Cilia and cell cycle re-entry

An Overview of the Cilia

Vertebrate cilia are divided into two groups: motile and non-motile (or primary) cilia. Motile cilia consist of nine bundles of doublet microtubules surrounding a central pair of singlet micro-tubules (9 + 2).^1,2 They are generally present in multiciliated bundles (with the exception of the sperm tail) and form synchronous, bilateral contours, generating a physical flow to move mucus in the respiratory epithelium or fluid in the female reproductive tract.³ In contrast, primary cilia lack the central pair of micro-tubules (9 + 0 configuration).³ With the exception of the 9 + 0 cilium found in the embryonic node, which generates a leftward fluid flow to establish left-right asymmetry during early development, the primary cilium is solitary and non-motile in almost all other vertebrate cell types.⁴ In this review, we focus on the role of primary cilia in cell cycle regulation. However, the reader is directed to many other excellent reviews on this topic.^5–12

Primary cilia consist of the axoneme, a microtubular structure sheathed by a specialized form of the plasma membrane and the basal body, also a microtubular structure forming the base of the cilium. The basal body originates from the centrosome, which is comprised of the mother and daughter centrioles, wrapped around a slurry of proteins that forms the pericentriolar matrix. The centrosome serves as a microtubule-organizing center or a spindle pole during active cell division. The basal body is considered a “differentiated” form of the centrosome. As cells exit the cell cycle, the centrosome differentiates into a basal body, enabling it to anchor to the plasma membrane. Following anchoring or docking to the plasma membrane, the basal body serves as a “template” in nucleating α- and β-tubulin heterodimers to form the ciliary axoneme.^3,13 Post-translational modifications in the axonemal tubulins, including acetylation, detyrosination, polyglutamylation and glycylation, confer structural rigidity of the nucleating microtubules.^14,15 During cilium biogenesis, several accessory structures are formed, one of which is the transition zone.^3,16 It consists of a fibrous material that is found at the verge of the basal body and axoneme, filtering ciliary proteins from the cytoplasm.^3,16 Other structures, such as striated rootlets and basal feet, are thought to provide structural support of the cilium, as basal bodies devoid of these appendages lead to increased structural fragility of the cilium.¹⁷

Since there is no de novo protein synthesis within the cilium, proteins move in and out of the cilium through an evolutionary conserved process called intraflagellar transport (IFT).^18,19 IFT is an active process based on the bidirectional movement of multiprotein particles that form two complexes, namely A and B, which are powered by motor proteins.¹⁸ IFT complex A is mainly associated with retrograde motor proteins, whereas IFT complex B is associated with anterograde motor proteins.¹⁸ Extensive work in the green algae Chlamydomonas reinhardtii has revealed that cargo proteins are loaded onto IFT complex B and directed to the tip of the axoneme.¹⁸ Turnover products on the other hand are picked up by IFT complex A and brought back to the cell body to be reused or degraded.^18,20 Consistently, many loss-of function mutations in core components of anterograde IFT lead to short cilia or lack of cilia, whereas mutations in components of the retrograde IFT lead to bulged cilia.

Cilia and the Cell Cycle

Cilia typically begin to form during the G₁ or G₀ phase of the cell cycle and begin to disassemble as cells re-enter the cell cycle.^21,22 The mechanism(s) by which entry into G₁/G₀ triggers the differentiation of the centrosome to a basal body, perhaps the earliest stage(s) of ciliogenesis, was extensively discussed in a recent review in reference ⁷. Here we emphasize the mechanism(s) by which cell cycle re-entry induces ciliary resorption and, conversely, how ciliary resorption can influence cell cycle re-entry.

