Ciliary Length Control Research Articles

Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control.

Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi-subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin-2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP. KAP dissociates from IFT at the ciliary tip and diffuses back to the cell body. This observation led to the diffusion-as-a-ruler model of ciliary length control, which postulates that KAP is progressively sequestered into elongating cilia because its return to the cell body will require increasingly more time, limiting motor availability at the ciliary base, train assembly, building block supply, and ciliary growth. Here, we show that Chlamydomonas FLA8 also returns to the cell body by diffusion. However, more than 95% of KAP and FLA8 are present in the cell body and, at a given time, just ~1% of the motor participates in IFT. After repeated photobleaching of both cilia, IFT of fluorescent kinesin subunits continued indicating that kinesin-2 cycles from the large cell-body pool through the cilia and back. Furthermore, growing and full-length cilia contained similar amounts of kinesin-2 subunits and the size of the motor pool at the base changed only slightly with ciliary length. These observations are incompatible with the diffusion-as-a-ruler model, but rather support an "on-demand model," in which the cargo load of the trains is regulated to assemble cilia of the desired length.

The Rab GTPase-binding protein EHBP1L1 and its interactors CD2AP/CIN85 negatively regulate the length of primary cilia via actin remodeling

Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of WT EHBP1L1, although this was not observed in the CD2AP/CIN85-binding-deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.

FBW7 couples structural integrity with functional output of primary cilia

Structural defects in primary cilia have robust effects in diverse tissues and systems. However, how disorders of ciliary length lead to functional outcomes are unknown. We examined the functional role of a ciliary length control mechanism of FBW7-mediated destruction of NDE1, in mesenchymal stem cell (MSC) differentiation. We show that FBW7 functions as a master regulator of both negative (NDE1) and positive (TALPID3) regulators of ciliogenesis, with an overall positive net effect on primary cilia formation, MSC differentiation to osteoblasts, and bone architecture. Deletion of Fbxw7 suppresses ciliation, Hedgehog activity, and differentiation, which are partially rescued in Fbxw7/Nde1-null cells. We also show that NDE1, despite suppressing ciliogenesis, promotes MSC differentiation by increasing the activity of the Hedgehog pathway by direct binding and enhancing GLI2 activity in a cilia-independent manner. We propose that FBW7 controls a protein-protein interaction network coupling ciliary structure and function, which is essential for stem cell differentiation.

CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels.

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

Functional exploration of heterotrimeric kinesin-II in IFT and ciliary length control in Chlamydomonas.

Heterodimeric motor organization of kinesin-II is essential for its function in anterograde IFT in ciliogenesis. However, the underlying mechanism is not well understood. In addition, the anterograde IFT velocity varies significantly in different organisms, but how this velocity affects ciliary length is not clear. We show that in Chlamydomonas motors are only stable as heterodimers in vivo, which is likely the key factor for the requirement of a heterodimer for IFT. Second, chimeric CrKinesin-II with human kinesin-II motor domains functioned in vitro and in vivo, leading to a ~ 2.8 fold reduced anterograde IFT velocity and a similar fold reduction in IFT injection rate that supposedly correlates with ciliary assembly activity. However, the ciliary length was only mildly reduced (~15%). Modeling analysis suggests a nonlinear scaling relationship between IFT velocity and ciliary length that can be accounted for by limitation of the motors and/or its ciliary cargoes, e.g. tubulin.

AC6 regulates the microtubule-depolymerizing kinesin KIF19A to control ciliary length in mammals

Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders.

Analysis of ciliary status via G-protein-coupled receptors localized on primary cilia.

G-protein-coupled receptors (GPCRs) comprise the largest and most diverse cell surface receptor family, with more than 800 known GPCRs identified in the human genome. Binding of an extracellular cue to a GPCR results in intracellular G protein activation, after which a sequence of events, can be amplified and optimized by selective binding partners and downstream effectors in spatially discrete cellular environments. Because GPCRs are widely expressed in the body, they help to regulate an incredible range of physiological processes from sensation to growth to hormone responses. Indeed, it is estimated that ∼ 30% of all clinically approved drugs act by binding to GPCRs. The primary cilium is a sensory organelle composed of a microtubule axoneme that extends from the basal body. The ciliary membrane is highly enriched in specific signaling components, allowing the primary cilium to efficiently convey signaling cascades in a highly ordered microenvironment. Recent data demonstrated that a limited number of non-olfactory GPCRs, including somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1), are selectively localized to cilia on several mammalian cell types including neuronal cells. Utilizing cilia-specific cell biological and molecular biological approaches, evidence has accumulated to support the biological importance of ciliary GPCR signaling followed by cilia structural changes. Thus, cilia are now considered a unique sensory platform for integration of GPCR signaling toward juxtaposed cytoplasmic structures. Herein, we review ciliary GPCRs and focus on a novel role of MCHR1 in ciliary length control that will impact ciliary signaling capacity and neuronal function.

