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Cyclin-dependent kinase control of motile ciliogenesis

  1. Eszter K Vladar  Is a corresponding author
  2. Miranda B Stratton
  3. Maxwell L Saal
  4. Glicella Salazar-De Simone
  5. Xiangyuan Wang
  6. Debra Wolgemuth
  7. Tim Stearns
  8. Jeffrey D Axelrod
  1. Stanford University School of Medicine, United States
  2. University of Colorado School of Medicine, United States
  3. Stanford University, United States
  4. Columbia University Medical Center, United States
Research Article
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Cite this article as: eLife 2018;7:e36375 doi: 10.7554/eLife.36375

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
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., 2009Figure 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).

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, thus separating Cdk2 activity from the MCC cell fate decision.

Next, we more closely investigated the relationship between Cdk2 and the MCC transcriptional regulators Mcidas, Myb and Foxj1. While the precise hierarchy remains unclear, Mcidas (as part of the EMD complex) is known act upstream of the other two, but there is also evidence for feedback regulation and overlap in targets (Brooks and Wallingford, 2014). As MCC TFs are repressed by Cdki treatment (Figure 1A), we asked if they can function downstream of Cdk2 to drive ciliogenesis when expressed ectopically in the presence of Cdki treatment. MTECs infected at d3 of culture with lentivirus containing the MCC TF Mcidas, then treated with NU6140 from ALI + 0 to 4d were able to carry out the complete motile ciliogenesis pathway as judged by Foxj1 expression and the presence of cilia (Figure 2C). Cultures expressing Myb or Foxj1 under these conditions did not undergo ciliogenesis. NU6140 treated cells expressing Foxj1 had nuclear or nucleo-cytoplasmic Foxj1 signal, but never made cilia (Figure 2C). This suggests that Cdk2 acts upstream of Mcidas and raises the possibility that Mcidas or one of its binding partners may be a target of Cdk2. Consistent with previous reports, the expression of Mcidas and Myb (Stubbs et al., 2012; Tan et al., 2013), but not Foxj1 (You et al., 2004), was sufficient to drive motile ciliogenesis in untreated MTECs (Figure 2C).

Our results place the Cdk2 requirement downstream of the Notch-dependent cell fate decision and upstream of EMD in the MCC gene expression pathway for initiating ciliogenesis. The clear temporal separation of the proliferation (preALI) and differentiation (beginning at ALI + 0 d) phases of the MTEC culture system already made it unlikely that the ciliogenesis block by Cdki treatment starting at ALI + 0 d stems from the disruption of a proliferative event. The fact that Cdk2 acts downstream of the Notch signaling event (Figure 2A) indicates that Cdki blocked cells have exited the cell cycle and undergone MCC fate selection, reinforcing the conclusion drawn from TRRAP expression (Figure 2B) in these cells. Identifying Cdk2 as an upstream regulator of the MCC transcriptional program fills an important gap in knowledge for this process and further cements a universal role for the cell cycle machinery in centriole generation.

Cdk2 is required to sustain the MCC gene expression program

Although understanding of the regulatory hierarchy of the program is still emerging, MCC gene expression appears to be a protracted, multi-step process with early and later transcriptional steps driving the sequential execution of cilium biogenesis events (Meunier and Azimzadeh, 2016). Results presented thus far indicate that Cdk2 activates MCC gene expression. To test if it is also required for the sustained expression of ciliary genes, we treated MTECs after the onset of ciliogenesis, from ALI + 3 to ALI + 4 d, with NU6140 (acute treatment). Acute Cdki treatment resulted in an immediate ciliogenesis arrest (Figure 3A–B), indicating that Cdk2 is not only required to initiate, but also to sustain the pathway. To characterize the Cdki arrest, we used centriole markers (γ-Tubulin and Odf2) to quantitate the fraction of MCCs at different stages of ciliogenesis (Figure 1—figure supplement 1B), as previously described (Vladar and Stearns, 2007). Untreated cultures consistently transition from a heterogeneous population of ciliating cells with some mature MCCs to a population with fewer ciliating and more mature MCCs between ALI + 3 to 4d (Figure 3A–B). We found that Cdki treatment blocked this transition and cells arrested at all stages of ciliogenesis (Figure 3A–B). The MCC TF Myb is expressed first during motile ciliogenesis; then it turns on Foxj1 (along with other TFs) and is then downregulated while Foxj1 stays on (Tan et al., 2013). Thus, the relative expression of these two MCC TFs can be used to monitor ciliogenesis progression. In untreated MTECs, we consistently detected a decrease in Myb+/Foxj1- and the increase in Myb-/Foxj1 +cells from ALI + 3 to 4d by antibody labeling (Figure 3A,C). However, we did not detect a statistically significant decrease in the Myb+/Foxj1- population and the Myb-/Foxj1+ population failed to increase under Cdki treatment (Figure 3C), indicating that cells failed to progress in the ciliogenesis pathway. We also found that the expression of structural and regulatory ciliary genes (Ccdc67, Plk4, Sass6 and Cetn2) declined upon Cdki treatment, which suggests that continued Cdk2 activity is required to maintain MCC gene expression (Figure 3D). We do not distinguish whether ciliogenesis arrests due to impairment of a signal or due to the depletion of ciliogenic components, although arrest is evident at the level of the transcriptional network. We again rule out that the NU6140 arrest is simply nonspecific injury to MCCs as treatment had no effect on mature MCCs (Figure 3—figure supplement 1).

Figure 3 with 1 supplement see all
Cdk2 activity is required to sustain motile ciliogenesis.

(A) MTECs were treated with NU6140 from ALI + 3 to 4d. They were fixed at ALI + 3 and+4 d and labeled with antibodies to monitor ciliogenesis: left, Odf2 (green), ac. α-Tub (red) and E-cadherin (blue); center, γ-Tubulin (green) Ccp110 (red) and Cep164 (blue); and right, DAPI (blue), Myb (green), Foxj1 (red) and ac. α-Tub (white). MTECs at ALI + 3 d contain predominantly ciliating cells. Many mature MCCs emerge in untreated ALI + 4 d MTECs, but not in the presence of NU6140. Left panels: Stage IV MCCs are marked by ac. α-Tub + cilia (arrow). Center panels: Stage I MCCs are Ccp110+, Cep164-; Stage II MCCs are Ccp110+, Cep164+; Stage III MCCs are Ccp110 low, Cep164 high and Stage IV MCCs are Ccp110-, Cep164+. Right panels: Stage I MCCs are Foxj1-, Myb+; Stage II MCCs are Foxj1+, Myb+; and Stage III-IV MCCs are Foxj1+, Myb-. Scale bar, 10 μm. n.s., not significant; *p<0.01, **p<0.001 (B) Quantitation of MCCs at Stages I-IV (see Figure 1—figure supplement 1B) with and without acute NU6140 treatment. n.s., not significant; *p<0.05 (C) Quantitation of Foxj1 and Myb positive cells with and without acute NU6140 treatment. n.s., not significant; *p<0.05, **p<0.01 (D) Realtime PCR showing that the expression of ciliary components Plk4, Sass6, Ccdc67 and Cetn2 decreases in cells treated with NU6140 from ALI + 3 to 4d. Note that as ciliogenesis peaks expression levels for some components also decrease in untreated MTECs from ALI + 3 to 4d. Levels were normalized to Gapdh expression and compared to values obtained for MTECs at ALI-1d. *p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.36375.006

Cdk2 is active and localizes to the nucleus and centrioles during motile ciliogenesis

