The emerging complexity of the vertebrate cilium: new functional roles for an ancient organelle

Cilia and flagella are found on the surface of a strikingly diverse range of cell types. These intriguing organelles, with their unique and highly adapted protein transport machinery, have been studied extensively in the context of cellular locomotion, sexual reproduction, or fluid propulsion. However, recent studies are beginning to show that in vertebrates particularly, cilia have been recruited to perform additional developmental and homeostatic roles. Here, we review advances in deciphering the functional components of cilia, and we explore emerging trends that implicate ciliary proteins in signal transduction and morphogenetic pathways.     

Autophagy and regulation of cilia function and assembly

Motile and primary cilia (PC) are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. Recent studies highlight the interplay between cilia and autophagy, a conserved cellular process responsible for intracellular degradation. Signaling from the PC recruits the autophagic machinery to trigger autophagosome formation. Conversely, autophagy regulates ciliogenesis by controlling the levels of ciliary proteins. The cross talk between autophagy and ciliated structures is a novel aspect of cell biology with major implications in development, physiology and human pathologies related to defects in cilium function.     

Control of vertebrate intraflagellar transport by the planar cell polarity effector Fuz

Cilia play key roles in development and homeostasis, and defects in cilia structure or function lead to an array of human diseases. Ciliogenesis is accomplished by the intraflagellar transport (IFT) system, a set of proteins governing bidirectional transport of cargoes within ciliary axonemes. In this paper, we present a novel platform for in vivo analysis of vertebrate IFT dynamics. Using this platform, we show that the planar cell polarity (PCP) effector Fuz was required for normal IFT dynamics in vertebrate cilia, the first evidence directly linking PCP to the core machinery of ciliogenesis. Further, we show that Fuz played a specific role in trafficking of retrograde, but not anterograde, IFT proteins. These data place Fuz in the small group of known IFT effectors outside the core machinery and, additionally, identify Fuz as a novel cytoplasmic effector that differentiates between the retrograde and anterograde IFT complexes.     

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 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.     

Regulation of Cilium Length and Intraflagellar Transport by the RCK-Kinases ICK and MOK in Renal Epithelial Cells

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.     

A proteomic analysis of human cilia: identification of novel components

Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains > or = 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axonemes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin I, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle.     

Proteomic analysis of mammalian primary cilia

The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium-shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia-specific proteins. These possible primary cilia-specific proteins include EVC2, INPP5E, and inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.     

Ultrastructural Aspects of Olfactory Signaling

The olfactory area of the nasal cavity is lined with olfactoryreceptor cell cilia that come in contract with incoming odormolecules. Ultrastructural immunocytochemical studies in rodentshave shown that these cilia contain all the proteins necessaryto transduce the odorous message into an electrical signal thatcan be transmitted to the brain. These signaling proteins includeputative odor receptors, GTP binding proteins, type III adenylylcyclase and cyclic nucleotide-gated channels. The rest of thecells, including dendrites and dendritic knobs, showed no discerniblelabeling with antibodies to these signaling proteins. Furthermore,freeze-fracture and freeze-etch studies have shown that themembrane morphology of olfactory cilia differs substantiallyfrom that of non-sensory cilia. Olfactory cilia have many moremembrane particles. Transmembrane signaling proteins, such asodor receptors, adenylyl cyclase and cyclic nucleotide-gatedchannels, conceivably appear as membrane particles. Thus, thelong-standing supposition that olfactory cilia are peculiarlyadapted to deal with the reception and initial transductionof odorous messages has now been verified in terms of both ultrastructuralmorphology and cytochemistry. Emerging studies on vomeronasalreceptor cell microvilli indicate that the same is true forthis organ, even though the actual signaling components differfrom those of the main olfactory system. Chem. Senses 22: 295–311,1997.     

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.     

A transgenic zebrafish for in vivo visualization of cilia

Cilia are organelles for cellular signalling and motility. Mutations affecting ciliary function are also associated with cilia-related disorders (ciliopathies). The identification of cilia markers is critical for studying their function at the cellular level. Due to the lack of a conserved, short ciliary localization motif, the full-length ARL13b or 5HT₆ proteins are normally used for cilia labelling. Overexpression of these genes, however, can affect the function of cilia, leading to artefacts in cilia studies. Here, we show that Nephrocystin-3 (Nphp3) is highly conserved among vertebrates and demonstrate that the N-terminal truncated peptide of zebrafish Nphp3 can be used as a gratuitous cilia-specific marker. To visualize the dynamics of cilia in vivo, we generated a stable transgenic zebrafish Tg (β-actin: nphp3N-mCherry)^sx1001. The cilia in multiple cell types are efficiently labelled by the encoded fusion protein from embryonic stages to adulthood, without any developmental and physiological defects. We show that the line allows live imaging of ciliary dynamics and trafficking of cilia proteins, such as Kif7 and Smo, key regulators of the Hedgehog signalling pathway. Thus, we have generated an effective new tool for in vivo cilia studies that will help shed further light on the roles of these important organelles.     

Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

Regulated trafficking of G protein–coupled receptors (GPCRs) controls cilium-based signaling pathways. β-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet–Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between β-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a β-arrestin–dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.     

Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.     

The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development

Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.     

BROMI/TBC1D32 together with CCRK/CDK20 and FAM149B1/JBTS36 contributes to intraflagellar transport turnaround involving ICK/CILK1

Primary cilia are antenna-like organelles that contain specific proteins, and are crucial for tissue morphogenesis. Anterograde and retrograde trafficking of ciliary proteins are mediated by the intraflagellar transport (IFT) machinery. BROMI/TBC1D32 interacts with CCRK/CDK20, which phosphorylates and activates the intestinal cell kinase (ICK)/CILK1 kinase, to regulate the change in direction of the IFT machinery at the ciliary tip. Mutations in BROMI, CCRK, and ICK in humans cause ciliopathies, and mice defective in these genes are also known to demonstrate ciliopathy phenotypes. We show here that BROMI interacts not only with CCRK but also with CFAP20, an evolutionarily conserved ciliary protein, and with FAM149B1/ Joubert syndrome (JBTS)36, a protein in which mutations cause JBTS. In addition, we show that FAM149B1 interacts directly with CCRK as well as with BROMI. Ciliary defects observed in CCRK-knockout (KO), BROMI-KO, and FAM149B1-KO cells, including abnormally long cilia and accumulation of the IFT machinery and ICK at the ciliary tip, resembled one another, and BROMI mutants that are defective in binding to CCRK and CFAP20 were unable to rescue the ciliary defects of BROMI-KO cells. These data indicate that CCRK, BROMI, FAM149B1, and probably CFAP20 altogether regulate the IFT turnaround process under the control of ICK.     

Localization of extracellular matrix receptors on the chondrocyte primary cilium.

A single primary cilium is found in chondrocytes and other connective tissue cells. We have previously shown that extracellular matrix (ECM) macromolecules such as collagen fibers closely associate with chondrocyte primary cilia, and their points of contact are characterized by electron-opaque plaques suggesting a direct link between the ECM and the cilium. This study examines the expression of receptors for ECM molecules on chondrocyte primary cilia. Embryonic chick sterna were fluorescently labeled with antibodies against alpha and beta integrins, NG2, CD44, and annexin V. Primary cilia were labeled using acetylated alpha-tubulin antibody. Expression of ECM receptors was examined on chondrocyte plasma membranes and their primary cilia using immunofluorescence and confocal microscopy. All receptors examined showed a punctate distribution on the plasma membrane. alpha2, alpha3, and beta1 integrins and NG2 were also present on primary cilia, whereas annexin V and CD44 were excluded. The number of receptor-positive cilia varied from 8/50 for NG2 to 43/50 for beta1 integrin. This is the first study to demonstrate the expression of integrins and NG2 on chondrocyte primary cilia. The data strongly suggest that chondrocyte primary cilia have the necessary machinery to act as mechanosensors, linking the ECM to cytoplasmic organelles responsible for matrix production and secretion.     

Proteomic analysis of a membrane preparation from rat olfactory sensory cilia

The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research.     

Genetic defects of pronephric cilia in zebrafish

Cilia play key roles in many aspects of embryogenesis and adult physiology in vertebrates. Past genetic screens in zebrafish identified numerous defects of ciliogenesis, including several mutations in the components of the intraflagellar transport machinery. In contrast to previous studies, here we describe a collection of mutants that affect subpopulations of cilia. Mutant embryos are characterized by a shortening and an abnormal movement of kidney cilia, and in one case also a reduction of cilia length in the Kupffer's vesicle. In contrast to that, the cilia of sensory neurons, including photoreceptor cells, hair cells, and olfactory sensory cells, appear grossly intact. Motility defects of pronephric cilia vary in mutant strains from complete paralysis to an increased frequency of movement, and are associated with left-right asymmetry defects. While ciliary ultrastructure is normal in most mutants, one of the mutant loci is essential for the formation of proper microtubule architecture in the axoneme of pronephric cilia. Mutants characterized in this study reveal intriguing genetic differences between subpopulations of embryonic cilia, and provide an opportunity to study several aspects of cilia structure and function.     

Induction of Ran GTP drives ciliogenesis

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin-Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport.