C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors

Granule neuron progenitors (GNPs) are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh) secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3), another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis.     

VDAC3 and Mps1 negatively regulate ciliogenesis

Centrosomes serve to organize new centrioles in cycling cells, whereas in quiescent cells they assemble primary cilia. We have recently shown that the mitochondrial porin VDAC3 is also a centrosomal protein that is predominantly associated with the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes. Here, we show that depletion of VDAC3 causes inappropriate ciliogenesis in cycling cells, while expression of GFP-VDAC3 suppresses ciliogenesis in quiescent cells. Mps1 also negatively regulates ciliogenesis, and the inappropriate ciliogenesis caused by VDAC3 depletion can be bypassed by targeting Mps1 to centrosomes independently of VDAC3. Thus, our data show that a VDAC3-Mps1 module at the centrosome promotes ciliary disassembly during cell cycle entry and suppresses cilia assembly in proliferating cells. Our data also suggests that VDAC3 might be a link between mitochondrial dysfunction and ciliopathies in mammalian cells.     

Basal body stability and ciliogenesis requires the conserved component Poc1

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.     

Basal Body Assembly in Ciliates: The Power of Numbers

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly and function. Nonetheless, at this stage, our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium , historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly.     

Centriole translocation and degeneration during ciliogenesis in Caenorhabditis elegans neurons

Neuronal cilia that are formed at the dendritic endings of sensory neurons are essential for sensory perception. However, it remains unclear how the centriole‐derived basal body is positioned to form a template for cilium formation. Using fluorescence time‐lapse microscopy, we show that the centriole translocates from the cell body to the dendrite tip in the Caenorhabditis elegans sensory neurons. The centriolar protein SAS‐5 interacts with the dynein light‐chain LC8 and conditional mutations of cytoplasmic dynein‐1 block centriole translocation and ciliogenesis. The components of the central tube are essential for the biogenesis of centrioles, which later drive ciliogenesis in the dendrite; however, the centriole loses these components at the late stage of centriole translocation and subsequently recruits transition zone and intraflagellar transport proteins. Together, our results provide a comprehensive model of ciliogenesis in sensory neurons and reveal the importance of the dynein‐dependent centriole translocation in this process.     

Keeping the balance between proliferation and differentiation: the primary cilium

Primary cilia are post-mitotic cellular organelles that are present in the vast majority of cell types in the human body. An extensive body of data gathered in recent years is demonstrating a crucial role for this organelle in a number of cellular processes that include mechano and chemo-sensation as well as the transduction of signaling cascades critical for the development and maintenance of different tissues and organs. Consequently, cilia are currently viewed as cellular antennae playing a critical role at the interphase between cells and their environment, integrating a range of stimuli to modulate cell fate decisions including cell proliferation, migration and differentiation. Importantly, this regulatory role is not just a consequence of their participation in signal transduction but is also the outcome of both the tight synchronization/regulation of ciliogenesis with the cell cycle and the role of individual ciliary proteins in cilia-dependent and independent processes. Here we review the role of primary cilia in the regulation of cell proliferation and differentiation and illustrate how this knowledge has provided insight to understand the phenotypic consequences of ciliary dysfunction.     

Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

The LIM protein Ajuba is required for ciliogenesis and left-right axis determination in medaka

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer’s vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.     

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.     

From Zero to Many: Control of Centriole Number in Development and Disease

Centrioles are essential for the formation of microtubule-derived structures, including cilia, flagella and centrosomes. These structures are involved in a variety of functions, from cell motility to division. In most dividing animal cells, centriole formation is coupled to the chromosome cycle. However, this is not the case in certain specialized divisions, such as meiosis, and in some differentiating cells. For example, oocytes loose their centrioles upon differentiation, whereas multiciliated epithelial cells make several of those structures after they exit the cell cycle. Aberrations of centriole number are seen in many cancer cells. Recent studies began to shed light on the molecular control of centriole number, its variations in development, and how centriole number changes in human disease. Here we review the recent developments in this field.     

Development of a multiciliated cell

Multiciliated cells (MCC) are evolutionary conserved, highly specialized cell types that contain dozens to hundreds of motile cilia that they use to propel fluid directionally. To template these cilia, each MCC produces between 30 and 500 basal bodies via a process termed centriole amplification. Much progress has been made in recent years in understanding the pathways involved in MCC fate determination, differentiation, and ciliogenesis. Recent studies using mammalian cell culture systems, mice, Xenopus, and other model organisms have started to uncover the mechanisms involved in centriole and cilia biogenesis. Yet, how MCC progenitor cells regulate the precise number of centrioles and cilia during their differentiation remains largely unknown. In this review, we will examine recent findings that address this fundamental question.     