To ensure the timely execution of S phase events, Cdk2 activation in dividing cells is under tight control by nucleo-cytoplasmic shuttling, posttranslational modification and cyclin binding. Although these regulatory events may not all be at play in a postmitotic cell, we sought evidence that Cdk2 is active in ciliating MCCs. Using lentivirally expressed wildtype HA-Cdk2, we observed that Cdk2 was enriched in the nucleus in ciliating cells (nuclear to cytoplasmic ratio = 1.96±0.26), but not in mature (nuclear to cytoplasmic ratio = 0.74±0.07) MCCs (Figure 4A–B). Although total Cdk2 levels did not change during ciliogenesis at the protein or transcript level, we detected a peak in Cdk2 bearing the activating Thr160 phosphorylation (Gu et al., 1992) by Western blot at ALI+2 to +4d, a time interval that is enriched for MCCs in early ciliogenesis (Figure 4C, Figure 4—figure supplement 1A–B and Figure 1—figure supplement 1C). The presence of nuclear Cdk2 in ciliating MCCs and the enrichment of phospho-Thr160 Cdk2 in ciliating MTECs indicates that Cdk2 is active during early ciliogenesis. These signs of Cdk2 activation, together with the requirement for Cdk2 activity to sustain MCC TF expression support a key role for Cdk2 in the initiation and maintenance of the motile ciliogenesis program. Interestingly, we found that HA-Cdk2 also localized to centrioles in both ciliating and mature MCCs (Figure 4D). As we were not able to observe endogenous Cdk2 at MCC centrioles due to the lack of effective antibodies, we could not confirm that this centriolar localization of ectopically expressed Cdk2 is representative of endogenous Cdk2 distribution. However, others have reported that Cdk2 can localize to the centrosome in cycling cells (Kodani et al., 2015), and that it has centrosomal phosphotargets (Chen et al., 2002; Okuda et al., 2000). Thus, both the nuclear and centriolar pools of Cdk2 may be involved in ciliogenesis regulation.

Ccna1 is upregulated during motile ciliogenesis

A- and E-type cyclins associate with Cdk2 and control its activity. Ccna1 is chiefly expressed in meiotic and cancer cell cycles, while A2 and E1 drive the G1 to S phase transition and the S phase events of centriole and DNA duplication (Heim et al., 2017). We examined the expression of Ccna1, Ccna2, Ccne1 and Ccne2 during motile ciliogenesis (Figure 5A) and detected a large increase, followed by a decline in expression for Ccna1 and a more modest increase in expression for Ccne1. The timing of their peak expression correlates with the indicators of Cdk2 activation during ciliogenesis (Figure 4A–C). Ccne2 did not show statistically significant change and consistent with a postmitotic state, only a negligible amount of Ccna2 was detected (Figure 5A). Our observations confirm results from previously published microarray data sets that also identified high Ccna1 expression during ciliogenesis (Stubbs et al., 2008; Hoh et al., 2012). The majority of ciliary genes show strong upregulation followed by a decline in expression during ciliogenesis (ex. Cetn2), and this pattern can be used to identify transcripts important for the process (Hoh et al., 2012). Although we cannot rule out a role for the other canonical cyclins, only Ccna1 matched this profile.

Figure 4 with 1 supplement see all
Cdk2 is nuclear and active during motile ciliogenesis.

(A) MTECs were infected with lentivirus encoding HA-tagged Cdk2 at ALI-2d, then labeled at ALI + 4 d with HA (green), Foxj1 (red) and ac. α-Tub (blue) antibodies. A single image slice through the nuclear region (top panel) shows that Cdk2-HA is present in the nucleus in ciliating MCCs (Foxj1+, ac. α-Tub-, arrow) and nuclear excluded in mature MCCs (Foxj1+, ac. α-Tub+, arrowhead). Ac. α-Tub signal visible on the maximum projection (bottom panel) shows cilia on the mature (arrowhead) or lack thereof on the ciliating cells (arrow). Scale bar, 10 μm. (B) Quantitation of nuclear to cytoplasmic ratio of Cdk2-HA signal intensity in MCCs indicating nuclear enrichment in ciliating cells. Ciliating vs. mature MCCs were identified based on ac. α-Tub and Foxj1 signal. *p<0.0005 C. Western blot of MTEC timecourse lysates shows that total Cdk2 levels are equally abundant at all times, but phospho-T160 Cdk2 is enriched during early ciliogenesis; ratio of phospho/total Cdk2 is indicated under each lane. α-Tubulin signal reflects increasing ciliogenesis and is not to be interpreted as loading control; see Figure 4—figure supplement 1 for Ponceau S stain for blot which serves loading control. (C) MTECs were infected with lentivirus encoding HA-tagged Cdk2 at ALI-2d, then labeled at ALI + 4 d with HA (green), Pericentrin (red) and ac. α-Tub (blue) antibodies. Image slices through the basal body/centriolar region (top panel) shows that Cdk2-HA is centriolar in both ciliating (arrow) and mature (arrowhead) MCCs. Pericentrin signal is strong on ciliating centrioles and weak on mature basal bodies. Ac. α-Tub signal indicating the mature MCC (arrowhead) is visible on the maximum projection (bottom panel). Scale bar, 5 μm.

https://doi.org/10.7554/eLife.36375.008
Figure 5 with 1 supplement see all
Ccna1 is enriched in MCCs and is a target of the MCC gene expression program during ciliogenesis.

(A) Quantitative realtime PCR was used to assess A and E-type cyclin gene expression during motile ciliogenesis. Similar to the ciliary component Cetn2, Ccna1 and Ccne1 are enriched during ciliogenesis. Ccna2 and Ccne2 are not enriched. Levels were normalized to Gapdh expression and compared to values obtained from confluent, ALI-1d samples. Brackets indicate comparison between ciliogenesis timepoints; asterisk above bar indicates significant increase in expression during mid-ciliogenesis compared to confluent, ALI-1d sample. n.s., not significant; *p<0.05 (B) Western blot with MTEC lysates using Ccna1 antibody indicates that Ccna1 is enriched in ciliating MTECs. Testis lysate serves as control of Ccna1 expression. Values normalized to early ciliogenesis indicated under each lane. Acetylated α-Tubulin signal reflects increasing ciliogenesis and should not be interpreted as loading control; see Figure 5—figure supplement 1 for Ponceau S stain for blot, which serves loading control. (C) Realtime PCR for Ccna1 expression in MCCs vs. non-MCCs (sorted from Foxj1-EGFP MTECs) shows that it is restricted to MCCs and Ccna1 expression is higher mid-ciliogenesis (ALI +5 d). (D) Luciferase reporter assay using the human FOXJ1 (left) or CCNA1 promoter (right) in 293T/17 cells shows that they are responsive to MCC transcriptional regulators compared to vector only control. Promoters are only responsive to E2F4 in the presence of the Mcidas transcriptional activator. *p<0.01 (E) 293T/17 cells were infected with lentivirus expressing MCC TFs. Realtime PCR indicates that at least one or more MCC TFs can activate endogenous FOXJ1 (left), CETN2 (center) and CCNA1 (right) gene expression. E2F4 can only activate MCC-specific gene expression in the presence of the Mcidas transcriptional activator. n.s., not significant; *p<0.05, **p<0.01. .

https://doi.org/10.7554/eLife.36375.011

In addition to Ccna1 transcripts, we also found an enrichment of the Ccna1 protein during ciliogenesis (Figure 5B and Figure 5—figure supplement 1B). To test if Ccna1 expression is restricted to MCCs, we carried out qRT-PCR in sorted MCCs and non-MCCs obtained by FACS from the Foxj1-EGFP mouse line (Ostrowski et al., 2003) and found that Ccna1 expression was restricted to MCCs (Figure 5C and Figure 5—figure supplement 1A). This analysis also revealed that the negligible amount of Ccna2 expression derives from non-MCCs and that Ccne1 was detected in both populations (Figure 5—figure supplement 1A). We hypothesize that a small population of proliferating cells, likely progenitor basal cells underlying the luminal MCCs or possibly contaminating fibroblasts sometimes observed in the basal regions of MTEC cultures may be the source of these non-MCC transcripts. The MCC-specific pool of Ccne1 also showed the characteristic rise and fall of expression during ciliogenesis (Figure 5—figure supplement 1A), suggesting that Ccne1 may also regulate ciliogenesis.