Ciliary resorption has been most extensively studied in cell culture, where cells are arrested in G₀ by serum starvation and then induced to re-enter the cell cycle using serum or defined growth factors.²¹ Recent work indicates that the retraction process of the cilia upon growth factor stimulation involves several proteins, such as human enhancer of filamentation 1 (HEF1), Aurora A kinase, Pitchfork (Pifo) and Tctex-1.^23–25 Pugacheva and colleagues first showed that growth factor stimulation of serum-deprived cells induces ciliary disassembly through the sequential activation of HEF1 and Aurora A, which, in turn, activates his-tone deacetylase 6 (HDAC6) at the axoneme.²³ HDAC6 deacetylates axonemal microtubules facilitating resorption.²³ Inhibition of HEF1 or Aurora A kinase blocks cilia disassembly, whereas constitutively active variants of these proteins accelerate this process.²³ Consistently, chemical inhibition of HDAC6 blocks serum-induced ciliary resorption.²³ These studies identified the components of a ciliary resorption program induced by cell cycle re-entry.

Pitchfork (Pifo) is a recently identified gene that is expressed in the pit cells of the embryonic node, the floor plate of the neural tube, the apical ectodermal ridge and growth zone of the embryonic limb bud.²⁴ All of these structures are extremely sensitive to cilia-based signaling during embryonic development. Kinzel and colleagues showed that Pifo accumulates at the basal body during the early stages of cilia disassembly and participates in the ciliary resorption process working along with Aurora A.²⁴ The authors reported defects in ciliary disassembly, centriole overduplication and multipolar spindle formation in cells lacking one allele of Pifo.^23,24 Remarkably, these cells retained cilia during mitosis.²⁴ Similar cellular phenotypes were seen when Aurora A kinase activity was inhibited by overexpression of a pathogenic mutant of Pifo, R80K.²⁴ Mice or humans heterozygous for mutations in Pifo were shown to have laterality defects, possibly due to structural defects of the cilia in the embryonic node,²⁴ suggesting that the pool of Pifo at the basal body during the initial course of cilia resorption may account for proper left-right patterning.

While mutations in human PIFO can block ciliary dissasembly, naturally occurring mutations in the inositol polyphosphate-5-phosphatase E (INPP5E), described in patients with Joubert syndrome, accelerate ciliary dissasembly.^26,27 INPP5E is a lipid phosphatase localized exclusively at the cilia and removes the 5-phosphate group from PI(3,4,5)P3 and PI(4,5)P2.^26,27 Loss of INPP5E results in an excess of these precursors in the axonemal membrane.^26,27 While the percentage of ciliated cells and ciliary length appeared unaffected in cells lacking INPP5E, ultrastructural analysis of the primary cilia showed dilations throughout the axoneme.^26,27 Further, ciliary resorption was accelerated in response to serum or a combination of defined growth factors resulting in a faster cell cycle reentry.^26,27 It was concluded that the ciliary pool of INPP5E maintains an appropriate balance of certain phospholipids, wherein a disturbance of this balance could lead to destabilization of the primary cilium in response to a mitogenic stimulus that could, in turn, result in faster ciliary resorption and faster cell cycle re-entry.^26,27 Mice lacking INPP5E show numerous developmental defects, ranging from kidney cysts to polydactyly and delayed ossification of metacarpals and phalanges to anencephaly and exencephaly,^26,27 highlighting the important role of ciliary resorption, per se, during embryonic development.

Additional evidence implicating ciliary resorption in cell cycle re-entry comes from Li et al., who identified a new role of Tctex-1 in both ciliary resorption and regulation of G₁/S transition.²⁵ Tctex-1 is a light chain component of the cytoplasmic dynein.²⁵ However, phosphorylation of Tctex-1 at T94 uncouples it from the dynein holocomplex, leading to an accumulation at the transition zone of the cilia upon serum stimulation.²⁵ Depletion of wild type or expression of a Tctex-1 mutant unable to be phosphorylated at T94 led to a block in ciliary resorption, which, in turn, suppressed cell cycle re-entry.²⁵ Accumulation of phospho(T94)Tctex-1 at the transition zone not only preceded ciliary resorption, but was also essential for ciliary resorption and entry into S. Because these effects were seen only with the phosphomimetic form of Tctex-1 and exclusively in ciliated cells, the authors proposed that Tctex-1 controlled ciliary resorption and entry into S through a function independent of its role as a component of the cytoplasmic dynein.²⁵ The results were more remarkable in vivo, where radial glial cells depleted of Tctex-1 prematurely differentiated to cortical neurons, resulting in the reduction of neural progenitor cell population.²⁵ Conversely, ectopic expression of a phosphomimetic mutant of the Tctex-1 led to a significant increase in the progenitor cell population.²⁵ Altogether, these data showed that cilia disassembly, per se, can directly regulate the G₁/S transition through an essential role in neocorticogenesis.²⁵

