Centrioles are microtubule-based, radially symmetric, cylindrical structures. A pair of centrioles, and surrounding pericentriolar material, comprise the centrosome (Vertii et al., 2016). In interphase, one of the centrioles can serve as basal body for a primary cilium while the pericentriolar material is a principal site for cytoplasmic microtubule nucleation and organization. In mitosis, centrosomes regulate the assembly and orientation of the mitotic spindle. In these roles, centrioles are vital to fundamental cellular processes including signal transduction, intracellular trafficking and cell division. Dysfunction of centrioles or associated structures results in developmental and adult tissue maintenance defects (Bettencourt-Dias et al., 2011), which have been linked to human disease, notably ciliopathies and cancer.

In dividing cells, centriole generation occurs in S phase of the cell cycle when exactly two new (daughter) centrioles assemble, each next to an existing (mother) centriole (Figure 1—figure supplement 1A) (Fırat-Karalar and Stearns, 2014). The resulting two centrosomes then segregate into the two daughter cells during mitosis to reduce the centriole number to two in each daughter. Control of centriole number is important for proper cellular function: extra centrioles can lead to abnormal mitoses (Yang et al., 2008) or give rise to extra primary cilia that results in defective signaling (Mahjoub and Stearns, 2012). Centriole generation is highly regulated to ensure the assembly of the correct number of structures by limiting duplication to S phase, by limiting assembly to one new daughter centriole alongside each mother centriole, by blocking the de novo (noncentriolar) generation of extra centrioles, and by coupling centriole and DNA duplication under common timing and regulation.

Multiciliated cells (MCCs) of the airway, ependymal, middle ear and oviduct epithelia break the rules that govern centriole formation in dividing cells, as they assemble, depending on cell type, dozens to hundreds of centrioles in a single postmitotic cytoplasm to act as basal bodies to motile cilia (Figure 1—figure supplement 1B) (Meunier and Azimzadeh, 2016). MCC fate is acquired in a Notch signaling-dependent manner, with cells experiencing Notch activation becoming secretory cells and cells not experiencing Notch activation progressing down the MCC pathway (Tsao et al., 2009). Ciliogenesis is initiated when nascent MCCs launch a MCC-specific transcriptional program to express hundreds of ciliary genes. Next, centrioles form in the cytoplasm, traffic to and dock with the apical plasma membrane and elongate a motile ciliary axoneme. In contrast to dividing cells, MCCs are capable of (1) generating centrioles in the postmitotic or G0 phase, (2) generating many daughter centrioles per mother centriole, (3) using unique structures termed deuterosomes for the de novo (noncentriolar) assembly of centrioles, and (4) uncoupling centriole and DNA duplication. Yet, both MCCs and dividing cells produce apparently structurally identical centrioles, and the MCC pathway appears to rely on many known cell cycle regulated centrosome duplication factors such as the Plk4 kinase and structural components including Sass6 and Centrin proteins (Vladar and Stearns, 2007). This suggests that there are both universal pathways as well as MCC specific alterations that permit large scale postmitotic centriole amplification.

Mechanisms that control centriole number in dividing cells consist of both centriole-intrinsic and cytoplasmic events. Limiting centriole duplication to S phase is ultimately under the control of the cell cycle machinery. Timely entry and progression through S phase is regulated by Cyclin-dependent kinase 2 (Cdk2) complexed with cyclins E or A2 (Hochegger et al., 2008). Distinct Cdk-cyclin pairs control cell cycle transitions through a highly orchestrated program of Cdk posttranslational modification and cyclin expression and degradation (Heim et al., 2017). The G1 to S phase transition occurs when mitogenic stimulation leads to activation of the Cdk4/6-cyclin D complex, which acts to dissociate the E2F1 transcription factor from the Dp1 and Retinoblastoma (Rb) proteins, leading to E2F1 activation. E2F1 turns on key S phase genes, notably cyclins E and A2 and DNA synthesis factors. Cyclin E binding leads to activation of Cdk2. Then Cdk2, coupled to cyclin A2 (which further phosphorylates E2F1) initiates entry into and controls progression through S phase and the twin events of centriole and DNA duplication (Hinchcliffe et al., 1999; Lacey et al., 1999; Matsumoto et al., 1999). The precise events that link the cell cycle machinery to centriole duplication are not yet clear, however, many regulators localize to the centrosome (Kodani et al., 2015) and may control Plk4 stability and activity (Korzeniewski et al., 2009).