Consistent with its MCC-specific expression, we demonstrated that a genomic fragment within the human CCNA1 promoter can drive Luciferase reporter gene expression in response to the MCC TFs E2F4 (but only in the presence of MCIDAS), FOXJ1 and MYB (Figure 5D and Figure 5—figure supplement 1C) in 293T/17 cells, similar to a FOXJ1 promoter fragment previously shown to display MCC-restricted expression (Tan et al., 2013). Furthermore, transfection of the same MCC TFs into 293T/17 cells turned on the expression of endogenous human CCNA1, and also FOXJ1 and CETN2 (Figure 5E). Ccna1 is therefore a target of the MCC gene expression program during ciliogenesis. We hypothesize that employing this A-type cyclin to drive a noncanonical somatic event may drive centriole assembly in MCCs, but not other Cdk2-driven events such as DNA replication.

Ccna1 localization depends on Cdk2

To investigate whether Ccna1 acts together with Cdk2, we examined the localization of endogenous and lentivirally expressed Ccna1-GFP. We found that similar to Cdk2, Ccna1-GFP was present in the nucleus during ciliogenesis. It was strongly nuclear in ciliating MCCs (Figure 6A) while mature MCCs had much lower overall levels of Ccna1-GFP in both nucleus and cytoplasm. Ccna2-GFP was localized in different compartments in different cells and the E-type cyclins showed only nuclear localization at all stages (Figure 6—figure supplement 1A). Using a Ccna1 antibody that specifically recognized Ccna1-GFP by Western blot and immunolabeling (Figure 6—figure supplement 1B–C), we confirmed the nuclear localization of endogenous Ccna1 in ciliating MCCs. Unlike with Ccna1-GFP, we did not detect endogenous signal in mature MCCs (Figure 6B), which may be due to antibody issues. Alternatively, it may indicate the proteasomal degradation of Ccna1 at the end of ciliogenesis, which may be overcome to some extent by continuous lentiviral expression.

Figure 6 with 1 supplement see all
Ccna1 is nuclear and localization depends on Cdk activity during ciliogenesis.

(A) MTECs were infected with lentivirus encoding GFP-tagged Ccna1 at ALI-2d, treated with NU6140 from ALI + 3 to+4 d (NU6140ac, acute treatment) or from ALI + 0 to+4 d (NU6140chr, chronic treatment) then labeled at ALI + 4 d with DAPI (blue), GFP (green), Foxj1 (red) and ac. α-Tub (white) antibodies. The ALI + 3 and 4d untreated panels shows that Ccna1-GFP is nuclear in ciliating MCCs (white arrow) and present in low amounts everywhere in mature MCCs (white arrowhead). Ccna1-GFP is retained in the nucleus in cells arrested during ciliogenesis due to acute NU6140 treatment (yellow arrow). Ccna1-GFP remains nucleo-cytoplasmic in cells blocked from ciliogenesis due to chronic NU6140 treatment (orange arrow). MCC fraction was quantitated based on ac. α-Tub antibody labeling. Scale bar, 10 μm. *p<0.001 (B) ALI + 4 d MTECs labeled with Ccna1 (green), Foxj1 (red) and ac. α-Tub (blue) antibodies shows nuclear Ccna1 (arrow) in ciliating MCCs and no discernible specific signal in mature MCCs (arrowhead). Scale bar, 10 μm. (C) MTECs were infected with equal amounts of lentivirus encoding Ccna1-GFP and Cdk2-HA at ALI-2d, then labeled at ALI + 4 d with GFP (green), HA (red) and Pericentrin (blue) antibodies. A single image slice through the nuclear region (top panel) shows that Ccna1-GFP and Cdk2-HA are nuclear in ciliating MCCs (arrow) and nucleo-cytoplasmic in mature MCCs (arrowhead). Centriolar Pericentrin signal shows a tight cluster of centrioles (arrow) in a ciliating MCC and centrioles distributed at the apical surface in a mature MCC. Scale bar, 10 μm. (D) ALI + 4 d MTECs labeled at ALI + 4 d with Ccna1 (green), Cep164 (red) and ac. α-Tub (blue) antibodies shows a mature MCC with centriolar Ccna1 signal. Scale bar, 5 μm. .

https://doi.org/10.7554/eLife.36375.013

We found that similar to other ciliary genes, endogenous Ccna1 gene expression is suppressed by both chronic (ALI + 0 to+4 d) and acute (ALI + 3 to+4 d) Cdki treatment (Figure 6—figure supplement 1D). Upon lentiviral co-expression, Cdk2-HA and Ccna1-GFP colocalized in the nucleus and Ccna1 was also detected at the centrioles (Figure 6C–D). Moreover, we found that Ccna1-GFP localization depends on Cdk2 activity (Figure 6A), as Ccna1-GFP failed to accumulate in the nucleus in MTECs under chronic Cdki treatment that blocks the initiation of motile ciliogenesis, nor was it able to exit the nucleus under acute Cdk inhibition that arrests MCCs at intermediate stages of ciliogenesis. These results support a model in which, as in cycling cells, Cdk2 and Ccna1 function as a complex to regulate motile ciliogenesis in MCCs.

Ccna1 mutant mice have fewer MCCs

Ccna1 knockout mice are viable, but male-sterile due to the requirement for Ccna1 in the meiotic divisions of the male germline (Liu et al., 1998). To assess a potential requirement for Ccna1 in the regulation of motile ciliogenesis, we observed MCCs by scanning electron microscopy and ac. α-Tub antibody labeling in the trachea and bronchi of adult Ccna1-/- mice. We consistently observed a 2.46-fold reduction in the fraction of MCCs in the mutant airways compared to wildtype litter mates (0.15 ± 0.01 vs. 0.36 ± 0.01). Ccna1-/- epithelia also contained an increased number of dome-shaped, possibly secretory cells, which may indicate further dysfunction in these airways (Figure 7A, Figure 7—figure supplement 1A). In comparison, we examined Ccne1; Ccne2 double knockout mice (Geng et al., 2003) and found no difference in the fraction of MCCs. (Figure 7B). Based on these observations, we conclude that Ccna1 is required for motile ciliogenesis. Although the ectopic expression of Ccna1 and Cdk2 either separately or together is not sufficient to drive the motile ciliogenesis pathway (Figure 1E, Figure 7—figure supplement 1B and not shown), our results are consistent with Cdk2 acting together with Ccna1 and possibly other cyclins upon MCC fate acquisition to activate the MCC gene expression program.

Figure 7 with 1 supplement see all
Ccna1, but not Ccne1;Ccne2 mutant mice have fewer MCCs.

(A) SEM of adult Ccna1-/- trachea (left panels) and ac. α-Tub (green) antibody labeling of bronchi (epithelium marked with E-cadherin antibody labeling, red) on cryosectioned lung tissue (right panels) show fewer MCCs with shorter, sparser cilia (bottom) compared to wildtype littermates (top). See Figure 7—figure supplement 1A for quantitation of MCC fraction for SEM. Scale bar, 10 μm for trachea, 20 μm for lung. (B) SEM of Ccne1-/-;Ccne2-/- trachea shows no difference in the number and distribution of MCCs (bottom) compared to wildtype littermates (top). Scale bar, 10 μm. (C) Schematic of Cdk2 function during cell cycle progression and proposed Cdk2-Ccna1 activity in the motile ciliogenesis pathway. Solid and dotted arrows linking the progenitor state to EMD activation represent alternative potential pathways that have yet to be distinguished. .

https://doi.org/10.7554/eLife.36375.015

Discussion

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.

Materials and methods

Mouse husbandry and MTEC culture

C57BL/6J (IMSR Cat# JAX:000664, RRID:IMSR_JAX:000664) mice were obtained from JAX. Ccna1 (MGI Cat# 2657243, RRID:MGI:2657243) (Liu et al., 1998), Ccne1; Ccne2 (MGI Cat# 2675493, RRID:MGI:2675493, gift from Peter Sicinski, Harvard Medical School, Cambridge, MA) (Geng et al., 2003) and Foxj1-EGFP (IMSR Cat# JAX:010827, RRID:IMSR_JAX:010827, gift from Larry Ostrowski, UNC Chapel Hill, Chapel Hill, NC) (Ostrowski et al., 2003) mice have been previously described. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Stanford University School of Medicine in accordance with established guidelines for animal care. MTEC culture and lentiviral infection were carried out as previously described (Vladar and Brody, 2013). In short, tracheas were isolated and incubated overnight in Pronase solution to release epithelial cells. Cells were seeded onto 24, 12 or 6 well size Transwell filters (Corning) and cultured submerged in proliferation medium until confluence. The air-liquid interface (ALI) was created by adding differentiation medium to only the bottom compartment of the culture dish. Cells were treated at ALI with 10 μM Roscovitine, 5 μM Purvalanol A, 10 μM NU6140, 10 μM Cdk2 Inhibitor III, 1 μM RO3306 (all from Tocris) and 1 μM DAPT (Abcam) in differentiation medium for various lengths of time. MTECs are fed fresh medium with or without drugs every two days.

Lentiviral vectors containing myc-Mcidas, Foxj1 and Myb have been previously described (Tan et al., 2013). Lentiviral vectors containing human Cdk2-HA and Cdk2D145N-HA (van den Heuvel and Harlow, 1993) were generated by transferring the Cdk2-HA open reading frames into the BamHI site of the pRRL.sin-18.PPT.PGK.pre lentiviral vector using PCR. The GFP-Ccna1/a2/e1 and e2 constructs were generated by PCR amplifying and inserting the cyclin open reading frames, obtained by RT-PCR from MTEC or NIH/3T3 cell cDNA, into the AgeI or BamHI site of the pRRL.sin-18.PPT.PGK.GFP.pre lentiviral vector to create a C-terminal GFP fusion. Lentivirus was prepared according to published methods using the psPAX2 and pMD2.G helper plasmids (Addgene) in the 293T/17 cell line (see below). MTECs were infected with lentivirus on day three of culture (ALI-2d) using spin infection following EGTA treatment to temporarily disrupt epithelial junctions (Vladar and Brody, 2013).

Cell lines

293T/17 (ATCC Cat# CRL-11268, RRID:CVCL_1926) cells were used for lentiviral preparation and the Luciferase and endogenous gene activation assays. mIMCD3 (ATCC Cat# CRL-2123, RRID:CVCL_0429) cells were used to test Ccna1 antibody specificity. Cells were purchased from ATCC and were assumed to be authenticated by the supplier. Cells were not specifically monitored for mycoplasma contamination, but routine DAPI staining would have revealed the presence of contamination.

Immunofluorescence and immunohistochemistry

MTECs grown on 24 well size Transwells were fixed in −20°C methanol or 4% paraformaldehyde for 10 min, blocked in 10% normal horse serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1–2 hr, then with secondary antibodies for 30 min at room temperature. Samples were mounted in Mowiol mounting medium containing 2% N-propyl gallate (Sigma). Lung tissues were fixed in 4% paraformaldehyde overnight at 4°C, rinsed in PBS, incubated in 30% sucrose, embedded in OCT compound and frozen. 10 μm cryosections were labeled as above. Samples were imaged with Leica LAS X software on a Leica SP5 or SP8 confocal microscope (Leica). For antibodies and fixation conditions, see Supplementary Table 1 in Supplementary file 1.

Cell lysates and western blots

MTEC lysates were generated in triplicate from MTECs cultured in 12- or 6-well Transwells to the desired stage and harvested by scraping the Transwell surface in 1x Laemmli sample buffer. Approximately 10,000 cells per lane were loaded for SDS-PAGE, then transferred to nitrocellulose membrane. For the MTEC timecourses equal loading was verified by Ponceau S staining of the membrane. Acetylated α-Tubulin antibody labeling shows increasing signal with increased ciliogenesis as MTECs mature and thus should not be interpreted as a loading control. Mouse testis lysate was prepared using published methods (Panigrahi et al., 2012) to serve as a control for Ccna1 expression.

Quantitative realtime PCR

cDNA was prepared from MTECs cultured on 12- or 6-well Transwells at various timepoints and with various treatments as indicated, and from MCCs (EGFP+) and non-MCCs (EGFP-) obtained by FACS from Foxj1-EGFP MTECs grown on 12 6-well Transwells as previously described (Vladar and Stearns, 2007). Early, mid and late ciliogenesis timepoints were obtained at ALI + 2, 4 and 8 days. Gapdh levels were used to normalize target gene expression values. Ciliogenesis timecourse gene expression levels were compared to levels in confluent, but not yet differentiated cells harvested at ALI-1d. MCC vs. non-MCC timecourse gene expression levels were compared to levels at ALI + 0 d. qPCR was performed in triplicate with Power SYBR Green Master Mix (Thermo Fisher) in a StepOnePlus Real-Time PCR System (Thermo Fisher), and gene expression was evaluated using the ΔΔCt method. For primer sequences, see Supplementary Table 2 in Supplementary file 1.

Cdk2 nucleo-cytoplasmic signal quantitation

The ratio of lentivirally expressed Cdk2-HA nuclear to cytoplasmic signal intensity in MCCs (identified by Foxj1 and ac. α-Tub signal) was quantitated on individual image slices using ImageJ (NIH) by measuring signal intensity for manually outlined nuclear and cytoplasmic areas (nuclear area identified by Foxj1 or DAPI signal) and normalizing to the total measured area.

Luciferase assay

Human FOXJ1 and CCNA1 promoter genomic DNA fragments (see Figure 5—figure supplement 1B) were cloned into the pGL4.20 (Promega) firefly luciferase expression vector and transfected into 293T/17 cells using FuGENE6 (Roche) along with plasmids containing human FOXJ1, MYB, E2F4 and/or MCIDAS cDNAs (Tan et al., 2013) and pRL-TK (Promega) Renilla luciferase expression vector. Reporter activity was assessed using the Dual-Glo Luciferase Assay System (Promega) with a FLUOStar Omega (BMG Labtech) luminescence plate reader. Relative reporter activity was calculated by normalization to the vector only transfection control in triplicate.

Electron microscopy

Adult mouse airway tissues were fixed in 2% glutaraldehyde, 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4 (all from Electron Microscopy Sciences) at 4°C overnight, osmicated, dehydrated and dried with a Tousimis Autosamdri-815 critical point dryer. Samples were then mounted luminal side up, sputter coated with 100 Å layer of Au/Pd and analyzed with a Hitachi S-3400N VP-SEM microscope (Hitachi) operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.

Transparent reporting

  1. Sample-size estimation: No explicit power analysis was used during the design of the study. Biological and experimental replicates (indicated in Supplementary Table 3 in Supplementary file 1) were completed to at least an n = 3 where possible.