Kim et al. also showed that when cells are induced to form longer cilia, they show a delay in G₁ to S transition, reinforcing the idea that cilia can influence cell cycle re-entry.²⁸ Using cell culture and zebrafish embryos as model systems, Kim et al. showed that the mother centriole protein, Nde1 suppressed ciliary formation.²⁸ Nde1 mediated such an effect by tethering another form of dynein light chain, LC8, at the basal body through a direct physical interaction.²⁸ Cells lacking Nde1 showed both abnormally long cilia and a delay in cell cycle re-entry, which was rescued in cells lacking cilia.²⁸ Conversely, pharmacological and/or genetic manipulations to induce abnormally long cilia, independently of Nde1, led to a delay in the G₁ to S transition that was further enhanced in cells lacking Nde1.²⁸ The effect of Nde1 on ciliary length was also shown in zebrafish embryos, where embryos depleted of Nde1 showed increased ciliary length and a reduction in the number of proliferating cells of the Kupffer's vesicle, an organ analogous to the embryonic node that determines left-right patterning in zebrafish.²⁸ Interestingly, naturally occurring mutations in NDE1 have been recently described in patients with severe forms of microcephaly,^29,30 a defect seen also in mice lacking Nde1.³¹ Microcephaly is a pathologic condition characterized by the severe thinning of the superficial cortical layers, leading to smaller brains.^32,33 It is often seen with mutations in genes encoding centrosomal proteins, and it is currently thought that it is caused by a mitotic arrest and/or defects in the asymmetric division of neuronal precursors.^33,34 The data by Kim et al.²⁸ suggest an alternative model in which microcephaly in patients carrying mutations in NDE1 may arise from the formation of abnormally long cilia, leading to a prolonged G₁ phase and premature exit of the cell cycle of neuronal progenitors. This hypothesis is consistent with the data of Li et al.^25,28 Thus, it is tempting to speculate that some forms of microcephaly could be caused by primary defects in ciliary biogenesis and/or resorption altering the balance between differentiation and proliferation of neural progenitor cells.

All of the above studies highlight the fact that cilia are not simply a consequence of the differentiated state of the cell, but they can actually influence cell cycle progression. The data on Tctex-1 and INPP5E suggest that ciliary resorption, per se, is part of the mechanism by which the cilium influences cell cycle progression and, in particular, the G₁ to S transition.^25–27 The data by Kim et al.²⁸ are consistent with this model but they can also suggest a broader model in which cilia, in general, can dampen down the overall strength of a mitogenic stimulus. This could be envisioned by the redistribution of growth factor receptors along the plasma and axonemal membrane, sequestering important cell cycle regulators in the cilium, etc. in addition to a delay in ciliary resorption due to abnormally long cilia.

