Components Of Cilia Research Articles

Mechanisms and genes in Drosophila hearing

Abstract The fruit fly Drosophila melanogaster com­municates acoustically and hears with its an­tennae. Fundamental aspects of hearing can be studied in these antennal ears, the audi­tory sensory cells of which are evolutionarily related to vertebrate hair cells and are spec­ified developmentally by homologous tran­scription factors. Like vertebrate hair cells, Drosophila auditory sensory cells are also mo­tile and actively amplify the mechanical vi­brations they transduce. The transduction and amplification mechanisms rely on the in­terplay between mechanically activated ion channels and motor proteins, whose move­ment impacts upon the macroscopic perfor­mance of the ear. The first molecular trans­ducer components have been identified and various auditory system-relevant proteins have been described. Several of these pro­teins are conserved components of cilia, sug­gesting the fly’s ear as a model for human cil­iopathies. The evolution of sensory signaling cascades can also be studied using the fly’s ear, as the fly employs key chemo- and pho­toreceptor proteins to hear. Evidence is al­so accumulating that the fly’s ear is a multi­functional sensory organ, which, in addition to mediating hearing, serves to detect wind, gravity and presumably temperature.

Mechanisms and genes in Drosophila hearing

The fruit fly Drosophila melanogaster communicates acoustically and hears with its antennae. Fundamental aspects of hearing can be studied in these antennal ears, the auditory sensory cells of which are evolutionarily related to vertebrate hair cells and are specified developmentally by homologous transcription factors. Like vertebrate hair cells, Drosophila auditory sensory cells are also motile and actively amplify the mechanical vibrations they transduce. The transduction and amplification mechanisms rely on the interplay between mechanically activated ion channels and motor proteins, whose movement impacts upon the macroscopic performance of the ear. The first molecular transducer components have been identified and various auditory system-relevant proteins have been described. Several of these proteins are conserved components of cilia, suggesting the fly’s ear as a model for human ciliopathies. The evolution of sensory signaling cascades can also be studied using the fly’s ear, as the fly employs key chemo- and photoreceptor proteins to hear. Evidence is also accumulating that the fly’s ear is a multifunctional sensory organ, which, in addition to mediating hearing, serves to detect wind, gravity and presumably temperature.

Drosophila Hören: Mechanismen und Gene

Abstract Drosophila hearing: mechanisms and genes. The fruit fly Drosophila melanogaster communicates acoustically and hears with its antennae. Fundamental aspects of hearing can be studied in these antennal ears. Their auditory sensory cells are evolutionarily related with vertebrate hair cells and are developmentally specified by homologous transcription factors. Like vertebrate hair cells, Drosophila auditory sensory cells are also motile and actively amplify the mechanical vibrations that they transduce. This transduction and amplification rely on the interplay between mechanically activated ion channels and motor proteins, whose movement impacts on the macroscopic performance of the ear. First molecular trans­ducer components have been identified and various auditory relevant proteins have been described. Several of these proteins are conserved components of cilia, putting forward the fly’s ear as a model for human ciliopathies. Also the evolution of sensory signalling cascades can be studied using the fly’s ear as the fly employs key Chemo-and Photoreceptor proteins to hear. Evidence is also accumulating that the fly’s ear is a multifunctional sensory organ that, in addition to mediating hearing, serves the detection of wind and gravity and, presumably, temperature.

Hedgehog Signaling Controls T Cell Killing at the Immunological Synapse

The centrosome is essential for cytotoxic T lymphocyte (CTL) function, contacting the plasma membrane and directing cytotoxic granules for secretion at the immunological synapse. Centrosome docking at the plasma membrane also occurs during cilia formation. The primary cilium, formed in nonhematopoietic cells, is essential for vertebrate Hedgehog (Hh) signaling. Lymphocytes do not form primary cilia, but we found and describe here that Hh signaling played an important role in CTL killing. T cell receptor activation, which "prearms" CTLs with cytotoxic granules, also initiated Hh signaling. Hh pathway activation occurred intracellularly and triggered Rac1 synthesis. These events "prearmed" CTLs for action by promoting the actin remodeling required for centrosome polarization and granule release. Thus, Hh signaling plays a role in CTL function, and the immunological synapse may represent a modified cilium.

Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase

The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched ∼150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency.

FOP Is a Centriolar Satellite Protein Involved in Ciliogenesis

Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G1, when FOP protein levels are lowest, and most labeled in G2. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.

Evc Regulates a Symmetrical Response to Shh Signaling in Molar Development

Tooth morphogenesis involves patterning through the activity of epithelial signaling centers that, among other molecules, secrete Sonic hedgehog (Shh). While it is known that Shh responding cells need intact primary cilia for signal transduction, the roles of individual cilia components for tooth morphogenesis are poorly understood. The clinical features of individuals with Ellis-van Creveld syndrome include various dental anomalies, and we show here that absence of the cilial protein Evc in mice causes various hypo- and hyperplasia defects during molar development. During first molar development, the response to Shh signaling is progressively lost in Evc-deficient embryos and, unexpectedly, the response consistently disappears in a buccal to lingual direction. The important role of Evc for establishing the buccal-lingual axis of the developing first molar is also supported by a displaced activity of the Wnt pathway in Evc mutants. The observed growth abnormalities eventually manifest in first molar microdontia, disruption of molar segmentation and symmetry, root fusions, and delayed differentiation. Analysis of our data indicates that both spatially and temporally disrupted activities of the Shh pathway are the primary cause for the variable dental anomalies seen in patients with Ellis-van Creveld syndrome or Weyers acrodental dysostosis.

Transcriptional Program of Ciliated Epithelial Cells Reveals New Cilium and Centrosome Components and Links to Human Disease

Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

Abstract 19282: NAT10 Mutaion Causes Ciliary Aplisia and Congenital Heart Disease Associated with Heterotaxy

Introduction: We recently showed patients with CHD associated with heterotaxy can exhibit respiratory ciliary dysfunction (CD) similar to that of patients with primary ciliary dyskinesia (PCD) - a ciliopathy with airway mucociliary clearance defects. Exome sequencing analysis revealed known PCD causing mutation and rare coding variants in known PCD genes were enriched in heterotaxy patients with CD, suggesting a common etiology for heterotaxy and motile cilia dysfunction. Further interrogation for novel variants in non-PCD genes have uncovered one patient with a homozygous mutation in the acetyltransferase NAT10 . In this study, we investigated the hypothesis that NAT10 may play an essential role in ciliogenesis and is required for left-right patterning. Methods and Results: Exome sequencing was carried out in heterotaxy patients with ciliary dysfunction. Coding variants were filtered against dbSNP and 1000 genome databases. Novel homozygous coding variant in NAT10 (M638V) was identified in one heterotaxy patient with ciliary aplasia. This mutation is in a highly conserved residue of the HAT domain and is predicted to be deleterious. As NAT10 is known to acetylate tubulin, a key structural component of cilia, this seems likely to be disease causing. Using human hertRPE cells, we showed shRNA knockdown of NAT10 resulted in the inhibition of primary cilia formation. We further did NAT10 shRNA knockdown in ciliated human airway epithelial cells and showed complete repression of ciliogenesis. In contrast, reciliating airway epithelial cells treated with control shRNA formed normal motile cilia. Further analysis of zebrafish embryos after Nat10 morpholino injections showed laterality defects consistent with heterotaxy, confirming a requirement for NAT10 in left-right patterning. Conclusion: We identified a novel homozygous NAT10 mutation in heterotaxy patient with ciliary aplasia. We showed NAT10 is required for both formation of nonmotile primary cilia and motile cilia in the airway. Studies in zebrafish embryos further suggest NAT10 is required for motile cilia function regulating left-right patterning. Together these findings support the conclusion that NAT10 mutation can cause heterotaxy and ciliary aplasia phenotypes.

