Author response: Cyclin-dependent kinase control of motile ciliogenesis (2018)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication. https://doi.org/10.7554/eLife.36375.001 Introduction 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. Results Cyclin-dependent kinase activity is required for motile ciliogenesis We used the MTEC culture system to test the requirement for Cdk activity during MCC differentiation (Figure 1—figure supplement 1A–C). MTECs are a faithful model of airway epithelial development and regeneration and permit the observation and manipulation of both the proliferative and differentiation phases of the process (You et al., 2002). At maturity, MTECs contain all the cell types of the donor tissue, including MCCs, secretory cells and basal stem cells. The cultures are initiated by seeding basal stem cells isolated from mouse trachea onto porous Transwell membranes (Vladar and Brody, 2013). Cells proliferate to confluence while submerged in medium, then are lifted to an air-liquid interface (ALI) which promotes the differentiation of airway epithelial cells (Figure 1—figure supplement 1C). Culture progression follows a timeline in which basal cells proliferate during days 1–3, the resulting confluent cell layer acquires a columnar, apically compacted morphology from days 3–5, ALI is created on day 5 (ALI + 0 days), and MTECs are mature by ALI + 14 d with MCCs and secretory cells at the luminal surface with underlying basal cells. MCC fate acquisition and motile ciliogenesis occur asynchronously in both the in vivo airway epithelium and in MTECs (Vladar and Stearns, 2007), but early ALI cultures are strongly enriched for MCCs in the initial stages of motile ciliogenesis. To test the requirement for Cdk activity, we treated differentiating MTECs with well-characterized, dose-dependent small molecule inhibitors that predominantly act on Cdk1 and Cdk2 (Cdkis). We verified the previously established ability of these Cdkis to produce cell cycle arrest in 293T/17 cells before using in MTECs (not shown). MTECs were treated from ALI + 0 to ALI + 4 d (chronic treatment) with the Cdkis Purvalanol A, Roscovitine, NU6140 and Cdk2 Inhibitor III, then labeled at ALI + 4 d with anti-acetylated α-Tubulin (ac. α-Tub) antibody to mark cilia. While untreated cultures contained many MCCs, we found that all four Cdkis blocked ciliogenesis (Figure 1—figure supplement 2). To characterize this phenotype, we labeled cells with antibodies against structural and regulatory components of the ciliogenesis pathway, and found that Cdki treated MTECs failed to form centrioles (ac. α-Tub, Odf2, Pericentrin, Sass6, and Plk4), deuterosomes (Ccdc67, also known as Deup1) or ciliary axonemes (ac. α-Tub), indicating an early ciliogenesis arrest (Figure 1A–B). Motile ciliogenesis is initiated and maintained by the expression of ciliary genes via the sequential activity of MCC transcription factors (MCC TFs, see Figure 1—figure supplement 1B) (Meunier and Azimzadeh, 2016). We found that Cdki treated cells did not express the MCC TFs Foxj1 and Myb (Figure 1A). Using qRT-PCR, untreated cells showed strong upregulation of both MCC TFs and ciliary genes at ALI + 4 d compared to confluent, but not yet differentiating cells (ALI-1d or day 4 of culture), whereas Cdki treated cells had no detectable levels of MCC TFs and ciliary genes were not upregulated (Figure 1C). Cdki treatment therefore blocks motile ciliogenesis at an early step of the pathway at the level of MCC gene expression. Figure 1 with 2 supplements see all Download asset Open asset Chronic Cdk inhibitor treatment blocks motile ciliogenesis. (A) MTECs were treated with NU6140 from ALI + 0 to 4d. They were fixed at ALI + 0 and +4 d and labeled with antibodies to monitor ciliogenesis: left, ac. α-Tub (green), Foxj1 (red) and Myb (blue); center left, Odf2 (green), ac. α-Tub (red) and E-cadherin (blue); center right, Plk4 (green), Pericentrin (red) and E-cadherin (blue); and right, Ccdc67 (green), Sass6 (red) and E-cadherin (blue). MTECs are confluent without any sign of motile ciliogenesis at ALI + 0 d. Untreated ALI + 4 d cells are robustly ciliating, but NU6140 treatment blocks all signs of motile ciliogenesis. Ac. α-Tub marks cytoplasmic microtubules in non-MCCs (white arrow) and motile ciliary axonemal tufts in MCCs (white arrowhead); when MCCs are present, the much weaker cytoplasmic signal is not discernible. Foxj1 and Myb mark both ciliating (yellow arrow) MCCs without ac. α-Tub + axonemes and Foxj1 marks mature MCCs (yellow arrowheads) with axonemes. NU6140 treatment has no effect on primary cilium formation (orange