Growth Factor-Induced Ciliary Resorption

Since ciliary resorption in response to growth factor stimulation appears to be central to the mechanism by which cilia influence cell cycle progression, here we discuss and speculate on how growth factor-mediated signaling and, in particular, Ca²⁺ signaling might affect ciliary disassembly. As early as 1979, Tucker and colleagues found that treatment of ciliated cells with PDGF or Ca²⁺ ionophores to increase intracellular Ca²⁺ concentration led to loss of cilia.²² A hypothetical role of growth factor-induced Ca²⁺ signaling in ciliary resorption is shown in Figure 1. Recently, it has been shown that ciliary resorption is triggered by the addition of a specific combination of growth factors, including PDGF-AA and either insulin-like growth factor1 (IGF-1), epidermal growth factor (EGF) or fibroblast growth factor (FGF).²⁶ Any of these growth factors applied individually did not lead to sufficient ciliary disassembly.²⁶ However, suppression of PDGF-AA signaling by adding neutralizing antibodies against PDGFR-α was capable of blocking cilia disassembly in the presence of PDGF-AA, IGF-1, EGF, FGF or all together in combination.²⁶ This suggests that PDGFR-α-mediated signaling is an essential step in resorbing the cilium, whereas IGF-1, EGF or FGF may play a rather permissive role for PDGF-AA in the disassembly process.²⁶ The fact that IGF-1, EGF or FGF can be used to facilitate PDGF-AA-induced ciliary resorption suggests that they share a common biological function in regard to ciliary resorption. Given the fact that any of these growth factors can induce an increase in intracellular Ca²⁺ concentration coupled with the fact that the addition of Ca²⁺ ionophores in the presence of PDGF facilitates ciliary disassembly,²² it is reasonable to propose that IGF-1, EGF and/or FGF signaling may facilitate the resorption process, perhaps by, activating Ca²⁺-permeable channels (Fig. 1).

There are three lines of evidence implicating members of the transient receptor potential (TRP) ion channels in the growth factor-induced ciliary disassembly program. First, several of these channels are known to be activated in response to growth factor stimulation.³⁵ Second, they are regulated by phospholipid metabolism,³⁶ a process in which INPP5E has a crucial role. And third, they are localized at the primary cilium. Specifically, TRPV2 is activated in response to IGF-1 receptor stimulation.³⁷ In resting cells, TRPV2 is localized in intracellular compartments, but upon IGF-1 stimulation, TRPV2 translocates to the plasma membrane to mediate IGF-1-induced Ca²⁺ entry.³⁷ Interestingly, replacing the TRPV ortholog in C. elegans, OCR-2, with human or mouse TRPV2 rescued the loss of chemosensory perception and showed a localization pattern along the sensory cilia and plasma membrane.³⁸ It is tempting to speculate that IGF-1 stimulation in ciliated mammalian cells may utilize TRPV2 at the cilium or plasma membrane to increase intracellular Ca²⁺ levels and possibly result in ciliary resorption.

Stimulation of EGFR by EGF activates the TRPP2 Ca²⁺-permeable channel (polycystin-2, PC2 or PKD2).³⁹ TRPP2 was originally identified as one of the genes responsible for autosomal-dominant polycystic kidney disease, a classic form of a ciliopathy.^40,41 EGF activates TRPP2 by releasing it from PI(4,5)P2-mediated inhibition.³⁹ This is achieved through the activation of phospholipase C (PLC-γ2) or PI3K that hydrolyze PI(4,5)P2 to inositol trisphosphate and diacylglycerol or convert PI(4,5)P2 to PI(3,4,5)P3, respectively.³⁹ Interestingly, TRPP2, PI(4,5)P2 and EGFR are all localized at the primary cilium, suggesting that the EGF-induced activation of TRPP2 could mediate Ca²⁺ entry at the cilium, thus contributing to the ciliary resorption process.³⁹ While it remains to be directly tested whether cells lacking TRPP2 have a defect in ciliary resorption, it has been shown that mouse embryonic fibroblasts derived from transgenic mice overexpressing or lacking TRPP2 show defects in mitotic spindle organization and have supernumerary centriole⁴² defects that were also seen in mice with cells derived from Pifo^+/− mice.²⁴

TRPC1 is also present at the primary cilium,⁴³ which has been shown to mediate Ca²⁺ entry in response to FGF and to physically associate with its receptor, FGFR-1, in the neuroepithelial progenitor cells.⁴⁴ Knockdown of TRPC1 inhibited FGF-stimulated Ca²⁺ influx and cell proliferation.⁴⁴ Neural stem cells have cilia, and they undergo ciliary disassembly prior to cell cycle re-entry.⁴⁵ This suggests that the induction of Ca²⁺ entry through the ciliary pool of TRPC1 may promote disassembly of the primary cilia and cell cycle re-entry in response to FGFR activation.