  2. Replicates: replicate information is found in Supplementary Table 3 in Supplementary file 1 with biological replicates indicated by underlining and technical replicates indicated by italicized font. Outliers were not encountered. Data were not excluded from analysis.

  3. Statistical reporting: the Prism7 software (GraphPad Software) was used to generate graphs and perform statistical analyses. Pairwise comparisons were made with a two-tailed Student’s t test or an ANOVA test. For multiple comparisons, a follow up test (Dunnett’s or Bonferroni’s) was applied to correct for multiple hypothesis testing. For all cases, a p value less than 0.05 was considered significant. Error bars on graphs represent standard error. See Supplementary Table 3 in Supplementary file 1 for information about replicates and statistical tests for data presented in figures.

  4. Group allocation: samples generated in an identical manner were allocated into groups based on treatment (drug, lentiviral gene expression, etc.) or genotype.

  5. Source data: not applicable

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Decision letter

  1. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands
  2. Jay Rajagopal
    Reviewing Editor; Harvard University, United States
  3. Steven Brody
    Reviewer; Washington University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Cyclin-dependent kinase control of motile ciliogenesis" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Steven Brody (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary

The manuscript submitted by Vladar and colleagues reports a novel role for the canonical mitotic regulator Cdk2 in the control of ciliogenesis in murine airway culture systems. They claim that Cdk2 is required to initiate and maintain the MCC gene expression program during ciliogenesis. They claim that Cdk2 is coupled with cyclin A1 to initiate centriole replication in MCCs. The authors begin by assessing the effects of pharmacologic Cdk inhibitors on differentiating MTEC ALI cultures, observing that inhibition of Cdk2 produces a reversible block in the initiation ciliogenesis revealed by the absence of MCC markers by immunofluorescence imaging. The specificity of this result was established with a dominant-negative allele of Cdk2, which phenocopies pharmacologic inhibition. Subsequent data suggests ongoing Cdk2 activity (after initiation of ciliogenesis) is also required for MCCs to complete maturation into "Stage IV" or Foxj1+/Myb- cells. In order to establish epistasis relationships, MTEC ALI were treated simultaneously with Cdk2 inhibitors and the Notch antagonist DAPT or with Cdk2 inhibitors in the setting of enforced Mcidas expression. The claim is that these experiments place Cdk2 downstream of Notch and upstream of Mcidas in specifying MCCs. In order to identify the cellular compartment in which Cdk2 functions, tagged overexpressed Cdk2 was localized at ALI+4d, a timepoint at which both mature and ciliating MCCs are present. The authors claim nuclear exclusion of HA-Cdk2 in mature (but not ciliating) MCCs and go on to co-localize HA-Cdk2 to the centriolar compartment. In order to identify the cognate cyclin with which Cdk2 associates during MCC development, the authors utilize qRT-PCR and note the potent induction of Ccna1 during the mid-ciliogenesis stage specifically in MCCs (defined here by Foxj1 expression). Reporter assays and endogenous qRT-PCR experiments upon enforced overexpression of MCC transcription factors suggest Ccna1 can be induced by E2F4 in the presence of Mcidas. The authors next examine the localization of ectopic and native Ccna1, concluding Ccna1 is nuclear localized during ciliogenesis and subsequently undetectable or nucleocytoplasmic in mature MCCs. Given the similarity of this pattern to that previously observed for Cdk2, they argue for the function of Cdk2/Ccna1 as a physically interacting complex. In the final series of experiments the authors demonstrate the in vivo importance of Ccna1 in ciliogenesis by examining the airways of previously generated Ccna1-/- mice.

All the reviewers agree that there is broad general interest in these findings. It is clear that Cdk2/Ccna1 are important regulators of airway ciliogenesis. The data are convincing that Cdk inhibitors interfere with both processes: centriologenesis and cilia formation. This is an important contribution. However, there are substantive concerns that need to be addressed about the interpretation and mechanism and the various steps of ciliogenesis and maintenance that are affected by Cdk2.

Major Concerns that must be addressed:

1) The actual role of Cdk2 in ciliated cell formation, ciliogenesis, and cilia maintenance per se is unclear. They need to clarify effects on cell fate specification and ciliogenesis and ciliated cell maintenance. Is Cdk2 directing specifically the initiation and (perhaps) maintenance of MCC centriologenesis or is there a broader role in the (co-)regulation of other steps of cilia assembly. A specific role in centriole replication needs to be clear. It may be that there are biological or technical limitation such that a failure of initiation of centriole amplification halts the entire subsequent program of ciliogenesis, this may be difficult to prove in a revision. However, the apparent later stage role of Ccna1, after centriologenesis, seems contradictory to the proposal that Cdk2-Ccna1 are regulating centriole replication and needs to be resolved. There might be some experimental limitations that cannot fully separate the roles of Cdk2 in regulating ciliated cell formation and centriologenesis. But the authors need to clarify the specific roles of Cdk in either dilated cell formation or centriologenesis and also discuss these limitations in the paper

2) The links between the celluar localization of Cdk2, the activation of Cdk2, and the expression of MCC TFs are inconclusive. It is difficult to appreciate the nuclear-cytoplasmic shuttling during ciliogenesis. In Figure 4, nuclear vs cytoplasmic lysates would be better than this IF (which isn't convincing for nuclear exclusion). One could immunoprecipitate tagged Cdk2 from nuclear and cytoplasmic compartments at various timepoints and show Ccna1 blots?

3) The experimental evidence is not sufficient to support that CdK2 acts downstream of Notch signaling. The authors cannot exclude the possibility that Cdk2 and Notch signaling function parallel but share the same downstream consequence.

Requirements for revision:

1) The authors should provide additional evidence that Cdk2 acts downstream of Notch signaling, rather than in a parallel pathway. A further assessment of the components of Notch signaling might be one approach.

2) The authors should provide more convincing evidence of nuclear-cytoplasmic shuttling with the HA-Cdk2, in control, basal cells and in cells undergoing centriologenesis and ciliogenesis. Identification of the fusion protein in Isolated nuclear and cytoplasmic fractions could be performed.

3) Biochemical evidence of the interaction of Cdk2 with Ccna2 in cyotoplasmic fractions during specific stages of centriologenesis and/or cilia assembly. Related to this strengthen evidence regarding the relationship of HA-Cdk2 and components of centrioles (biochemically or by IF).

4) A better characterization of pT160 Cdk2 to identify which specific stages of MCC differentiation are associated with this activity. For example, is there coincident biochemical evidence of high levels of the massive centriole replication (and the presence of other markers of centriologenesis) or it later, post centriole replication/docking and instead coincident with cilia assembly? Or, during both processes?

5) There are several relatively minor points that ought to be addressed related to clarifying the use of specific parameters in graphs, providing statistics, controls for western blots, and quantifying ciliated vs non-ciliated cells in Ccna1 ko mice.

