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.     

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.     

Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons

Cilia play diverse roles in vertebrate and invertebrate sensory neurons. We show that a mutation of the zebrafish oval (ovl) locus affects a component of the ciliary transport (IFT) mechanism, the IFT88 polypeptide. In mutant retina, cilia are generated but not maintained, producing the absence of photoreceptor outer segments. A loss of cilia also occurs in auditory hair cells and olfactory sensory neurons. In all three sense organs, cilia defects are followed by degeneration of sensory cells. Similar phenotypes are induced by the absence of the IFT complex B polypeptides, ift52 and ift57, but not by the loss of complex A protein, ift140. The degeneration of mutant photoreceptor cells is caused, at least partially, by the ectopic accumulation of opsins. These studies reveal an essential role for IFT genes in vertebrate sensory neurons and implicate the molecular components of intraflagellar transport in degenerative disorders of these cells.     

Intraflagellar transport is required in Drosophila to differentiate sensory cilia but not sperm

BACKGROUND: Intraflagellar transport (IFT) uses kinesin II to carry a multiprotein particle to the tips of eukaryotic cilia and flagella and a nonaxonemal dynein to return it to the cell body. IFT particle proteins and motors are conserved in ciliated eukaryotes, and IFT-deficient mutants in algae, nematodes, and mammals fail to extend or maintain cilia and flagella, including sensory cilia. In Drosophila, the only ciliated cells are sensory neurons and sperm. no mechanoreceptor potential (nomp) mutations have been isolated that affect the differentiation and function of ciliated sense organs. The nompB gene is here shown to encode an IFT protein. Its mutant phenotypes reveal the consequences of an IFT defect in an insect. RESULTS: Mechanosensory and olfactory neurons in nompB mutants have missing or defective cilia. nompB encodes the Drosophila homolog of the IFT complex B protein IFT88/Polaris/OSM-5. nompB is expressed in the ciliated sensory neurons, and a functional, tagged NOMPB protein is located in sensory cilia and around basal bodies. Surprisingly, nompB mutant males produce normally elongated, motile sperm. Neuronally restricted expression and male germline mosaic experiments show that nompB-deficient sperm are fully functional in transfer, competition, and fertilization. CONCLUSIONS: NOMPB, the Drosophila homolog of IFT88, is required for the assembly of sensory cilia but not for the extension or function of the sperm flagellum. Assembly of this extremely long axoneme is therefore independent of IFT.     

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a "canonical" IFT motor, whereas OSM-3 is an "accessory" IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.     

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.     

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.     

Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.     

Identification and characterization of a long isoform of human IFT80, IFT80-L

Intraflagellar transport (IFT) proteins are evolutionarily conserved throughout all ciliated organisms and are essential for the assembly and maintenance of cilia. IFT80, a component of the IFT complex, was linked recently to a human developmental disorder, Jeune asphyxiating thoracic dystrophy. We report here identification and characterization of a human IFT80 long isoform (namely IFT80-L), the carboxyl terminus of which shares the protein sequence of IFT80. Sequence analysis indicates that IFT80-L is likely an evolutionarily merged product of genes IFT80 and TRIM59, a RING finger gene we reported previously. Expression analysis of IFT80-L demonstrates that IFT80-L is ubiquitously expressed in humans. By using the nerve growth factor-induced cell differentiation assays, we reveal that IFT80-L is highly expressed in the rapidly proliferating cells but not in differentiated cells, which withdraw from the cell cycle. Our findings suggest that IFT80-L, like other IFT proteins, plays an important role in cell proliferation and differentiation.     

The cilia protein IFT88 is required for spindle orientation in mitosis

Cilia dysfunction has long been associated with cyst formation and ciliopathies. More recently, misoriented cell division has been observed in cystic kidneys, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells. Several IFT proteins also localize to spindle poles in mitosis, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737(orpk) and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies.     

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.     

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis

Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.     

IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella. Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed. To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20. We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at approximately 17 S, similar to IFT proteins extracted from mouse testis. Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina. We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex. The internal architecture of the IFT complex was investigated using the yeast two-hybrid system. IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B. Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types. Furthermore, IFT20 appears to bridge kinesin II with the IFT complex.     

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.     

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.     

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.     

Intraflagellar transport is required for the vectorial movement of TRPV channels in the ciliary membrane

The membranes of all eukaryotic motile (9 + 2) and immotile primary (9 + 0) cilia harbor channels and receptors involved in sensory transduction (reviewed by). These membrane proteins are transported from the cytoplasm onto the ciliary membrane by vesicles targeted for exocytosis at a point adjacent to the ciliary basal body. Here, we use time-lapse fluorescence microscopy to demonstrate that select GFP-tagged sensory receptors undergo rapid vectorial transport along the entire length of the cilia of Caenorhabditis elegans sensory neurons. Transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 move in ciliary membranes at rates comparable to the intraflagellar transport (IFT) machinery located between the membrane and the underlying axonemal microtubules. OSM-9 motility is disrupted in certain IFT mutant backgrounds. Surprisingly, motility of transient receptor potential polycystin (TRPP) channel PKD-2 (polycystic kidney disease-2), a mechano-receptor, was not detected. Our study demonstrates that IFT, previously shown to be necessary for transport of axonemal components, is also involved in the motility of TRPV membrane protein movement along cilia of C. elegans sensory cells.