arrow) or on apical cell junctions. Scale bar, 10 μm. (B) Quantitation of the Cdki block and the release for Cdki treatment. MCCs were identified by ac. α-Tub labeling. n.s., not significant; *p<0.0001 (C) Realtime PCR results show that the expression of MCC TFs (Foxj1, Myb, Gmnc and Mcidas) is suppressed and the expression of ciliary components (Plk4, Sass6, Ccdc67 and Cetn2) is not upregulated in cells treated with NU6140 from ALI + 0 to 4d. Levels were normalized to Gapdh expression and compared to values obtained for MTECs at ALI-1d (n.d. = none detected). n.s., not significant, *p<0.05, **p<0.0001 (D) MTECs were treated with NU6140 from ALI + 0 to 4d, then cultured without Nu6140 until ALI + 8 d. Cells were fixed at ALI + 4 and+8 d and labeled with Odf2 (green), ac. α-Tub (red) and E-cadherin (blue) antibodies to show that MTECs ciliate robustly after release from Cdki treatment. Scale bar, 20 μm. (E) MCCs were quantitated based on ac. α-Tub labeling in MTECs infected with GFP, Cdk2-HA or Cdk2D145N-HA lentivirus. Cdk2D145N, but not wildtype Cdk2 expression blocks ciliogenesis. Ectopic wildtype Cdk2 expression in MTECs is not sufficient to drive motile ciliogenesis. n.s., not significant; *p<0.000. https://doi.org/10.7554/eLife.36375.002 Ciliogenesis arrest was fully reversible for all Cdkis, as MTECs released from ALI + 0 to +4 d Cdki treatment were robustly ciliated by ALI + 8 d (Figure 1B,D for NU6140 and Purvalanol A, others not shown). Cdki treatment had no effect on overall epithelial morphology as judged by E-cadherin antibody labeling of apical cell-cell junctions or on the presence of primary cilia (Figure 1A). These results suggest that the ciliogenesis arrest is a specific inhibition of the motile ciliogenesis pathway and not a nonspecific detrimental effect on the differentiation or overall health and integrity of the MTECs. The Cdkis used are known to have dose-dependent activity on multiple Cdk-cyclin complexes, and at higher doses they can also inhibit unrelated kinases (Knockaert et al., 2002; Peyressatre et al., 2015). We found the while the Cdk1/2-specific Purvalanol A and Roscovitine and the Cdk2-specific NU6140 and Cdk2 Inhibitor III compounds robustly inhibited ciliogenesis, the Cdk1 inhibitor RO3306 had no effect (Figure 1B and Figure 1—figure supplement 2). Moreover, lentiviral expression of a dominant negative HA-Cdk2 (D145N) construct also blocked ciliogenesis, while the expression of wildtype HA-Cdk2 or GFP had no effect (Figure 1E). Thus, we conclude that Cdk2 is required for initiation of the motile ciliogenesis pathway, consistent with its previously described role in centriole assembly in S phase. Cdk2 acts downstream of MCC specification and upstream of the MCC gene expression program Upon airway epithelialization, MCC and secretory cell fates are acquired in a Notch signaling-dependent mechanism. Precursor cells in which Notch signaling is activated are diverted to the secretory cell fate, whereas cells in which Notch is not activated continue toward MCC differentiation (Tsao et al., 2009) Figure 1—figure supplement 1A–B). In the prospective MCCs (those that avoid Notch activation), MCC differentiation may be a default occurrence, though we cannot rule out that some additional external initiating event is required. Subsequent to this cell fate decision point, cells remaining in the MCC pathway activate the MCC gene expression program to turn on ciliary genes (Brooks and Wallingford, 2014; Meunier and Azimzadeh, 2016). To test the relationship between Cdk2 and the Notch signaling event, we treated MTECs from ALI + 0 to 4d with NU6140 to block Cdk activity and with the γ-secretase inhibitor DAPT to block Notch signaling (Stubbs et al., 2012). We monitored ciliogenesis using antibody labeling for ac. α-Tub to mark cilia and Foxj1 to mark MCCs at earlier stages of ciliogenesis (nascent MCCs without mature cilia already express Foxj1) (You et al., 2004). As expected, NU6140 treated MTECs lacked MCCs. MTECs treated with DAPT alone had a two-fold increase in MCCs compared to untreated cells as more cells evaded Notch activation and remained in the MCC pathway (Figure 2A). However, DAPT treatment was not able to induce MCCs in the presence of NU6140 (Figure 2A), indicating that even cells directed toward the MCC fate by Notch inhibition still require Cdk2 activation to continue this progression. Because the Notch decision point occurs before Cdk2 activation, we refer to Cdk2 activation as acting ‘downstream’ of the Notch decision, although strictly speaking, it is peculiar to argue that Cdk2 activation acts downstream of something that