Interestingly, recent insights into MCC differentiation revealed that multiple cell cycle regulators also play important roles in motile ciliogenesis (Meunier and Azimzadeh, 2016). The initiation of the MCC gene expression program depends on a transcriptional complex (EMD complex) comprising the E2F4 or E2F5 transcription factor, a Geminin family transcriptional activator (Mcidas or Gmnc) and Dp1 (Ma et al., 2014). This complex is highly similar in make up to the E2F1/Rb/Dp1 complex that controls G1 to S phase progression, which suggests the existence of universal cell cycle regulatory mechanisms to create a permissive environment for centriole assembly. Downstream of EMD, the Myb and p73 transcription factors, also well known for their cell cycle functions (Allocati et al., 2012), turn on further MCC genes (Tan et al., 2013; Marshall et al., 2016). Finally, the G2 to M phase cell cycle machinery, including Cdk1 was shown regulate intermediate stages of ciliogenesis in ependymal MCCs (Al Jord et al., 2017).

To investigate the extent to which the G1 to S phase cell cycle machinery is involved in motile ciliogenesis, we tested the role of Cdk2 using primary mouse tracheal epithelial cell (MTEC) culture, which recapitulates basal (stem) cell proliferation and subsequent MCC differentiation (You et al., 2002). Here, we show that downstream of MCC fate acquisition, Cdk2 is required to initiate and maintain the MCC gene expression program during ciliogenesis. Consistently, MCCs driven to differentiate in the absence of Cdk2 activity fail to undergo ciliogenesis. Unlike in dividing cells, Cdk2 appears to function during ciliogenesis together with Cyclin A1 (Ccna1), a cognate cyclin thought to activate Cdk2 in meiotic cell cycles (Joshi et al., 2009). In sum, our results indicate that Cdk2 activity is universal regulator of centriole assembly, and the involvement of Ccna1, a noncanonical binding partner in somatic cells, and likely other factors enable it to function in a postmitotic cell and drive centriole amplification.

Previously, the earliest known event in the motile ciliogenesis pathway was the transcriptional upregulation of ciliary genes by MCC TFs that are known to also regulate the cell cycle in dividing cells. Here, we show that Cdk2, the key regulator of cell cycle entry and centrosome and DNA duplication in S phase is responsible for activation of the MCC gene expression program (Figure 7C), further supporting the notion that an alternative cell cycle program controls MCC differentiation. Seeking to understand how differentiated MCCs can overcome the strict regulatory limits on centriole duplication that exist in cycling cells to generate hundreds of centrioles, we tested the requirement for Cdk2 during motile ciliogenesis. We found that it is required for initiating and sustaining the MCC gene expression program, and that it likely works together with Ccna1. These results uncover surprising roles for both Cdk2 and Ccna1 in a quiescent somatic cell, further establish the role of cell cycle regulators in motile ciliogenesis, and suggest that centriole generation and number control are regulated by common cell cycle-associated mechanisms in which MCC-specific alterations can drive amplification.

Centriole generation is normally restricted to the S phase of the cell cycle. The involvement of multiple cell cycle-related proteins in the motile ciliogenesis pathway led to the hypothesis that MCCs enter a unique cell cycle state, the so called S* phase (Tan et al., 2013) that shares characteristics with both cycling and quiescent states and simultaneously facilitates both centriole generation and maintenance of a postmitotic state. Thus, we speculated that Cdk2, the regulator of S phase entry may be involved in ciliogenesis. We report that Cdk2 is localized to the nucleus in ciliating MCCs, and that Cdk2 inhibition arrests the pathway at its earliest known step, the initiation of the MCC gene expression program. Nuclear localization is consistent with an active kinase (Pagano et al., 1993) and a role in regulating MCC transcription.

MCC TFs represent attractive candidate substrates for Cdk2 during ciliogenesis. Such a direct action mechanism would be consistent with both the lack of transcriptional activation under Cdki treatment and with known functions of Cdk2 in dividing cells. Cdk2-Ccne1 propels cells towards S phase, in part, by phosphorylating the Rb protein, which maintains the E2F1 transcription factor in an inactive complex with Dp1 (Heim et al., 2017). Related complexes, comprising E2F4/5, Dp1 and a Geminin family member (Geminin, Mcidas and Gmnc) act early during ciliogenesis to turn on MCC gene expression (Vladar and Mitchell, 2016). Recent studies on the role of the Geminin family proteins in MCCs suggest that Geminin is initially complexed with E2F4/5 and Dp1 to inhibit ciliogenesis; subsequently, complexes that include Mcidas and Gmnc emerge to turn on ciliary genes. We favor the hypothesis that this process is regulated by Cdk2 phosphorylation of one or more of these complex components. E2F5 is a known Cdk2 target (Morris et al., 2000), and studies are underway to test whether this interaction occurs during ciliogenesis.

