieve their sensory and motile functions. Not only the bidirectional trafficking of specific proteins within cilia but also their selective entry and exit across the ciliary gate is mediated by the intraflagellar transport (IFT) machinery with the aid of motor proteins. The IFT-B complex, which is powered by the kinesin-2 motor, mediates anterograde protein trafficking from the base to the tip of cilia, whereas the IFT-A complex together with the dynein-2 complex mediates retrograde protein trafficking. The BBSome complex connects ciliary membrane proteins to the IFT machinery. Defects in any component of this trafficking machinery lead to abnormal ciliogenesis and ciliary functions, and results in a broad spectrum of disorders, collectively called the ciliopathies. In this review article, we provide an overview of the architectures of the components of the IFT machinery and their functional interplay in ciliary protein trafficking.     

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.     

IFT52 plays an essential role in sensory cilia formation and neuronal sensory function in Drosophila

Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. Here we investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation in Drosophila. We show that Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects.     

Primary ciliary dyskinesia relative protein ZMYND10 is involved in regulating ciliary function and intraflagellar transport in Paramecium tetraurelia

Cilia are highly conserved in most eukaryotes and are regarded as an important organelle for motility and sensation in various species. Cilia are microscopic, hair-like cytoskeletal structures that protrude from the cell surface. The major focus in studies of cilia has been concentrated on the ciliary dysfunction in vertebrates that causes multisymptomatic diseases, which together are referred to as ciliopathies. To date, the understanding of ciliopathies has largely depended on the study of ciliary structure and function in different animal models. Zinc finger MYND-type containing 10 (ZMYND10) is a ciliary protein that was recently found to be mutated in patients with primary ciliary dyskinesia (PCD). In Paramecium tetraurelia, we identified two ZMYND10 genes, arising from a whole-genome duplication. Using RNAi, we found that the depletion of ZMYND10 in P. tetraurelia causes severe ciliary defects, thus provoking swimming dysfunction and lethality. Moreover, we found that the absence of ZMYND10 caused the abnormal localization of the intraflagellar transport (IFT) protein IFT43 along cilia. These results suggest that ZMYND10 is involved in the regulation of ciliary function and IFT, which may contribute to the study of PCD pathogenesis.     

Characterization of Tetratricopeptide Repeat-Containing Proteins Critical for Cilia Formation and Function

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.     

Rabl2 GTP hydrolysis licenses BBSome‐mediated export to fine‐tune ciliary signaling

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome‐mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2‐GTP enters cilia by binding to IFT‐B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT‐B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP‐locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine‐tune cilia‐dependent signaling for normal embryonic development and organismic homeostasis.     

Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25

In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.     

Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits

Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.     

Intraflagellar Transport 80 Is Required for Cilia Construction and Maintenance in Paramecium tetraurelia

Intraflagellar transport (IFT) represents a bidirectional dynamic process that carries cargo essential for cilia building and the maintenance of ciliary function, which is important for the locomotion of single cells, intracellular and intercellular signalling transduction. Accumulated evidence has revealed that defects in IFT cause several clinical disorders. Here, we determined the role of IFT80, an IFT‐B protein that is mutated in Jeune asphyxiating thoracic dystrophy. Using the RNAi method in the ciliate Paramecium as model, we found that loss of IFT80 prevents cilia biogenesis and causes strong cell lethality. A specific antibody against IFT80 was also prepared in our study, which labelled IFT80 in cilia of Paramecium. GFP fusion experiments were performed to illustrate the dynamic movement of IFT‐A and IFT‐B proteins in cilia of Paramecium; then, we found that the depletion of IFT80 in cells prevents IFT‐A and IFT‐B proteins from entering the cilia. Our results showed the distribution change of other IFT proteins in cells that were depleted of IFT80, and we discuss the possible roles of IFT80 in Paramecium.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

Crystal structure of the intraflagellar transport complex 25/27

The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub‐complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure‐guided mutagenesis. IFT27 displays the fold of Rab‐like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase‐activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP‐binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high‐resolution structural understanding of the IFT complex.     

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery

Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high‐resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C‐terminal 320 residues adopt the fold of an eight‐bladed β‐propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N‐terminal domain, which in Chlamydomonas ODA16 is located on top of the β‐propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull‐down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.     

IFT54 directly interacts with kinesin‐II and IFT dynein to regulate anterograde intraflagellar transport

The intraflagellar transport (IFT) machinery consists of the anterograde motor kinesin‐II, the retrograde motor IFT dynein, and the IFT‐A and ‐B complexes. However, the interaction among IFT motors and IFT complexes during IFT remains elusive. Here, we show that the IFT‐B protein IFT54 interacts with both kinesin‐II and IFT dynein and regulates anterograde IFT. Deletion of residues 342–356 of Chlamydomonas IFT54 resulted in diminished anterograde traffic of IFT and accumulation of IFT motors and complexes in the proximal region of cilia. IFT54 directly interacted with kinesin‐II and this interaction was strengthened for the IFT54^Δ342–356 mutant in vitro and in vivo. The deletion of residues 261–275 of IFT54 reduced ciliary entry and anterograde traffic of IFT dynein with accumulation of IFT complexes near the ciliary tip. IFT54 directly interacted with IFT dynein subunit D1bLIC, and deletion of residues 261–275 reduced this interaction. The interactions between IFT54 and the IFT motors were also observed in mammalian cells. Our data indicate a central role for IFT54 in binding the IFT motors during anterograde IFT.     

Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52_330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52_281–329 in a complex that interacts directly with IFT70/IFT52_330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.     

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.     

The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone

In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow and which assembles a gate to maintain the specific ciliary proteome. Here we characterize the function of a novel centriolar satellite protein, synovial sarcoma X breakpoint–interacting protein 2 (SSX2IP), in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using small interfering RNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body. Cep290 takes a central role in gating proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, somatostatin receptor 3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome, and Rab8.