Elucidating CCRK’s function in ciliary assembly and cilium‐dependent signaling

Our studies are focused on the questions of how ciliary length and ciliary import of cell signaling regulators are controlled. Despite many advances, these questions have yet to be fully answered. One clue as to the mechanistic basis of these processes comes from studies on Cell Cycle‐Related Kinase (CCRK). Work on the Clamydomonas homolog, LF2, indicates that this kinase ensures that flagella are neither too long nor too short, likely mediated by regulated transport of ciliary tubulin ( Craft et al., 2015). We previously showed that mammalian CCRK is similarly required for ciliary length control and that it is needed for cells to achieve appropriate responses to Hedgehog (Hh) signals ( Snouffer et al., 2017). Additional work indicated that CCRK promotes the efficient transport of Hh pathway regulatory factors, such as Smoothened and Gli2, into cilia. However, the manner by which the kinase directs these processes is unknown. One target of CCRK phosphorylation is the ciliary length regulator Intestinal Cell Kinase (ICK); CCRK‐mediated phosphorylation in ICK’s TDY motif promotes ICK activity. Our analysis of Ccrk/Ick double mutants indicates that CCRK must also function via ICK‐independent mechanisms and that ICK promotes functions in the absence of CCRK‐mediated phosphorylation. To gain insight into the ICK‐independent CCRK functions in ciliary length control and ciliary cargo import, we performed phosphoproteomic screens for potential mediators of CCRK function. This screen has pointed to candidate mediators that regulate microfilament and microtubule dynamics. Consistent with a role for CCRK’s action via microfilament dynamics, Ccrk mutant, but not wild‐type, cells showed greatly reduced import of ciliary cargo upon treatment with sub‐threshold doses of the actin polymerization inhibitor Cytochalasin D. Our ongoing work on this mechanism will be presented.Support or Funding InformationThis work was supported by National Institute of Child Health and Human Development grant number R01HD050761 and by the Office of the Vice President of Research at the University of Georgia.

A CCRK and a MAK Kinase Modulate Cilia Branching and Length via Regulation of Axonemal Microtubule Dynamics in Caenorhabditis elegans

The diverse morphologies of primary cilia are tightly regulated as a function of cell type and cellular state. CCRK- and MAK-related kinases have been implicated in ciliary length control in multiple species, although the underlying mechanisms are not fully understood. Here, we show that in C.elegans, DYF-18/CCRK and DYF-5/MAK act in a cascade to generate the highly arborized cilia morphologies of the AWA olfactory neurons. Loss of kinase function results in dramatically elongated AWA cilia that lack branches. Intraflagellar transport (IFT) motor protein localization, but not velocities, in AWA cilia is altered upon loss of dyf-18. We instead find that axonemal microtubules are decorated by the EBP-2 end-binding protein along their lengths and that the tubulin load is increased and tubulin turnover is reduced in AWA cilia of dyf-18 mutants. Moreover, we show that predicted microtubule-destabilizing mutations in two tubulin subunits, as well as mutations in IFT proteins predicted to disrupt tubulin transport, restore cilia branching and suppress AWA cilia elongation in dyf-18 mutants. Loss of dyf-18 is also sufficient to elongate the truncated rod-like unbranched cilia of the ASH nociceptive neurons in animals carrying a microtubule-destabilizing mutation in a tubulin subunit. We suggest that CCRK and MAK activity tunes cilia length and shape in part via modulation of axonemal microtubule stability, suggesting that similar mechanisms may underlie their roles in ciliary length control in other cell types.

HDAC3 and HDAC8 are required for cilia assembly and elongation.

ABSTRACTCilia are extended from mother centrioles in quiescent G0/G1 cells and retracted in dividing cells. Diverse post-translational modifications play roles in the assembly and disassembly of the cilium. Here, we examined class I histone deacetylases (HDACs) as positive regulators of cilia assembly in serum-deprived RPE1 and HK2 cells. We observed that the number of cells with cilia was significantly reduced in HDAC3- and HDAC8-depleted cells. The ciliary length also decreased in HDAC3- and HDAC8-depleted cells compared to that in control cells. A knockdown-rescue experiment showed that wild-type HDAC3 and HDAC8 rescued the cilia assembly and ciliary length in HDAC3- and HDAC8-depleted cells, respectively; however, deacetylase-dead HDAC3 and HDAC8 mutants did not. This suggests that deacetylase activity is critical for both HDAC3 and HDAC8 function in cilia assembly and ciliary length control. This is the first study to report that HDACs are required for the assembly and elongation of the primary cilia.