Our results raise a number of fascinating questions about Cdk2 specifically and about cell cycle regulation in general. How does Cdk2 become active in a quiescent cell? How does Cdk2 activation in MCCs not lead to cell cycle reentry or to DNA replication? We hypothesize that Cdk2 activation during ciliogenesis occurs in the proposed S* phase that shares characteristics with both G1 and S. Moreover, we propose that events downstream of Cdk2 activation are modular and subject to MCC-specific regulation to allow centriole amplification without DNA replication or cell cycle progression. As the key regulator of the G1 to S phase transition in cycling cells, Cdk2 activity is under a multitude of regulatory constraints, and our results indicate that some of these regulatory events are also active in MCCs. We demonstrated that Cdk2 bearing the activating Thr160 phosphorylation peaks around the time of ciliogenesis initiation in MTECs. In dividing cells, this modification is imparted by Cdk-activating kinase (CAK) downstream of cyclin binding (Jeffrey et al., 1995), and it is essential for the G1 to S phase transition. In the future, we will test the potential activity and role of additional Cdk2 regulatory measures, including the removal of the inhibitory Thr14 and Tyr15 phosphorylations by the Cdc25A phosphatase (Heim et al., 2017).

Our data point to the involvement of Ccna1 as a cognate cyclin to Cdk2 in motile ciliogenesis regulation based on its MCC restricted expression, colocalization with Cdk2 and the finding of diminished MCCs in the Ccna1 mutant mice. Although technical limitations (the inability to sort ciliating cells from heterogeneous cultures, and the small culture size) prevent us from demonstrating direct association in ciliating cells, their colocalization during ciliogenesis, similar loss of function phenotypes and the existence of Cdk2-Ccna1 complexes in other tissues (Joshi et al., 2009) are consistent with Ccna1 binding to activate Cdk2 to control ciliogenesis. Finally, we show that Cdk2 and Ccna1 are both enriched in nuclei of ciliating but not mature MCCs. Of note, Ccna1 regulates meiosis in mammals and plants (Liu et al., 1998; d'Erfurth et al., 2010), another event in which centrioles are generated in the absence of DNA replication.

We demonstrate that Ccna1 is a target of multiple MCC transcription factors, which accounts for its sustained and robust expression during ciliogenesis. As we propose that Ccna1 acts together with Cdk2 to initiate the MCC gene expression program, it remains to be understood how Ccna1 gets turned at the onset of ciliogenesis. By analogy to cell cycle progression, we speculate that Ccna1 may be degraded at or near the conclusion of motile ciliogenesis, possibly as a regulatory step in the pathway. Our inability to detect Ccna1 by antibody labeling in mature MCCs raises the possibility of a regulated cyclin destruction event, which requires closer examination. Ccna1 mice showed a reduction in MCC number but not the complete block to ciliogenesis observed with Cdki treatment. The different phenotypes could represent different responses to a chronic loss of Ccna1 in vivo compared to the acute inhibition of Cdk activity in vitro. We also speculate that it may be due to partial redundancy with other cyclins. In spite of its broader expression and the lack of an MCC phenotype, we cannot rule out a role for Ccne1. Recent studies identified Ccno as an important regulator of ciliogenesis downstream of the EMD complex (Wallmeier et al., 2014), and although it is not known whether Ccno can activate Cdk2, it could conceivably also act partially redundantly with Ccna1.