Tubulin posttranslational modifications as regulators of microtubule-based functions in cells

Microtubules (MT) form a dynamic intracellular network that plays essential roles in cellular function and development. They are also the structural components of cilia and flagella. MTs maintain cilia structure and dynamics by the specific interaction of a large set of MT-associated proteins, including molecular motors and MAPs. How the interactions between MTs and their multiple associated proteins are spatio-temporally is not known. An increasing body of evidence supports that tubulin posttranslational-modifications (PTMs) have a key role in regulating the recruitment of protein complexes in the MT because they allow for a rapid, reversible and locally restricted generation of MT diversity. In particular, two PTMs, glutamylation and glycylation are strongly enriched in the cilium. Glycylation is restricted to axonemal MTs, suggesting a highly specialised function, whereas glutamylation is also present in centrioles and basal bodies. Our hypothesis is that differentially glutamylated/glycylated MTs can distinguish MT subpopulations in the cilium allowing for the recruitment of specific proteins. Here we address the role of glutamylation and glycylation in cilia, by modulating the extent of each modification using the ependymal cell system as a model. We study the role of both modifications in cilia physiology, including cilia assembly, maintenance and beating. Our data demonstrate that glycylation is essential for cilia maintenance, whereas glutamylation is required for cilia beating. Taken together, tubulin PTMs are likely to play an important role for cilia and could possibly been linked to human disorders known to be associated with defects in MT-based traffic, such as ciliary dysfunctions (ciliopathies).

Genetic variation in SPAG16 regions encoding the WD40 repeats is not associated with reduced sperm motility and axonemal defects in a population of infertile males

BackgroundSPAG16 is a critical structural component of motile cilia and flagella. In the eukaryotic unicellular algae Chlamydomonas, loss of gene function causes flagellar paralysis and prevents assembly of the “9 + 2” axoneme central pair. In mice, we have previously shown that loss of Spag16 gene function causes male infertility and severe sperm motility defects. We have also reported that a heterozygous mutation of the human SPAG16 gene reduces stability of the sperm axonemal central apparatus.MethodsIn the present study, we analyzed DNA samples from 60 infertile male volunteers of Western European (Italian) origin, to search for novel SPAG16 gene mutations, and to determine whether increased prevalence of SPAG16 single nucleotide polymorphisms (SNPs) was associated with infertility phenotypes. Semen parameters were evaluated by light microscopy and sperm morphology was comprehensively analyzed by transmission electron microscopy (TEM).ResultsFor gene analysis, sequences were generated covering exons encoding the conserved WD40 repeat region of the SPAG16 protein and the flanking splice junctions. No novel mutations were found, and the four SNPs in the assessed gene region were present at expected frequencies. The minor alleles were not associated with any assessed sperm parameter in the sample population.ConclusionsAnalysis of the SPAG16 regions encoding the conserved WD repeats revealed no evidence for association of mutations or genetic variation with sperm motility and ultrastructural sperm characteristics in a cohort of Italian infertile males.

Proteomic analysis of multiple primary cilia reveals a novel mode of ciliary development in mammals