does not occur (Notch signaling). Figure 2 Download asset Open asset Cdk2 acts downstream of Notch signaling and upstream of the MCC gene expression program. (A) MTECs were treated with NU6140 and/or DAPT from ALI + 0 to 4d. They were fixed at ALI + 0 and +4 d and labeled with ac. α-Tub (green) and Foxj1 (red) antibodies. DAPT induces MCC formation but not in the presence of NU6140. Scale bar, 10 μm. (B) MTECs were treated with NU6140 from ALI + 0 to 4d. They were fixed at ALI + 0 and +4 d and labeled with TRRAP (green) and Foxj1 (red) antibodies. Scale bar, 20 μm. (C) MTECs were infected with lentivirus encoding GFP, or Foxj1 or Myb and GFP from separate promoters, or myc-tagged Mcidas at ALI-2d, then treated with NU6140 from ALI + 0 to 4d. They were fixed at ALI + 4 d and labeled with GFP or myc (green), Foxj1 (red) and ac. α-Tub (white) antibodies and stained with DAPI (blue) to mark nuclei. Only Mcidas, but not GFP, Foxj1 or Myb expression can drive the complete motile ciliogenesis pathway (arrows indicate GFP+ cells without ac. α-Tub+ cilia, arrowheads point to mature myc+ MCCs) in NU6140 treated MTECs. Foxj1 expression leads to nuclear Foxj1 accumulation, but not ac. α-Tub+ cilia. MCCs were quantitated based on Foxj1 and ac. α-Tub expression. Scale bar, 10 μm. *p<0.001, **p<0.0001. https://doi.org/10.7554/eLife.36375.005 TRRAP, a component of multiple histone acetyltransferase complexes was recently shown to be required for the initiation of the MCC gene expression program ‘downstream’ of the Notch signaling event (downstream in the same sense as described for Cdk2 above) (Wang et al., 2018). TRRAP nuclear expression arises in prospective MCCs prior to Foxj1 expression. We asked whether TRRAP might act downstream of Cdk2 by assessing TRRAP expression in Cdki treated MTECs. We found that while Nu6140 treated MTECs lacked Foxj1+ MCCs, they contained as many TRRAP+ cells as untreated MTECs at the same stage (Figure 2B). This indicates that in prospective MCCs, TRRAP does not act downstream of Cdk2 activation. Cdk2 therefore acts either downstream of or in parallel to TRRAP in the initiation of the MCC gene expression program (Figure 2C). Furthermore, the appearance of TRRAP expression in the absence of Cdk2 activity indicates the adoption of the MCC cell fate, Cdk2 activity from the MCC cell fate Next, we more the relationship between Cdk2 and the MCC transcriptional regulators Mcidas, Myb and While the precise Mcidas part of the complex) is known act upstream of the other but there is also for and in (Brooks and Wallingford, 2014). As MCC TFs are by Cdki treatment (Figure we asked they can function downstream of Cdk2 to drive ciliogenesis when in the presence of Cdki treatment. MTECs infected at of culture with lentivirus the MCC Mcidas, then treated with NU6140 from ALI + 0 to 4d were able to out the complete motile ciliogenesis pathway as judged by Foxj1 expression and the presence of cilia (Figure 2C). Myb or Foxj1 under these did not undergo ciliogenesis. NU6140 treated cells Foxj1 had nuclear or Foxj1 but cilia (Figure 2C). This suggests that Cdk2 acts upstream of Mcidas and the that Mcidas or one of its binding may be a of Cdk2. with the expression of Mcidas and Myb (Stubbs et al., et al., but not Foxj1 (You et al., was sufficient to drive motile ciliogenesis in untreated MTECs (Figure 2C). results the Cdk2 requirement downstream of the cell fate decision and upstream of in the MCC gene expression pathway for initiating ciliogenesis. The of the proliferation and differentiation at ALI + 0 phases of the MTEC culture system already it that the ciliogenesis block by Cdki treatment at ALI + 0 d from the of a proliferative The that Cdk2 acts downstream of the Notch signaling event (Figure indicates that Cdki blocked cells have the cell cycle and MCC fate the from TRRAP expression (Figure in these cells. Cdk2 as an upstream regulator of the MCC transcriptional program an important in for this process and further a universal role for the cell cycle machinery in centriole Cdk2 is required to the MCC gene expression program Although of the regulatory of the program is still MCC gene expression appears to be a process with early and transcriptional the sequential of cilium events (Meunier and Azimzadeh, 2016). Results indicate that Cdk2 MCC gene expression. To test it is also required for the expression of ciliary genes, we treated MTECs after the of ciliogenesis, from ALI + to ALI + 4 with NU6140 Cdki treatment in an ciliogenesis arrest (Figure indicating that Cdk2 is not required to but also to the To characterize the Cdki we used centriole and to the of MCCs