Ciliary Length Sensing Regulates IFT Entry via Changes in FLA8/KIF3B Phosphorylation to Control Ciliary Assembly

The length of cilia is robustly regulated [1]. Previous data suggest that cells possess a sensing system to control ciliary length [2-5]. However, the details of the mechanism are currently not known [6, 7]. Such a system requires a mechanism that responds to ciliary length, and consequently, disruption of that response system should alter ciliary length [1]. The assembly rate of cilium mediated by intraflagellar transport (IFT) gradually decreases as the cilium elongates and eventually is balanced by the constant rate of disassembly, at which point cilium elongation stops [8, 9]. Because the rate of IFT entry into the cilium also decreases as the cilium elongates [10], regulation of IFT entry could provide the mechanism for length control. Previously, we showed that phosphorylation of the FLA8/KIF3B subunit of the anterograde kinesin-II IFT motor blocks IFT entry and flagellar assembly in Chlamydomonas [11]. Here, we show in Chlamydomonas that cellular signaling in response to alteration of flagellar length regulates phosphorylation of FLA8/KIF3B, which restricts IFT entry and, thus, flagellar assembly to control flagellar length. Cellular levels of phosphorylated FLA8 (pFLA8) are tightly linked to flagellar length: FLA8 phosphorylation is reduced in cells with short flagella and elevated in cells with long flagella. Depletion of the phosphatases CrPP1 and CrPP6 increases the level of cellular pFLA8, leading to short flagella due to decreased IFT entry. The results demonstrate that ciliary length control is achieved by a cellular sensing system that controls IFT entry through phosphorylation of the anterograde IFT motor.

TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-β (TGF-β) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1−/− and Tsc2−/− cells, and we found that Tsc1 is required for TGF-β induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1−/− cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2−/− cells. Furthermore, we observed that Tsc1−/− MEFs display significantly elongated cilia, whereas cilia in Tsc2−/− MEFs were shorter than normal. The elongated cilium phenotype of Tsc1−/− MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1−/− MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.

Cellular Mechanisms of Ciliary Length Control.

Cilia and flagella are evolutionarily conserved, membrane-bound, microtubule-based organelles on the surface of most eukaryotic cells. They play important roles in coordinating a variety of signaling pathways during growth, development, cell mobility, and tissue homeostasis. Defects in ciliary structure or function are associated with multiple human disorders called ciliopathies. These diseases affect diverse tissues, including, but not limited to the eyes, kidneys, brain, and lungs. Many processes must be coordinated simultaneously in order to initiate ciliogenesis. These include cell cycle, vesicular trafficking, and axonemal extension. Centrioles play a central role in both cell cycle progression and ciliogenesis, making the transition between basal bodies and mitotic spindle organizers integral to both processes. The maturation of centrioles involves a functional shift from cell division toward cilium nucleation which takes place concurrently with its migration and fusion to the plasma membrane. Several proteinaceous structures of the distal appendages in mother centrioles are required for this docking process. Ciliary assembly and maintenance requires a precise balance between two indispensable processes; so called assembly and disassembly. The interplay between them determines the length of the resulting cilia. These processes require a highly conserved transport system to provide the necessary substances at the tips of the cilia and to recycle ciliary turnover products to the base using a based microtubule intraflagellar transport (IFT) system. In this review; we discuss the stages of ciliogenesis as well as mechanisms controlling the lengths of assembled cilia.

Current topics of functional links between primary cilia and cell cycle.

Primary cilia, microtubule-based sensory structures, orchestrate various critical signals during development and tissue homeostasis. In view of the rising interest into the reciprocal link between ciliogenesis and cell cycle, we discuss here several recent advances to understand the molecular link between the individual step of ciliogenesis and cell cycle control. At the onset of ciliogenesis (the transition from centrosome to basal body), distal appendage proteins have been established as components indispensable for the docking of vesicles at the mother centriole. In the initial step of axonemal extension, CP110, Ofd1, and trichoplein, key negative regulators of ciliogenesis, are found to be removed by a kinase-dependent mechanism, autophagy, and ubiquitin–proteasome system, respectively. Of note, their disposal functions as a restriction point to decide that the axonemal nucleation and extension begin. In the elongation step, Nde1, a negative regulator of ciliary length, is revealed to be ubiquitylated and degraded by CDK5-SCFFbw7 in a cell cycle-dependent manner. With regard to ciliary length control, it has been uncovered in flagellar shortening of Chlamydomonas that cilia itself transmit a ciliary length signal to cytoplasm. At the ciliary resorption step upon cell cycle re-entry, cilia are found to be disassembled not only by Aurora A-HDAC6 pathway but also by Nek2-Kif24 and Plk1-Kif2A pathways through their microtubule-depolymerizing activity. On the other hand, it is becoming evident that the presence of primary cilia itself functions as a structural checkpoint for cell cycle re-entry. These data suggest that ciliogenesis and cell cycle intimately link each other, and further elucidation of these mechanisms will contribute to understanding the pathology of cilia-related disease including cancer and discovering targets of therapeutic interventions.