SummaryCilia are structurally and functionally diverse organelles, whose malfunction leads to ciliopathies. While recent studies have uncovered common ciliary transport mechanisms, limited information is available on the proteome of cilia, particularly that of sensory subtypes, which could provide insight into their functional and developmental diversities. In the present study, we performed proteomic analysis of unique, multiple 9+0 cilia in choroid plexus epithelial cells (CPECs). The analysis of juvenile swine CPEC cilia identified 868 proteins. Among them, 396 were shared with the proteome of 9+0 photoreceptor cilia (outer segment), whereas only 152 were shared with the proteome of 9+2 cilia and flagella. Various signaling molecules were enriched in a CPEC-specific ciliome subset, implicating multiplicity of sensory functions. The ciliome also included molecules for ciliary motility such as Rsph9. In CPECs from juvenile swine or adult mouse, Rsph9 was localized to a subpopulation of cilia, whereas they were non-motile. Live imaging of mouse choroid plexus revealed that neonatal CPEC cilia could beat vigorously, and the motility waned and was lost within 1–2 weeks. The beating characteristics of neonatal CPEC cilia were variable and different from those of typical 9+2 cilia of ependyma, yet an Efhc1-mediated mechanism to regulate the beating frequency was shared in both types of cilia. Notably, ultrastructural analysis revealed the presence of not only 9+0 but also 9+2 and atypical ciliary subtypes in neonatal CPEC. Overall, these results identified both conserved and variable components of sensory cilia, and demonstrated a novel mode of ciliary development in mammals.

Primary cilia and Gli3 activity regulate cerebral cortical size

During neural development patterning, neurogenesis, and overall growth are highly regulated and coordinated between different brain regions. Here, we show that primary cilia and the regulation of Gli activity are necessary for the normal expansion of the cerebral cortex. We show that loss of Kif3a, an important functional component of primary cilia, leads to the degeneration of primary cilia, marked overgrowth of the cortex, and altered cell cycle kinetics within cortical progenitors. The G1 phase of the cell cycle is shortened through a mechanism likely involving reduced Gli3 activity and a resulting increase in expression of cyclin D1 and Fgf15. The defects in Gli3 activity alone are sufficient to accelerate cell cycle kinetics and cause the molecular changes seen in brains that lack cilia. Finally, we show that levels of full-length and repressor Gli3 proteins are tightly regulated during normal development and correlate with changes in expression of two known Shh-target genes, CyclinD1 and Fgf15, and with the normal lengthening of the cell cycle during corticogenesis. These data suggest that Gli3 activity is regulated through the primary cilium to control cell cycle length in the cortex and thus determine cortical size.

The structural heterogeneity of radial spokes in cilia and flagella is conserved

Radial spokes (RSs) are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms, the 96 nm axonemal repeat unit contains only a pair of spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pairs in Chlamydomonas flagella have been well characterized, while spoke triplets have received less attention. Here, we used cryoelectron tomography and subtomogram averaging to visualize the three-dimensional structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3, and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable RS heterogeneity is conserved and that organisms with RS pairs contain the basal part of RS3. This conserved RS heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility.

Identification of Signaling Pathways Regulating Primary Cilium Length and Flow-Mediated Adaptation

The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

Polycystins play a key role in the modulation of cholangiocyte proliferation

Background Polycystin-1 and -2 (PC-1 and PC-2) are critical components of primary cilia, which act as mechanosensors and drive cell response to injury. PC-1 activation involves the cleavage/processing of PC-1 cytoplasmic tail, driven by regulated intramembrane proteolysis or ubiquitine/proteasome, translocation in the nucleus and activation of transcription factors. Mutations of PC-1 or PC-2 occur in polycystic liver where cholangiocyte proliferation is enhanced. Aim We evaluated the involvement of PC-1 and PC-2 in modulating cholangiocyte proliferation. Methods We investigated rat cholangiocytes induced to proliferate by 17β-oestradiol. Proliferation was evaluated by PCNA immunoblotting or [ 3H]-thymidine incorporation into DNA. PC-1 silencing was performed by siRNA, while inhibition of regulated intramembrane proteolysis or proteasome by γ-secretase inhibitor, leupeptin or MG115. Results Cholangiocyte proliferation was associated with decreased PC-1 and PC-2 expression, which was inversely correlated with enhanced PCNA. The selective silencing of PC-1 induced activation of cholangiocyte proliferation in association with decreased PC-1 expression. Two different regulated intramembrane proteolysis inhibitors, γ-secretase-inhibitor and leupeptin, and the proteasome inhibitor, MG115, abolished the 17β-oestradiol proliferative effect. Conclusions PC-1 and PC-2 play a major role as modulators of cholangiocyte proliferation suggesting that primary cilia may act as sensors of cell injury driving, when activated, a proliferative cholangiocyte response to trigger the reparative processes.

