The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.     

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.     

Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control

BACKGROUND: Intraflagellar transport (IFT) is a motility process operating between the ciliary/flagellar (interchangeable terms) membrane and the microtubular axoneme of motile and sensory cilia. Multipolypeptide IFT particles, composed of complexes A and B, carry flagellar precursors to their assembly site at the flagellar tip (anterograde) powered by kinesin, and turnover products from the tip back to the cytoplasm (retrograde) driven by cytoplasmic dynein. IFT is essential for the assembly and maintenance of almost all eukaryotic cilia and flagella, and mutations affecting either the IFT motors or the IFT particle polypeptides result in the inability to assemble normal flagella or in defects in the sensory functions of cilia. RESULTS: We found that the IFT complex B polypeptide, IFT27, is a Rab-like small G protein. Reduction of the level of IFT27 by RNA interference reduces the levels of other complex A and B proteins, suggesting that this protein is instrumental in maintaining the stability of both IFT complexes. Furthermore, in addition to its role in flagellar assembly, IFT27 is unique among IFT polypeptides in that its partial knockdown results in defects in cytokinesis and elongation of the cell cycle and a more complete knockdown is lethal. CONCLUSION: IFT27, a small G protein, is one of a growing number of flagellar proteins that are now known to have a role in cell-cycle control.     

Structural studies of ciliary components

Cilia are organelles found on most eukaryotic cells, where they serve important functions in motility, sensory reception, and signaling. Recent advances in electron tomography have facilitated a number of ultrastructural studies of ciliary components that have significantly improved our knowledge of cilium architecture. These studies have produced nanometer-resolution structures of axonemal dynein complexes, microtubule doublets and triplets, basal bodies, radial spokes, and nexin complexes. In addition to these electron tomography studies, several recently published crystal structures provide insights into the architecture and mechanism of dynein as well as the centriolar protein SAS-6, important for establishing the 9-fold symmetry of centrioles. Ciliary assembly requires intraflagellar transport (IFT), a process that moves macromolecules between the tip of the cilium and the cell body. IFT relies on a large 20-subunit protein complex that is thought to mediate the contacts between ciliary motor and cargo proteins. Structural investigations of IFT complexes are starting to emerge, including the first three-dimensional models of IFT material in situ, revealing how IFT particles organize into larger train-like arrays, and the high-resolution structure of the IFT25/27 subcomplex. In this review, we cover recent advances in the structural and mechanistic understanding of ciliary components and IFT complexes.     

Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in chlamydomonas

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.     

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.     

Protein transport in growing and steady‐state cilia

Cilia and eukaryotic flagella are threadlike cell extensions with motile and sensory functions. Their assembly requires intraflagellar transport (IFT), a bidirectional motor‐driven transport of protein carriers along the axonemal microtubules. IFT moves ample amounts of structural proteins including tubulin into growing cilia likely explaining its critical role for assembly. IFT continues in non‐growing cilia contributing to a variety of processes ranging from axonemal maintenance and the export of non‐ciliary proteins to cell locomotion and ciliary signaling. Here, we discuss recent data on cues regulating the type, amount and timing of cargo transported by IFT. A regulation of IFT‐cargo interactions is critical to establish, maintain and adjust ciliary length, protein composition and function.     

Evolution of intraflagellar transport from coated vesicles and autogenous origin of the eukaryotic cilium

The cilium/flagellum is a sensory-motile organelle ancestrally present in eukaryotic cells. For assembly cilia universally rely on intraflagellar transport (IFT), a specialised bidirectional transport process mediated by the ancestral and conserved IFT complex. Based on the homology of IFT complex proteins to components of coat protein I (COPI) and clathrin-coated vesicles, we propose that the non- vesicular, membrane-bound IFT evolved as a specialised form of coated vesicle transport from a protocoatomer complex. IFT thus shares common ancestry with all protocoatomer derivatives, including all vesicle coats and the nuclear pore complex (NPC). This has major implications for the evolutionary origin of the cilium. First, it reinforces the tenet that duplication and divergence of pre-existing structures, rather than symbiosis, were the major themes during cilium evolution. Second, it suggests that the initial step in the autogenous origin of the cilium was the establishment of a membrane patch with transmembrane proteins transported by the ancestral vesicle-coating IFT complex. We propose a scenario for how the initial membrane patch gradually protruded to enhance exposure to the environment, then started to move, and finally compartmentalised to render receptor signalling and ciliary beating more efficient.     

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.     

The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.     

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 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.     

Ciliogenesis: building the cell's antenna

The cilium is a complex organelle, the assembly of which requires the coordination of motor-driven intraflagellar transport (IFT), membrane trafficking and selective import of cilium-specific proteins through a barrier at the ciliary transition zone. Recent findings provide insights into how cilia assemble and disassemble in synchrony with the cell cycle and how the balance of ciliary assembly and disassembly determines the steady-state ciliary length, with the inherent length-dependence of IFT rendering the ciliary assembly rate a decreasing function of length. As cilia are important in sensing and processing developmental signals and directing the flow of fluids such as mucus, defects in ciliogenesis and length control are likely to underlie a range of cilium-related human diseases.     

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88^Tg737Rpw mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88^−/− mice are completely sterile. They produce ∼350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.     

The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.     

Sensory ciliogenesis in Caenorhabditis elegans: assignment of IFT components into distinct modules based on transport and phenotypic profiles

Sensory cilium biogenesis within Caenorhabditis elegans neurons depends on the kinesin-2-dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs (Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as "modules." We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegans IFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis.     

RABL4/IFT27 in a nucleotide-independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip

Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.