6) Finally, the last four paragraphs of the Discussion could be condensed since it is speculation and not directly focused on the data. I think that one major emphasis of the discussion should be a comparison to Al Jord et al., 2017, who show that Cdk1 (rather than Cdk2) in balance with APC is required for centriole amplification-ciliation and inhibition of mitosis, respectively. The Vlader discussion should further explain why Al Jord implicates Cdk1 and Vlader Cdk2 beyond what is written in paragraph two. The authors should discuss specifically if they believe that their data indicate that Cdk2 is directly a program or has specific protein targets, these issues are discussed loosely in paragraphs three, four and five. Consider inhibition of APC and examine Cdk2-dependent centriole amplification, this would address the discussion point in paragraph six.

https://doi.org/10.7554/eLife.36375.021

Author response

Major Concerns that must be addressed:

1) The actual role of Cdk2 in ciliated cell formation, ciliogenesis, and cilia maintenance per se is unclear. They need to clarify effects on cell fate specification and ciliogenesis and ciliated cell maintenance. Is Cdk2 directing specifically the initiation and (perhaps) maintenance of MCC centriologenesis or is there a broader role in the (co-)regulation of other steps of cilia assembly. A specific role in centriole replication needs to be clear. It may be that there are biological or technical limitation such that a failure of initiation of centriole amplification halts the entire subsequent program of ciliogenesis, this may be difficult to prove in a revision. However, the apparent later stage role of Ccna1, after centriologenesis, seems contradictory to the proposal that Cdk2-Ccna1 are regulating centriole replication and needs to be resolved. There might be some experimental limitations that cannot fully separate the roles of Cdk2 in regulating ciliated cell formation and centriologenesis. But the authors need to clarify the specific roles of Cdk in either dilated cell formation or centriologenesis and also discuss these limitations in the paper

This is a complex multi-part question, which we attempt to break down and address systematically.

First, it is apparent that we failed to adequately convey the currently accepted understanding of these events. We hope that the following is a clearer description of the events and regulation of cell fate specification, motile ciliogenesis and cilium maintenance during multiciliated cell (MCC) differentiation. To amend the manuscript, we have added two new schematics to Figure 1—figure supplement 1 (new Figure 1—figure supplement 1A-B), updated Figure 1—figure supplement 1C and Figure 7B (now 7C) and clarified the text in the Introduction as well as the Discussion.

To summarize: upon airway epithelialization, MCC and secretory cell fates are acquired in a Notch signaling-dependent mechanism. Cells in which Notch signaling is activated are diverted to the secretory cell fate, whereas cells in which Notch is not activated differentiate into MCCs. Therefore, it is the absence of Notch signaling that leads to the MCC fate. We note that typically, Notch signaling entails a feedback, in which neighboring cells emerge as either Notch expressing cells, in which Notch is activated (here, the secretory cells), or ligand expressing cells, in which Notch is not activated (here, the MCCs). In the prospective MCCs in which Notch is not activated, it appears that MCC differentiation is a default occurrence, though we cannot rule out that some additional initiating event is required that might or might not be linked to having expressed ligand.

Thus, the first key point that we make is that the idea of being downstream of an ABSENCE of another event (the Notch signal) is a bit slippery. If having expressed the ligand, or receiving another signal, is not necessarily required, one might alternately describe this scenario as the progenitors retaining a pre-existing MCC fate unless they are diverted to the secretory fate through Notch activation. Of note, published data suggest that cells diverted to the secretory fate can be transdifferentiated back to the MCC fate by inhibiting Notch, indicating that the potential to become an MCC is not eliminated, but rather is merely suppressed, by ongoing Notch signaling. Because the opportunity to divert to the secretory cell fate exists prior to MCC differentiation (see below), we choose to retain the language existing in the literature of MCC fate occurring ‘downstream’ of Notch signaling, while expressing this caveat in the amended text.

The second key point is that the entire morphogenetic program of MCC differentiation, including not only centriologenesis, but all the subsequent events including docking, axonemogenesis, and likely numerous other events, are driven by the activation of a MCC-specific transcriptional complex comprised of E2F4/5, Mcidas and DP1 (the EMD complex) to initiate and drive motile ciliogenesis. EMD turns on multiple other secondary transcription factors (including Myb, Rfx3 and Foxj1) and they cooperate to turn on hundreds of structural and regulatory ciliary genes. This leads to activation of the motile cilium biogenesis pathway, which consists of centriole amplification followed by centriole apical transport and membrane docking and then axoneme elongation. These transcription factors are expressed throughout differentiation, and most (with the exception of Foxj1 and possibly Foxn4) are downregulated only after differentiation is complete. Therefore, activating the EMD complex, a step requiring Cdk2, drives not just centriologenesis, but the entirety of the differentiation program. Ciliogenesis is a linear pathway, and a failure to initiate centriole assembly will absolutely preclude subsequent steps in the ciliogenesis program. This in itself does not distinguish between an initiation and a maintenance function for Cdk2. However, our result showing that cells in which Cdk2 is inhibited after the centriole replication has begun nonetheless are arrested in differentiation (Figure 3C) demonstrates an ongoing requirement for Cdk2 in addition to a role in starting the process. This ongoing requirement may reflect a continuing transcriptional activity, but might also, or in addition, be associated with a more direct role of Cdk2 in a downstream process, a possibility suggested by the centriolar pool, for example. We suggest that investigation of the distinct pools of Cdk2 and roles other than initiating and sustaining MCC TF activity are beyond the scope of this manuscript.

With this conceptual framework in mind, we can address some specific queries:

Is Cdk2 directing specifically the initiation and (perhaps) maintenance of MCC centriologenesis or is there a broader role in the (co-)regulation of other steps of cilia assembly.

Our data support a role for Cdk2 in nascent MCCs (in the absence of, or ‘downstream of,’ the Notch signaling event) upstream of the EMD transcriptional complex. Because EMD drives the whole constellation of differentiation events, we propose that Cdk2 is required in nascent MCCs to initiate ciliogenesis. Our data noted above (Figure 3C) also supports an ongoing role for Cdk2 during differentiation.

However, the apparent later stage role of Ccna1, after centriologenesis, seems contradictory to the proposal that Cdk2-Ccna1 are regulating centriole replication and needs to be resolved.

We do not view these roles as contradictory. Both centriole replication and subsequent events are dependent on EMD activation. As described above, both functions might be mediated by Cdk2-Ccna1 acting on EMD, or the later maintenance of ciliogenesis could potentially be mediated by non-transcriptional functions instead of, or in addition to, a transcriptional function.

They need to clarify effects on cell fate specification and ciliogenesis and ciliated cell maintenance.

We have added new data, requested by the reviewers, that incidentally allows us to address this question more precisely. The new data (new Figures 2B and 7C) regards the role of Cdk2 in relation to TRRAP in this process. TRRAP, a histone deacetylase complex member (Wang et al., 2018) was identified as an early regulator of motile ciliogenesis and was shown to act in the nucleus to turn on the EMD complex downstream of (in the absence of) the Notch signaling event. We show that TRRAP expression is induced in the absence of Cdk activity, indicating that TRAPP functions either upstream of, or in parallel to, Cdk2 activation (see new Figure 7C). We allowed MTEC cultures to differentiate for four days in the presence of the Cdk inhibitor Nu6140. As shown before, these cultures are not able to initiate EMD activity and undergo motile ciliogenesis. However Nu6140 treatment had no effect on the cells’ ability to turn on nuclear TRRAP expression. Thus, we while Cdk2 is required to initiate ciliogenesis, we can conclude it is not involved in the MCC fate decision as evidenced by the expression of TRRAP even in the absence of Cdk2 activity.

There might be some experimental limitations that cannot fully separate the roles of Cdk2 in regulating ciliated cell formation and centriologenesis.

We presume here that the reviewer means distinguishing initiation of the differentiation program from a specific and direct function in centriologenesis. Indeed, answering this question would require biochemical analyses to complement the genetic manipulations performed thus far. As described in our response to point 2 below, the asynchrony of differentiation and the limiting amounts of material render these types of studies infeasible. In case we are interpreting the comment incorrectly, and the reviewer means separating the roles of Cdk2 in MCC cell fate specification versus centriologenesis, we argue that the TRRAP result shows that Cdk2 is not required for acquisition of the MCC cell fate.