We consistently observed Cdk2 at centrioles in both MTECs and in human ALI cultures (unpublished results). Cdks, including Cdk2, have been reported at the centrosome in dividing cells, so this is likely a localization pattern shared with MCCs. Centrosomal Cdk2 targets include Ccp110, which dissociates from the mother centriole distal end upon phosphorylation to allow primary cilium growth (Chen et al., 2002). Ccp110 is present and required for motile ciliogenesis (Song et al., 2014), and the centriolar pool of Cdk2 may be regulating motile axoneme elongation in MCCs. Nucleophosmin 1 (Npm1), a Cdk2 substrate that shows both nuclear and centriolar localization, has been suggested as a link between Cdk2 activity and centrosome duplication (Okuda et al., 2000). Although we can detect Npm1 in MCCs (unpublished results), its role in ciliogenesis remains untested. We note that forced expression of Mcidas in the presence of inhibited Cdk2 can sustain ciliogenesis, suggesting that putative Cdk2 activity at replicating centrioles may not be absolutely required.

Our results, and the analogy between the G1 to S phase transition and motile ciliogenesis, indicate that Cdk2 is a common regulator of the two processes, however, it is important to consider the possibility of redundant factors and mechanisms. The near normal viability of the Cdk2 knockout mouse (Berthet et al., 2003) revealed that other Cdks and Cdk-independent events are capable of driving cell cycle progression, at least in the context of chronic Cdk2 absence. Similarly, we cannot rule out the contribution of other Cdks to motile ciliogenesis. Cdk4 and Cdk6 play important roles in G1 exit and should be investigated in MCCs. Our Cdkis likely do not inhibit Cdk4/6 at the concentrations we employed, but some might conceivably target Cdk5 (Peyressatre et al., 2015). Although Cdk5 is not a canonical cell cycle regulator (Shupp et al., 2017), we cannot exclude that it has a role in motile ciliogenesis.

The Cdk1-APC/C mitotic oscillator was recently shown to promote intermediate phases of centriole replication in MCCs without stimulating mitosis (Al Jord et al., 2017). We demonstrate that Cdk2 plays a role in initiating the MCC gene expression program, the precursor to centriole assembly. We support our placement of Cdk2 at this point in the regulatory hierarchy by demonstrating that it acts downstream of the Notch signaling event and either downstream of, or in parallel with, TRRAP, an early regulator of ciliogenesis, and upstream of the EMD complex that initiates and sustains MCC gene expression. In addition to its nuclear localization, we observe Cdk2 at centrioles, and thus we cannot rule out that, similar to Cdk1, it is also involved more directly in ciliogenesis. Cdk1 and Cdk2 redundancy at centrioles may explain why we did not detect a ciliogenesis phenotype with the Cdk1-specific RO3306 inhibitor as observed by Al Jord et al. As that study was chiefly carried out in ependymal MCCs, which make many fewer MCCs, it may also reflect an alternative Cdk requirement.

We set out to increase our understanding about how MCCs are able to break the rules of centriole assembly and number control faithfully observed by dividing cells. In sum, our results show that Cdk2 activity is required to create a permissive environment for centriole generation in either context. Emerging studies point to the involvement of universal centriole assembly factors and events, including common regulators and biogenesis steps, and MCC-specific features, like the use of deuterosomes and transcriptional regulation. We add the universal requirement for Cdk2 activity to this model, and underscore the importance of cell cycle regulation to centriole assembly in any context. It will be interesting to compare MCC mechanisms to those operating in other cells with alternative cell cycles or where DNA and centriole duplication are uncoupled. These include male germ cells in which meiosis is regulated by Cdk2-Ccna1 (Liu et al., 1998) and endocycling cells that undergo many rounds of DNA replication without mitosis (Lu and Roy, 2014). With growing attention to MCCs in various tissues, improved understanding of the motile ciliogenesis pathway is an important goal.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Eszter K Vladar

   1. Department of Pathology, Stanford University School of Medicine, Stanford, United States
   2. Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine, Aurora, United States
   3. Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   eszter.vladar@ucdenver.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4160-8894

2. Miranda B Stratton

   Department of Biology, Stanford University, Stanford, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

3. Maxwell L Saal

   1. Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine, Aurora, United States
   2. Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Glicella Salazar-De Simone

   Center for Radiological Research, Columbia University Medical Center, New York, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

5. Xiangyuan Wang

   Department of Genetics & Development, Columbia University Medical Center, New York, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Debra Wolgemuth

   Department of Genetics & Development, Columbia University Medical Center, New York, United States

   Contribution

   Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

7. Tim Stearns

   1. Department of Biology, Stanford University, Stanford, United States
   2. Department of Genetics, Stanford University School of Medicine, Stanford, United States

   Contribution

   Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0671-6582

8. Jeffrey D Axelrod

   Department of Pathology, Stanford University School of Medicine, Stanford, United States

   Contribution

   Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

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