The centrosome duplication cycle in health and disease

Centrioles function in the assembly of centrosomes and cilia. Structural and numerical centrosome aberrations have long been implicated in cancer, and more recent genetic evidence directly links centrosomal proteins to the etiology of ciliopathies, dwarfism and microcephaly. To better understand these disease connections, it will be important to elucidate the biogenesis of centrioles as well as the controls that govern centriole duplication during the cell cycle. Moreover, it remains to be fully understood how these organelles organize a variety of dynamic microtubule-based structures in response to different physiological conditions. In proliferating cells, centrosomes are crucial for the assembly of microtubule arrays, including mitotic spindles, whereas in quiescent cells centrioles function as basal bodies in the formation of ciliary axonemes. In this short review, we briefly introduce the key gene products required for centriole duplication. Then we discuss recent findings on the centriole duplication factor STIL that point to centrosome amplification as a potential root cause for primary microcephaly in humans. We also present recent data on the role of a disease-related centriole-associated protein complex, Cep164-TTBK2, in ciliogenesis.     

A molecular marker for centriole maturation in the mammalian cell cycle

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.     

DISCO is key to successful centriole maturation

Centriole maturation is essential for ciliogenesis, but which proteins and how they regulate ciliary assembly is unclear. In this issue, Kumar et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202011133) shed light on this process by identifying a ciliopathy complex at the distal mother centriole that restrains centriole length and supports the formation of distal appendages.