Cilium-autonomous regulation of tubulin transport by IFT

Results GFP-tagged a-tubulin entered Chlamydomonas cilia as a cargo of IFT and by diffusion. IFT-based transport of GFP-tubulin occurred at a low frequency in full-length steady-state cilia and was strongly increased during ciliary growth when IFT trains carried more tubulin. Cells possessing both non-growing and growing cilia selectively targeted GFP-tubulin into the latter indicating that cells regulate tubulin influx individually for each cilium. The preferential delivery of tubulin boosted the concentration of soluble tubulin in the matrix of growing cilia. Cilia length mutants showed abnormal kinetics of tubulin transport, suggesting that ciliary length control involves a regulation of the occupancy of IFT trains by tubulin cargoes.

Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas.

Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, α-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly.

TGF-β Signaling Regulates the Differentiation of Motile Cilia

The cilium is a small cellular organelle with motility- and/or sensory-related functions that plays a crucial role during developmental and homeostatic processes. Although many molecules or signal transduction pathways that control cilia assembly have been reported, the mechanisms of ciliary length control have remained enigmatic. Here, we report that Smad2-dependent transforming growth factor β (TGF-β) signaling impacts the length of motile cilia at the Xenopus left-right (LR) organizer, the gastrocoel roof plate (GRP), as well as at the neural tube and the epidermis. Blocking TGF-β signaling resulted in the absence of the transition zone protein B9D1/MSKR-1 from cilia in multi-ciliated cells (MCCs) of the epidermis. Interestingly, this TGF-β activity is not mediated by Mcidas, Foxj1, and RFX2, the known major regulators of ciliogenesis. These data indicate that TGF-β signaling is crucial for the function of the transition zone, which in turn may affect the regulation of cilia length.

Organelle Size: A Cilium Length Signal Regulates IFT Cargo Loading

Cilia grow by assembling structural precursors delivered to their tips by intraflagellar transport. New work on ciliary length control indicates that, during ciliary growth, cilia send a length signal to the cytoplasm that regulates cargo loading onto the constitutively trafficking intraflagellar transport machinery.

KIF19A Is a Microtubule-Depolymerizing Kinesin for Ciliary Length Control

Cilia control homeostasis of the mammalian body by generating fluid flow. It has long been assumed that ciliary length-control mechanisms are essential for proper flow generation, because fluid flow generation is a function of ciliary length. However, the molecular mechanisms of ciliary length control in mammals remain elusive. Here, we suggest that KIF19A, a member of the kinesin superfamily, regulates ciliary length by depolymerizing microtubules at the tips of cilia. Kif19a(-/-) mice displayed hydrocephalus and female infertility phenotypes due to abnormally elongated cilia that cannot generate proper fluid flow. KIF19A localized to cilia tips, and recombinant KIF19A controlled the length of microtubules polymerized from axonemes in vitro. KIF19A had ATP-dependent microtubule-depolymerizing activity mainly at the plus end of microtubules. Our results indicated a molecular mechanism of ciliary length regulation in mammals, which plays an important role in the maintenance of the mammalian body.

The primary cilium as a multiple cellular signaling scaffold in development and disease

Primary cilia, single hair-like appendage on the surface of the most mammalian cells, were once considered to be vestigial cellular organelles for a past century because of their tiny structure and unknown function. Although they lack ancestral motility function of cilia or flagella, they share common ground with multiciliated motile cilia and flagella on internal structure such as microtubule based nine outer doublets nucleated from the base of mother centrioles called basal body. Making cilia, ciliogenesis, in cells depends on the cell cycle stage due to reuse of centrioles for cell division forming mitotic spindle pole (M phase) and assembling cilia from basal body (starting G1 phase and maintaining most of interphase). Ciliary assembly required two conflicting processes such as assembly and disassembly and balance between these two processes determines the length of cilia. Both process required highly conserved transport system to supply needed substance to grow tip of cilia and bring ciliary turnover product back to the base of cilia using motor protein, kinesin and dynein, and transport protein complex, IFT particles. Disruption of ciliary structure or function causes multiple human disorder called ciliopathies affecting disease of diverse ciliated tissues ranging from eye, kidney, respiratory tract and brain. Recent explosion of research on the primary cilia and their involvement on animal development and disease attracts scientific interest on how extensively the function of cilia related to specific cell physiology and signaling pathway. In this review, I introduce general features of primary cilia and recent progress in understanding of the ciliary length control and signaling pathways transduced through primary cilia in vertebrates.