Although an increase in the intracellular Ca²⁺ concentration can occur during the initiation of the disassembly progress in response to growth factor stimulation, the exact mechanism by which this rise in intracellular Ca²⁺ may lead to ciliary resorption is unknown. Ca²⁺ has been recognized to induce destabilization of microtubule strands.⁴⁶ It was speculated that Ca²⁺ depolymerizes microtubules by competing with the GTP binding site of tubulins, which stabilizes microtubule strands.⁴⁶ In addition, Ca²⁺ binds to calmodulin (CaM), which undergoes a conformational change, activating a serine-threonine kinase, Ca²⁺/CaM-dependent protein kinase II (CaMKII).⁴⁷ CaMKII has been shown to depolymerize meiotic spindles in Xenopus egg extracts⁴⁸ and localizes at the mammalian olfactory cilia,^49,50 suggesting that the increase in Ca²⁺ and subsequent activation of CaMKII at the primary cilia may participate in the dissasembly process. It is important to note that a reduction in intracellular Ca²⁺ concentration resulted in abnormally long cilia in epithelial and mesenchymal cells,⁵¹ supporting the notion that Ca²⁺ directly or indirectly can regulate ciliary retraction. Further studies are necessary to understand whether and how Ca²⁺ and/or its binding partners participate in the depolymerization of axonemal microtubules during cell cycle re-entry.

Sensory Role of the Cilia in Proliferation

All of the previously described studies connecting cilia and cell cycle re-entry seemed to be in agreement with the fact that ciliary resorption is vital to cell cycle re-entry. Therefore, the critical question that arises is how ciliary retraction influences the transition from quiescence to proliferation. The answer is unknown. However, one thought is that critical cell cycle regulators are sequestered within the cilium and, upon ciliary disassembly, are released (or specifically targeted) to the cell body to signal the transition from quiescence to proliferation. In this section, we review signaling components housed in the cilium that have also been shown to regulate the G₁ to S transition (Fig. 2).

Wnt Signaling

The Wnt pathway has two major limbs, the canonical Wnt pathway and the Wnt-PCP/Ca²⁺ (or non-canonical) pathway (reviewed in ref. 52–54). These subpathways function in an antagonistic way, whereby hyperactivation of one pathway suppresses the other.⁵⁵ The canonical Wnt pathway is initiated by the binding of a Wnt protein (Wnt3a is considered a prototype) to its cognate Frizzled (Fz)'s receptor and the low-density lipoprotein-related protein 5 or 6 (LRP5/6) co-receptor (reviewed in ref. 52). Activation of canonical Wnt pathway leads to the accumulation of β-catenin and has been associated with oncogenesis and cell cycle progression.⁵⁶ In contrast, activation of Wnt-PCP/Ca²⁺ is not coupled to cell cycle progression, but rather to cell polarity and tissue morphogenesis.⁵³ The Wnt-PCP/Ca²⁺ pathway seems to mediate planar cell polarity (PCP), which is defined as the organization of cells within a plane perpendicular to the apical-basal axis.⁵⁷