at stages of ciliogenesis (Figure 1—figure supplement as previously described (Vladar and Stearns, 2007). Untreated cultures transition from a of ciliating cells with some mature MCCs to a with ciliating and more mature MCCs between ALI + to 4d (Figure We found that Cdki treatment blocked this transition and cells at all stages of ciliogenesis (Figure The MCC Myb is during motile then it turns on Foxj1 with other and is then while Foxj1 on (Tan et al., 2013). Thus, the expression of these two MCC TFs can be used to monitor ciliogenesis progression. In untreated we a in and the increase in from ALI + to 4d by antibody labeling (Figure However, we did not a in the and the failed to increase under Cdki treatment (Figure indicating that cells failed to in the ciliogenesis We also found that the expression of structural and regulatory ciliary genes (Ccdc67, Sass6 and Cetn2) Cdki which suggests that Cdk2 activity is required to maintain MCC gene expression (Figure We not whether ciliogenesis to of a signal or to the of although arrest is at the level of the transcriptional We rule out that the NU6140 arrest is nonspecific to MCCs as treatment had no effect on mature MCCs (Figure supplement Figure with 1 supplement see all Download asset Open asset Cdk2 activity is required to motile ciliogenesis. (A) MTECs were treated with NU6140 from ALI + to 4d. They were fixed at ALI + d and labeled with antibodies to monitor ciliogenesis: left, Odf2 (green), ac. α-Tub (red) and E-cadherin (blue); (green) (red) and (blue); and right, DAPI Myb (green), Foxj1 (red) and ac. α-Tub MTECs at ALI + d contain predominantly ciliating cells. mature MCCs in untreated ALI + 4 d but not in the presence of NU6140. MCCs are by ac. α-Tub + cilia MCCs are MCCs are III MCCs are and MCCs are MCCs are MCCs are and MCCs are Scale bar, 10 μm. n.s., not significant; (B) Quantitation of MCCs at Figure 1—figure supplement 1B) with and without NU6140 treatment. n.s., not significant; (C) Quantitation of Foxj1 and Myb cells with and without NU6140 treatment. n.s., not significant; *p<0.05, (D) Realtime PCR that the expression of ciliary components Sass6, Ccdc67 and in cells treated with NU6140 from ALI + to 4d. that as ciliogenesis expression levels for some components also in untreated MTECs from ALI + to 4d. Levels were normalized to Gapdh expression and compared to values obtained for MTECs at Cdk2 is and to the and centrioles during motile ciliogenesis To ensure the of S phase Cdk2 activation in dividing cells is under control by posttranslational modification and cyclin Although these regulatory events may not all be at play in a postmitotic we that Cdk2 is in ciliating MCCs. Using wildtype we that Cdk2 was enriched in the in ciliating cells to cytoplasmic = but not in mature to cytoplasmic = MCCs (Figure Although Cdk2 levels did not during ciliogenesis at the or we a in Cdk2 the et al., by at to a that is enriched for MCCs in early ciliogenesis (Figure Figure supplement and Figure 1—figure supplement 1C). The presence of nuclear Cdk2 in ciliating MCCs and the of Cdk2 in ciliating MTECs indicates that Cdk2 is during early ciliogenesis. These signs of Cdk2 activation, together with the requirement for Cdk2 activity to MCC expression a key role for Cdk2 in the initiation and maintenance of the motile ciliogenesis program. Interestingly, we found that HA-Cdk2 also to centrioles in both ciliating and mature MCCs (Figure As we were not able to Cdk2 at MCC centrioles to the of we not that this of Cdk2 is of Cdk2 However, others have that Cdk2 can localize to the centrosome in cells (Kodani et al., and that it has et al., 2002; et al., Thus, both the nuclear and of Cdk2 may be involved in ciliogenesis regulation. is upregulated during motile ciliogenesis and cyclins with Cdk2 and control its is in meiotic and cell while A2 and drive the G1 to S phase transition and the S phase events of centriole and DNA duplication (Heim et al., 2017). We the expression of Ccna1, and during motile ciliogenesis (Figure and a large by a in expression for and a more increase in expression for The timing of their expression with the of Cdk2 activation during ciliogenesis (Figure did not show and consistent with a postmitotic a of was (Figure results from previously data that also identified expression during ciliogenesis (Stubbs et al., et al., 2012). The of ciliary genes show strong upregulation by a in expression during ciliogenesis and this can be used to important for the process et al., 2012). Although we cannot rule out a role for the other this Figure 4 with 1 supplement see all Download asset Open asset Cdk2 is nuclear and