2) The links between the celluar localization of Cdk2, the activation of Cdk2, and the expression of MCC TFs are inconclusive. It is difficult to appreciate the nuclear-cytoplasmic shuttling during ciliogenesis. In Figure 4, nuclear vs cytoplasmic lysates would be better than this IF (which isn't convincing for nuclear exclusion). One could immunoprecipitate tagged Cdk2 from nuclear and cytoplasmic compartments at various timepoints and show Ccna1 blots?

Two ideas appear to be embedded in this comment. First, “The links between the cellular localization of Cdk2, the activation of Cdk2, and the expression of MCC TFs are inconclusive.” An abundance of prior data establishes that Cdk2 activation and its nuclear localization (and phosphorylation) are coincident. We have shown that its activity is required for EMD complex (MCC TF) activation, and we demonstrate the correlated nuclear localization and Thr160 phosphorylation of Cdk2. These conclusions are based on inhibitor and dominant negative studies, IF assays of nuclear localization (newly quantitated, as described below) and immunoblotting. Thus, the requirement for Cdk2 to activate EMD, and the markers of active Cdk2 are conclusive.

We acknowledge that we do not demonstrate that the EMD complex (MCC TF) is a direct target of Cdk2, and that we provide only immunofluorescence evidence that Cdk2 (and Ccna1) (co)localize in the nucleus in ciliating MCCs. Technical limitations and the nature of MCC differentiation prevent us from addressing these questions biochemically. MTECs (as well as the in vivo airways) undergo MCC fate acquisition and motile ciliogenesis in a highly asynchronous manner (Figure 3). Although younger MTECs are more enriched for cells in the early stages of ciliogenesis and older MTECs contain mostly mature MCCs, MTECs at all times contain MCCs at different levels. Moreover, MTECs contain a large fraction of nonMCCs, which include basal stem cells and secretory cells (see new Figure 1—figure supplement 1C). Our data suggest that Cdk2-Ccna1 is required for ciliogenesis, but not in mature MCCs, and for Ccna1 we specifically show that its expression is restricted to MCCs. This means that any biochemical fractionation or IP would require purification of MCCs in the process of ciliating from a complex cell population. We have the ability to FACS sort MCCs vs. nonMCCs using the Foxj1-EGFP mouse line (Vladar and Stearns, 2007), but this preferentially isolates mature MCCs and cannot distinguish between MCCs at different stages of ciliogenesis (see Figure 1—figure supplement 1 and Figure 3). There are currently no FACS markers specifically for ciliating MCCs or specific ciliogenesis stages. Finally, even if we could sort the desired cells, it is currently not technically feasible to scale up the MTEC culture (limited by the number of basal stem cells isolated from airway tissues) to produce a sufficient quantity of cell lysates for the required fractionation and IP experiments. Experiments from unsorted MTECs would not address these questions or would be impossible to interpret due to the presence of nonMCCs or mature MCCs. It is already well-known from work in other cells and tissues that Cdk2 can undergo nucleo-cytoplasmic shuttling and that it can interact with Ccna1. To add additional value to our manuscript, testing this specifically in ciliating MCCs would be required, but we are unable to isolate these cells.

“It is difficult to appreciate the nuclear-cytoplasmic shuttling during ciliogenesis.” As we are left with only IF assays, we strengthened our observation of nuclear localization by providing careful quantitation of our Cdk2-HA localization in MTECs, the results of which support the presence of Cdk2 in the nucleus in ciliating, but not in mature MCCs (new Figure 4B).

In the manuscript, we are careful to temper our overall conclusions in this revision in light of these limitations. We hope it will be possible to address these points more fully as technology advances. However, the remarkable conservation of Cdk2 localization and function (nuclear localization, partnering with an A-type cyclin and acting on an E2F family TF containing transcriptional complex) between MCCs and cycling cells complements our data and strongly supports our proposed mechanism.

3) The experimental evidence is not sufficient to support that CdK2 acts downstream of Notch signaling. The authors cannot exclude the possibility that Cdk2 and Notch signaling function parallel but share the same downstream consequence.

As discussed above, it is absence of Notch signaling that allows MCC differentiation. To reword the reviewer’s idea, s/he is asking whether Cdk2 might function in parallel and have common downstream consequences with the absence of Notch signaling. Said this way, the idea makes little sense. To reiterate, we argue that progenitors must both NOT have Notch activation, and have activated Cdk2. Since Notch activation suppresses MCC differentiation, temporally the Notch mediated event must occur prior to Cdk2 activation or no secretory cells would be produced. This idea is reinforced by the observation that TRRAP expression, an indicator of entering the MCC pathway (and therefore of having NOT been activated by Notch), occurs in the absence of Cdk2 activity (new Figure 2B). Thus, Notch signaling occurs before Cdk2 activation temporally, but strictly speaking, it is incorrect to say that absence of Notch signaling is upstream of Cdk2 activation.

The other challenging notion in this comment is that there is a downstream consequence of not being activated by Notch. As far as we know, the consequence might be to retain the same state that the cell was in prior to or in the absence of any Notch signaling at all.

Requirements for revision:

1) The authors should provide additional evidence that Cdk2 acts downstream of Notch signaling, rather than in a parallel pathway. A further assessment of the components of Notch signaling might be one approach.

We hope that the conceptual challenge in the idea of an activity being downstream of the absence of another signal has been adequately explained above. Strictly speaking, our data are consistent with a model in which both the absence of the Notch signal and the activation of Cdk2 are required in a given cell to promote the MCC fate. Because diversion to the secretory cell fate by Notch activity suppresses Cdk2 activation and the MCC fate, the decision about entering the secretory pathway (active Notch signaling) vs the MCC fate (absence of Notch signaling) must typically occur prior to activation of MCC TFs, and this (together with a lack of complete rigor) is probably the origin of the ‘upstream’ and ‘downstream’ descriptors currently in the literature. We have attempted to convey this admittedly somewhat subtle concept both here and in the revised text (see also the new Figure 1—figure supplement 1A-B), but have chosen to keep the language of ‘upstream’ and ‘downstream’ as shorthand.

Because it is absence of Notch signaling that is required for the MCC cell fate, investigating components of Notch signaling such as Notch intracellular domain (N-ICD) nuclear translocation and resulting gene expression would be relevant to the secretory cell rather than the MCC, and therefore not useful. We attempt to clarify the role of Notch signaling in MCC specification in the text as well as in the new Figure 1—figure supplement 1A-B.

2) The authors should provide more convincing evidence of nuclear-cytoplasmic shuttling with the HA-Cdk2, in control, basal cells and in cells undergoing centriologenesis and ciliogenesis. Identification of the fusion protein in Isolated nuclear and cytoplasmic fractions could be performed.

As stated above, technical limitations and the nature of MCC differentiation prevent us from addressing Cdk2-HA nuclear vs. cytoplasmic enrichment using nuclear and cytoplasmic fraction lysates. Like the in vivo airway surface, MTECs contain MCCs and nonMCCs, and MCCs ciliate asynchronously; thus a bulk extract would contain nuclear and cytoplasmic fractions from cells that would be expected to show multiple Cdk2 enrichment patterns (ciliating vs. mature cells) and thus make it impossible to interpret the results. As described above, we do not have the ability to isolate ciliating MCCs specifically due to the lack of appropriate FACS markers and the low number of these cells in our small scale primary cultures. To support our findings, we now provide quantitation of our Cdk2-HA subcellular localization in MTECs, and the results support the nuclear enrichment of Cdk2 in ciliating, but not in mature MCCs (new Figure 4B).