Several studies support the idea that the primary cilium can affect the Wnt pathway by regulating the balance between the two limbs of the pathway.^58–60 The general idea is that suppression of the non-canonical Wnt pathway can lead to hyperactivation of canonical Wnt, resulting in faster proliferation and suppression of morphogenesis.^59,61,62 While the connection between of the primary cilium and the Wnt pathway is under debate,^63,64 a recent publication provided evidence for a direct role of the primary cilium in restraining the canonical Wnt/β-catenin. Jouberin (Jbn) is the protein product of one of the genes mutated in patients with Joubert syndrome, a classic ciliopathy. In resting cells, Jbn is sequestered in the cilia (Fig. 2A). However, upon canonical Wnt activation, Jbn is released from the cilia via retrograde IFT, which then physically interacts with β-catenin to facilitate its translocalization to the nucleus and activation of β-catenin-dependent transcription of target genes^65,66 (Fig. 2B). One of the known target genes of β-catenin/TCF is cyclin D, which is required for the G₁/S transition. Therefore, it is tempting to speculate that during the ciliary disassembly process, the gradual loss of cilia may promote the formation of the Jbn-mediated β-catenin complex to facilitate G₁/S transition through the transcription of the cyclin D gene.^66,67

Hedgehog Signaling

Hedgehog (Hh) is a secreted protein that acts on its receptor Patched (Ptc) to mediate most of its effects.¹² In the absence of Hh, Ptc suppresses the activity of Smoothened (Smo) (Fig. 2A), whereas in the presence of Hh, Ptc relieves the inhibition of Smo and allows the activation of the pathway⁶⁸ (Fig. 2B). It has been recently shown in mammals that in the absence of the Hh ligand, Smo is localized at the cytoplasm, while Ptc is present at the cilium.⁶⁹ Upon binding of Hh to Ptc at the cilium, Ptc translocates to the cell body, while Smo moves into the cilium via IFT, where it activates the Gli transcription factors⁶⁹ (Fig. 2B). Loss or disfigured primary cilium impairs Hh signal transduction in mice.⁷⁰

The downstream effector molecules of Hh include a subset of transcription factors, namely Gli1-3, which are localized at the primary cilia.⁷¹ Gli2 and 3 retain an N-terminal repressor domain and a C-terminal activator domain surrounding the DNA binding site, whereas Gli1 lacks the repressor domain.⁷² Gli1 and 2 primarily function as active transcription factors, while Gli3 is proteolytically processed to its repressor form (Gli3R) or, otherwise, kept in an active form (Gli3A).⁷² In the absence of Hh, both Gli2 and 3 (Gli3R) are inactive, but in the presence of Hh, Smo protects Gli3 from being processed into Gli3R and activates Gli2, which translocates to the nucleus to promote transcription of Gli1 and other target genes.^70,71,73

The genes activated by the Gli family of transcription factors participate in cellular processes such as proliferation, survival and metastasis.¹² Cyclin D, cyclin E and E2F1 are among these genes, which are of particular interest, since they are key regulators of G₁ to S phase progression.⁷⁴ As mentioned earlier, ciliary resorption triggers cell cycle re-entry, specifically during G₁/S transition,^25,28 suggesting that the Gli proteins may serve as the “communicators” between the cilia and cell body to promote proper cell division. This idea is in part supported by the observation that the ability of Gli2 to induce medulloblastoma is enhanced in cells engineered to lack cilia.⁷⁵ Therefore, it is possible that during ciliary resorption in response to a mitogenic signal, there is an accumulation of active Gli2 in the nucleus driving the activation of target genes required for the transition from G₁ to S phase. Moreover, the accumulation of Gli2 at the nucleus could be further facilitated by the reduction in the Gli3 to Gli3R conversion (which suppresses Gli2-mediated transcription) that occurs within the cilium. Ectopic expression of Gli2 leads to an accumulation at the ciliary tip,¹² suggesting that the cilia can tether Gli2 from transcriptionally inducing unregulated proliferation. Given that Gli3 primarily works as a repressor (Gli3A is a transcription factor with relatively weak activity) for Gli2, which induces the transcription of Gli1, it is tempting to speculate that Gli2 might be one of effector molecules mediating the G₁/S transition in response to growth factor stimulation.