The primary cilium plays a crucial role in embryonic development by allowing the integration of a variety of inputs, including chemical and mechanical signals. Primary cilia are found on most cell types; thereby, mutations in genes encoding cilia components may perturb many cellular functions, including airway mucus clearance, mechanosensation, and cell signaling, which are central regulators of organ function and homeostasis. Numerous mutations leading to ciliary dysfunction have been identified in recent years and thus linked to human cilia-related diseases, called ciliopathies (1, 2). Some of these mutations affect components of the centrioles, which are cytoplasmic cylindrical structures composed by triplets of microtubules arranged in a ninefold symmetry.Cilia originate from centrioles and are anchored to the cell surface. In most mammalian cells, centrioles are present within the centrosome, the main organizing center of microtubules. During G1 phase, cells have one centrosome containing two centrioles of different ages. The older mother centriole is distinguished from the younger daughter centriole by the presence of two sets of appendages organized around its circumference. The centrosome duplicates in S phase and, as a result, a new centriole is formed orthogonally to each parent centriole. The new centrioles subsequently elongate during S and G2 phases, and each daughter cell inherits a parent and a newly formed centriole after mitosis. During this transition, new centrioles become daughter centrioles, and the daughter centriole from the previous cycle acquires appendages to mature into a mother centriole. Distal appendages (DAs) are essential for anchoring the mother centriole to the plasma membrane and for the formation of a cilium (2). The formation of a mature centriole competent for ciliogenesis is therefore a complex process taking place over three successive cell cycles.Different molecular factors required for the progressive maturation of centrioles and the assembly of DAs have been identified in the past, and perturbation of their function has been linked to ciliopathies (2, 3). However, the precise mechanism by which DAs are assembled onto centrioles remains elusive. In this issue, Kumar et al. focused their attention on CEP90, a poorly characterized protein whose mutations have been implicated in several ciliopathies (4). CEP90 is a component of centriolar satellites, which are proteinaceous granules located at the periphery of the centrosome (5, 6). Using a combination of expansion microscopy and structured illumination super-resolution microscopy techniques, the authors found that CEP90 also localized to centrioles, where it formed a discontinuous ring with a ninefold symmetry. CEP90 localized near a well-characterized DA component, CEP164, which was consistent with CEP90 being present at the base of these appendages. Then, they searched for CEP90 interactors. For that, the researchers first had to circumvent the shortcoming of discriminating between interactions that may take place at the centrosome from those occurring within centriolar satellites. To get around this, Kumar et al. used a cell line in which satellite assembly is inhibited. Among the candidates they found interacting with CEP90 at the centrosome were OFD1 and Moonraker (MNR), which are two proteins previously associated with multiple ciliopathies. OFD1 is a centriole component required to restrict centriole elongation and assemble DAs (7). MNR, also called OFIP or KIAA073, is a satellite component necessary for cilia formation (8). Making again use of super-resolution microscopy, the authors showed that all three proteins colocalized at the centriole distal end, with the MNR protein being the closest to the centriole wall, so they named this newly identified complex after DISCO (distal centriole complex).Next, Kumar et al. elegantly demonstrated that, as previously shown for OFD1 (7), inactivating either CEP90 or MNR led to the absence of cilia in cells. In mice, deficiency of any of these proteins resulted in Hedgehog signaling inhibition and early arrest of embryonic development. As reported for OFD1-deficient cells, loss of MNR in human cells resulted in overly long centrioles. However, centriole length was normal in CEP90-deficient cells, suggesting partially distinct functions between members of the DISCO complex. The authors noted that ciliogenesis was blocked at an early stage in CEP90^−/− and MNR^−/− cells and, given that DAs are essential for centriole anchoring and ciliogenesis, they decided to examine DA organization in these cells (4). Indeed, they found that DA components, such as CEP83, were not recruited during centriole maturation in MNR^−/− or CEP90^−/− cells, and DAs were not detected by electron microscopy. These findings pointed out that CEP90 and MNR, like OFD1, were required for the assembly of DAs.Since CEP90 is required for satellite accumulation around the centrosome, and satellites are, in turn, essential for ciliogenesis (6), one possible explanation to their results is that CEP90 might affect DA assembly indirectly via its role in satellite localization. To answer this question, the authors again used cells lacking centriolar satellites. CEP90 was correctly localized at centriole distal ends in these cells, and DAs were formed, supporting a direct requirement for the centriolar pool of CEP90 in DA assembly. Putting all their data together, Kumar et al. proposed the following model: First, MNR is recruited to elongating centrioles, which, in turn, triggers the recruitment of OFD1 to arrest elongation at the end of the first cell cycle. MNR and OFD1 then recruit CEP90, which initiates the recruitment of DA components, including CEP83, at the end of the following cell cycle (Fig. 1). Thus, the DISCO complex allows for coupling the arrest of centriole elongation to centriole maturation across successive cell cycles.Open in a separate windowFigure 1.The DISCO complex restrains centriole elongation and initiates DA assembly. (1) The DISCO complex member MNR is recruited first at the distal end of assembling centrioles. MNR then recruits other members of the complex, including OFD1, which inhibits centriole elongation at the end of the first cell cycle, i.e., when newly formed centrioles become daughter centrioles (DCs). Other members of the complex include CEP90 and possibly also FOPNL. (2) At the end of the following cell cycle, as the daughter centriole matures into a mother centriole (MC), CEP90 initiates the recruitment of CEP83, the most upstream component in DA assembly. A previously identified interaction between OFD1 and another DA component, CEP89, might also contribute to DA organization (10). Proteins are drawn in contact with each other when an interaction or hierarchical recruitment was described (3, 4, 8, 11).Besides OFD1 and MNR proteins, Kumar et al. also identified a protein called FGFR1OP N-Terminal Like (FOPNL or FOR20) as a potential CEP90 interactor (4). Interestingly, this interaction was confirmed in a recent study describing that a complex containing CEP90, OFD1, and FOPNL localizes at the distal end of Paramecium centrioles and is necessary for the recruitment of DA components and centriole docking in Paramecium and human cells (9). FOPNL was previously found in complex with MNR and OFD1 and shown to facilitate their interaction (8). Together, these data suggest that the DISCO complex could also include FOPNL. The functional similarities of some of the components of the DISCO complex between Paramecium and humans strongly suggest that the role of DISCO in centriole maturation and ciliogenesis is broadly conserved across species.Previous studies in different organisms have underpinned the relevance of ciliopathy-associated proteins to ensure normal organism development and tissue function (1, 2). Overall, the findings by Kumar et al. highlight the critical role of a ciliopathy-associated protein complex at distal centrioles in building distal appendages, thus supporting centriole maturation and ciliogenesis in rodents and human cells (4).     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

Mitochondrial origin of centrioles and cilia in some human endocrine neoplasms.

Fine structural investigation of surgically removed human pituitary and parathyroid adenomas, pheochromocytomas and bronchial carcinoids revealed a hitherto undetected sequence of events in the formation of centrioles and cilia indicating that mitochondria may serve as their progenitors. The first steps seem to be the disappearance of mitochondrial cristae and a polar accumulation of a fibrillar-granular material with a subsequent increase of electron density of the double mitochondrial membranes and deposition of more electron opaque substance within and around these procentriolar bodies. This process is followed by the disintegration of the double membranes and an asymmetrical division of the electron dense aggregate. The larger part seems to be the precursor of the primary centriole (basal body) whereas the smaller one that of the secondary centriole. Formation of centrioleand rudimentary cilium-like structures was disclosed within the unaltered mitochondrial membranes of oncocytic cells present in two pituitary adenomas and in one pheochromocytoma. Accumulation of procentriolar bodies and mature centrioles, noted in some tumors, may be due to a defect in the process of centriolo- and ciliogenesis. It is conceivable that the mitochondrial genome plays an important role in formation of centrioles and cilia.     

Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1^−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1^−/− cells.