Radial Spoke Protein 3 Is a Mammalian Protein Kinase A-anchoring Protein That Binds ERK1/2

Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIalpha and RIIbeta, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes.

Mass spectrometric analysis of microtubule co‐sedimented proteins from rat brain

Microtubules (MTs) play crucial roles in a variety of cell functions, such as mitosis, vesicle transport and cell motility. MTs also compose specialized structures, such as centrosomes, spindles and cilia. However, molecular mechanisms of these MT-based functions and structures are not fully understood. Here, we analyzed MT co-sedimented proteins from rat brain by tandem mass spectrometry (MS) upon ion exchange column chromatography. We identified a total of 391 proteins. These proteins were grouped into 12 categories: 57 MT cytoskeletal proteins, including MT-associated proteins (MAPs) and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins. Of these uncharacterized proteins, six proteins were expressed in cultured cells, resulting in the identification of three novel components of centrosomes and cilia. Our present method is not specific for MAPs, but is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures.

Cilia and mitogen‐activated protein kinase signaling

Our lab identified mouse Radial Spoke Protein 3 (RSP3) through a yeast two‐hybrid screen designed to identify interactors that have a higher affinity for the phosphorylated, active form of ERK2 (pERK2). RSP3 is a structural component of motile cilia and an A‐Kinase Anchoring Protein (AKAP) for cAMP‐dependent Protein Kinase (PKA). To confirm our finding, I showed that the human homolog, RSP3H, co‐immunoprecipitates with ERK2 in overexpression studies. Furthermore, I demonstrated, through immunoprecipitation kinase assays, that RSP3H is phosphorylated by pERK2. Additionally, I showed through RT‐PCR that an mRNA transcript of RSP3H is present in HEK293 and HeLa cells, neither of which are thought to contain motile cilia. I also performed immunofluorescence studies on overexpressed RSP3H, and discovered that not only does it localize to cilia‐like structures but also may induce the formation of multiple cilia‐like projections from the cell, thus functioning as a regulator of ciliogenesis. RSP3H may serve to localize ERK1/2 to cilia and function as a point of convergence of cAMP‐dependent and PKA‐mediated action upon ERK1/2 downstream signaling.

The Conserved Proteins CHE-12 and DYF-11 Are Required for Sensory Cilium Function in Caenorhabditis elegans

Sensory neuron cilia are evolutionarily conserved dendritic appendages that convert environmental stimuli into neuronal activity. Although several cilia components are known, the functions of many remain uncharacterized. Furthermore, the basis of morphological and functional differences between cilia remains largely unexplored. To understand the molecular basis of cilia morphogenesis and function, we studied the Caenorhabditis elegans mutants che-12 and dyf-11. These mutants fail to concentrate lipophilic dyes from their surroundings in sensory neurons and are chemotaxis defective. In che-12 mutants, sensory neuron cilia lack distal segments, while in dyf-11 animals, medial and distal segments are absent. CHE-12 and DYF-11 are conserved ciliary proteins that function cell-autonomously and are continuously required for maintenance of cilium morphology and function. CHE-12, composed primarily of HEAT repeats, may not be part of the intraflagellar transport (IFT) complex and is not required for the localization of some IFT components. DYF-11 undergoes IFT-like movement and may function at an early stage of IFT-B particle assembly. Intriguingly, while DYF-11 is expressed in all C. elegans ciliated neurons, CHE-12 expression is restricted to some amphid sensory neurons, suggesting a specific role in these neurons. Our results provide insight into general and neuron-specific aspects of cilium development and function.