Ciliary Proteins Implicated in Cell Cycle Regulation

An essential component of the IFT, IFT88, was shown to affect cell cycle progression in both ciliated and non-ciliated cell types.^76,77 In HeLa cells, which lack cilia, IFT88 was shown to regulate G₁/S transition through a physical interaction with Che1, an inhibitor of the DNA damage checkpoint protein, retinoblastoma.^76,78 Depletion of IFT88 in HeLa cells promoted G₁/S progression, while overexpression of IFT88 led to a G₁ arrest and induced apoptosis.⁷⁶ Recently, Doxsey and colleagues reported a new role of IFT88 in maintaining proper spindle orientation during cell division through the regulation of the astral microtubules.⁷⁷ Depletion of IFT88 in HeLa cells and zebrafish embryos as well as kidney cells derived from Ift88^+/− mice all showed mitotic misorientation and a delay in mitotic progression.⁷⁷ Depletion of IFT27, another major component of IFT, which shows sequence similarity to small G-proteins, not only suppresses cilia formation but also blocks cytokinesis.⁷⁹ Other IFT proteins, such as IFT20 and IFT52, are localized at the mitotic spindle apparatus,^80,81 collectively suggesting that these core components of IFT can have additional roles in cell cycle progression.

A genetic screen in Chlamydomonas reinhardtii identified Fa2p as a major player for flagella retraction.⁸² Fa2p, which is a member of the NIMA-related kinase (NEK) family, is localized at the proximal end of the cilium and basal body, where, through its kinase domain, it regulates ciliary retraction.⁸² NEKs play important roles in G₂ to M phase transition, suggesting that Fa2p coordinates ciliary turnover and subsequent cell cycle progression. Cenexin1 is an additional example of a protein that plays dual roles during ciliogenesis and cell cycle progression.⁸³ Cenexin1 is localized at the mother centriole, where it interacts with Polo-like kinase1 (Plk1) during G₂/M phase of the cell cycle or ninein during quiescence.⁸³ Plk1 plays essential roles in centrosome maturation,⁸⁴ whereas ninein maintains the integrity of the basal body during ciliogenesis.⁸⁵ These data suggest that Cenexin1 may regulate the two processes through mutually exclusive protein-protein interactions.

Conclusions and Speculations

The primary cilium is a dynamic organelle, whose cyclic formation and disassembly is inherently related to the cell cycle. New data provide evidence that ciliary resorption not only precedes but also influences cell cycle re-entry. These observations lead us to suggest that the cilium may behave as a “physical checkpoint” in cell cycle progression. For a process to qualify as a cell cycle checkpoint, it has to fulfill a set of three basic criteria: (1) to sense the error(s), (2) to transduce the signal and (3) to activate an effector to allow the cell to fix the error or self-destruct.⁸⁶ One could argue that the numerous receptors (sensors) present in the cilium can most certainly sense external cues for proliferation, while activation of the ciliary resorption program (transducers) can relay the signal to the cell by “releasing” cell cycle regulators into the cell (effectors) to initiate G₁/S transition. However, it is not immediately apparent whether the cilium can sense an “intrinsic” error or mistake, thus failing criterion 1. In this context, it is rather part of the signaling pathway that overcomes the G₁ to S “restriction point.” Thus, it appears that cilia can sense the environment and influence the decision to enter or abort the cell cycle. This type of signal transduction has immediate implications for processes and diseases dependent on the transition from quiescence to a proliferative state, such as cancer, polycystic kidney disease or neocortical development. While cilia are absent in most cancers, they have been shown to have dual effects on cancers induced by mutations in components of the Hedgehog signaling pathway.^75,87 Consistently, several forms of polycystic kidney disease associated with increased proliferation show defective ciliary structure and function.⁸⁸ Last but not least, the role of cilia-dependent regulation of the length of G₁ phase may be responsible for neocortical development, as put forth by studies of Kim et al. and Li et al.^25,28 While further investigation is needed to substantiate the role of cilia in cell cycle, the evidence for a causal role of this organelle in cell cycle re-entry seems to be gaining support.