3) Biochemical evidence of the interaction of Cdk2 with Ccna2 in cyotoplasmic fractions during specific stages of centriologenesis and/or cilia assembly. Related to this strengthen evidence regarding the relationship of HA-Cdk2 and components of centrioles (biochemically or by IF).

This experiment requires demonstration that Cdk2 interacts with Ccna1 (we assume that the Reviewer/Editor is asking about Ccna1 and not Ccna2) specifically in ciliating MCCs. Cdk2 is already known to physically interact with Ccna1 based on work in other systems. However, the technical limitations preclude isolation of this sub-population of cells. We raise and discuss this limitation to our study in the discussion. Our hypothesized mechanism involving a Cdk2-Ccna1 complex in early ciliogenesis regulation is bolstered by analogy to a vast trove of exiting data from other studies that indicate that Cdk2 requires A or E-type cyclin binding for activation.

4) A better characterization of pT160 Cdk2 to identify which specific stages of MCC differentiation are associated with this activity. For example, is there coincident biochemical evidence of high levels of the massive centriole replication (and the presence of other markers of centrioliogenesis) or it later, post centriole replication/docking and instead coincident with cilia assembly? Or, during both processes?

Again, technical limitations do not permit the isolation of the specific cells necessary for a biochemical approach to this question. It might have been possible to address this using IF if the T160 Cdk2 antibody were effective in IF experiments. Unfortunately, we were not able to obtain any immunofluorescence signal with the Cdk2 antibody using multiple fixation methods, suggesting either that the antibody does not work in IF experiments or it is not sufficiently sensitive for the amounts of phospho-Cdk2 expressed. Therefore, we are limited to inference from the increased pT160 Cdk2 signal in early ALI timepoints from bulk isolates that are strongly enriched for MCCs at the earliest stages of ciliogenesis that active Cdk2 is more abundant in these cells.

5) There are several relatively minor points that ought to be addressed related to clarifying the use of specific parameters in graphs, providing statistics, controls for western blots, and quantifying ciliated vs non-ciliated cells in Ccna1 ko mice.

Please note: on 5/2/18 we requested and received the following clarifications on this point by email from Maria Guerreiro, Journal Development Editor, eLife:

Concerning Figure 1C/Figure 4—figure supplement 1, the label on the y-axis is not described in the figure legend and is vague. Is confluence of cells binary or quantified? Is this instead confluence/density of MCC? It doesn't change after ALI is established, so how the denominator changes during different stages is unclear. Please clarify.

MTECs proliferate to confluence after about 3-4 days of submerged culture. Confluence is evaluated either visually (no membrane, only uninterrupted epithelial cell surface with cobblestone morphology visible) or it can be inferred from increased transepithelial electrical resistance as measured by an epithelial voltohmmeter. We changed the y-axis labels on both figures to ALI-1d (one day before ALI). The text indicates that this corresponds to a confluent, but not yet differentiating culture. We use cDNA from this time point as a comparison for MCC-related gene expression at later time points as it reflects an intact epithelium with no MCCs.

Figure 5A: the mRNA expression level of Ccna1 at the late ciliogenesis stage is higher than that at the early ciliogenesis stage. But the protein expression levels of Ccna1 in the Figure 5B show an opposite result.

We speculate that at the conclusion of ciliogenesis Ccna1 protein may be actively degraded. This is supported by our inability to detect it by immunofluorescence in individual mature MCCs (Figure 6B) and by analogy with data from other systems and processes that demonstrate that A-type cyclin levels are under strict control by gene expression as well as proteasomal degradation. MTEC lysates and cDNA preps from the three timepoints contain material from a complex mix of MCCs and nonMCCs, although the MCC fraction is enriched for cells at the early, mid and late stages of ciliogenesis. Late ciliogenesis samples also contain many mature and nearly mature MCCs, and it is possible that active degradation of Ccna1 in these cells results in the overall lower signal on the Western blot compared to the corresponding qPCR experiment. It is also possible that the cells that were used for protein lysate and cDNA prep contained a somewhat different proportion of MCCs vs. nonMCCs or ciliating (with higher Ccna1 gene and protein levels) vs. mature (with lower Ccna1 gene and protein levels) MCCs. In either case, both the qPCR and the Western blot results support a trend indicating that actively ciliating MCCs have high Ccna1 transcript and protein levels, which is downregulated as ciliogenesis concludes – supporting a role for Ccna1 in regulating this process.

In addition to quantifying ciliated vs. non-ciliated cells in Ccna1ko mice and including Western blot loading controls, there are two other minor things that need to be clarified: 1. Figure 3: The authors stated that they did not detect a statistically significant difference in the Myb+/Foxj1- population under the Cdki treatment. However, in the Figure 3C, the fraction of Myb+/Foxj1- is significantly decreased (p<0.05) in the Nu6140 acute treated ALI at D4 as compared with untreated ALI at D3.

Quantification of the Ccna1KO results is now included in the manuscript (new Figure 7—figure supplement 7A).

Ponceau-S stained membranes serving as loading control for Western blots are now show in the following: new Figure 4—figure supplement 4B for Figure 4C and new Figure 5—figure supplement 5B for Figure 5B.

We thank the Reviewers for catching this, as it is an error. We have now corrected the figure to indicate that there was no significant change detected in the Myb+/Foxj1- population in the Nu6140 acute treated ALI+4d as compared with untreated ALI+3d (t=0).

6) Finally, the last four paragraphs of the Discussion could be condensed since it is speculation and not directly focused on the data. I think that one major emphasis of the discussion should be a comparison to Al Jord et al., 2017, who show that Cdk1 (rather than Cdk2) in balance with APC is required for centriole amplification-ciliation and inhibition of mitosis, respectively. The Vlader discussion should further explain why Al Jord implicates Cdk1 and Vlader Cdk2 beyond what is written in paragraph two. The authors should discuss specifically if they believe that their data indicate that Cdk2 is directly a program or has specific protein targets these issues are discussed loosely in paragraphs three, four and five. Consider inhibition of APC and examine Cdk2-dependent centriole amplification, this would address the discussion point in paragraph six.

The Discussion has been condensed and we now discuss and compare in more detail our study regarding the role of Cdk2 and the Al Jord study on Cdk1. The APC inhibition experiment is interesting but we feel that it is beyond the scope of this study.

https://doi.org/10.7554/eLife.36375.022

Article and author information

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
    ORCID icon "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
    ORCID icon "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

Funding

National Institutes of Health (R01GM052022)

  • Tim Stearns

National Institutes of Health (R01GM121424)

  • Tim Stearns

National Institutes of Health (R01GM098582)

  • Jeffrey D Axelrod

National Institutes of Health (1R01HD034915)

  • Debra Wolgemuth

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Klara Fekete for help with animal husbandry, Koshi Kunimoto for help with histology, Lydia Joubert (Stanford Cell Sciences Imaging Facility) for help with electron microscopy, Aron Jaffe (Novartis) for the TRRAP antibody, and Chris Kintner (Salk) and members of the Axelrod and Stearns labs for helpful discussions. Work was supported by R01GM098582 (NIH) to JDA, R01GM052022 and R01GM121424 (NIH) to TPS and 1R01HD034915 (NIH) to DJW. EKV is a Boettcher Foundation Webb-Waring Early Career Investigator.

Ethics

Animal experimentation: All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Stanford University School of Medicine (#17926) in accordance with established guidelines for animal care.

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Jay Rajagopal, Harvard University, United States

Reviewer

  1. Steven Brody, Washington University, United States

Publication history

  1. Received: March 3, 2018
  2. Accepted: August 26, 2018
  3. Accepted Manuscript published: August 28, 2018 (version 1)
  4. Version of Record published: September 19, 2018 (version 2)

Copyright

© 2018, Vladar et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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