RESEARCH ARTICLE

The Plk1 kinase negatively regulates the Hedgehog signalingpathway by phosphorylating Gli1Tingting Zhang, Guangwei Xin, Mingkang Jia, Tenghan Zhuang, Shicong Zhu, Boyan Zhang, Gang Wang,Qing Jiang and Chuanmao Zhang*

ABSTRACTHedgehog (Hh) signaling is a highly conserved cell signalingpathway important for cell life, development and tumorigenesis.Increasing evidence suggests that the Hh signaling pathwayfunctions in certain phases of the cell cycle. However, thecoordination between Hh signaling and cell cycle control remainspoorly understood. Here, we show that polo-like kinase-1 (Plk1), acritical protein kinase regulating many processes during the cellcycle, also regulates Hh signaling by phosphorylating and inhibitingGli1, a downstream transcription factor of the Hh signaling pathway.Gli1 expression increases along with Hh signaling activation, leadingto upregulation of Hh target genes, including cyclin E, during the G1and S phases. Gli1 is phosphorylated at S481 by Plk1, and thisphosphorylation facilitates the nuclear export and binding of Gli1 withits negative regulator Sufu, leading to a reduction in Hh signalingactivity. Inhibition of Plk1 kinase activity led to Gli1 maintaining is rolein promoting downstream gene expression. Collectively, our datareveal a novel mechanism regarding the crosstalk between Hhsignaling and cell cycle control.

KEY WORDS: Gli1, Hedgehog signaling, Plk1 kinase, Cell cycle,Phosphorylation

INTRODUCTIONHedgehog (Hh) signaling is a highly conserved pathway importantfor cell life, metabolism, individual development and tumorigenesis(Corbit et al., 2005; Corrales et al., 2004; Forbes et al., 1996;Rohatgi et al., 2007; Tukachinsky et al., 2010; Wang et al., 2010).The Hh signaling pathway consists of several key components,including patched (Ptc), smoothened (Smo), suppressor of fused(Sufu) and glioma-associated oncogene (Gli) proteins. Ptc1 (alsoknown as PTCH1) is a cell membrane-localized 12-transmembraneprotein that has been extensively studied as an Hh ligand receptor.Smo is a G protein-coupled receptor-like seven-transmembraneprotein that functions as a transducer of Hh signaling (Corbit et al.,2005; Møller et al., 2017; Yang et al., 2012). Most Smo protein isstored in membrane vesicles within the cell. In the absence of Hhligands, Ptc1 prevents and inhibits cell surface accumulation andactivation of Smo. This inhibition is lifted by ligand binding tothe receptor, which promotes endocytosis of Ptc from the cellmembrane, and enhances the accumulation of Smo on the cell

membrane and its activation (Hui and Angers, 2011). The activatedSmo then transduces the upstream signal to the downstream Glifamily transcription factors. Once activated, these Gli transcriptionfactors translocate to the nucleus and initiate the transcription oftheir target genes (Ingham et al., 2011; Lum and Beachy, 2004;Robbins et al., 2012; Youn et al., 2016). Three members of the Glifamily, Gli1, Gli2 and Gli3, have been identified in mammals, andonly one Gli member, Cubitus interruptus (Ci), was identified in theflyDrosophila melanogaster, with this protein mediating all aspectsof the transcription of Drosophila Hh target genes (Méthot andBasler, 2001). Among the three Gli members in mammals, Gli2shows both transcriptional activator and repressor activities, Gli3serves mainly as a transcriptional repressor, and Gli1 is thought to bedriven by Hh signaling as a sensitive readout of this pathwayactivation (Ahn and Joyner, 2004; Altaba, 1999; Dessaud et al.,2008; Samanta et al., 2015; Sasaki et al., 1999).

Hh signaling is mainly transduced in the primary cilium, aslender organelle emanating from the cell surface, in ciliated cells ofvertebrates. The primary cilium consists of a basal body, amicrotubule-based axoneme generated from the basal body, and asignaling-receptor-enriched ciliary membrane sheet extending fromthe cell membrane. Between the ciliary membrane sheet and the cellmembrane is a periciliary diffusion barrier (PDB), a transition zonethat forms a selective barrier to prevent the membrane proteins fromundertaking lateral transportation between the ciliary and cellmembranes. Under the resting conditions of Hh signaling, Ptc1localizes to the primary cilium and prevents the localization of Smowithin the cilia or limits its residency time (Hui and Angers, 2011).Once bound with Hh ligands, Ptc1 is removed from the membrane,and its inhibitory effect on the relocation of Smo to the primary ciliamembrane is lifted. This leads to the relocation of Smo into theprimary cilia membrane and the activation of Hh signaling.

Transportation of the ciliary proteins between the cytoplasm andthe cilium is bi-directional along the microtubule-based axoneme andis mediated by a multiprotein complex, the intraflagellar transport(IFT) complex. Once translocated to the cilia and activated, Smomodulates the activity of the Gli family transcription factors for theirtarget genes.

The primary cilium is dynamic, and undergoes disassembly orresorption during G2-M phase transition and reassembly at the endof mitosis in a cell cycle regulation-dependent manner (Plotnikovaet al., 2008; Pugacheva et al., 2007; Wang et al., 2013; Zhang et al.,2017, 2015). This not only indicates that ciliogenesis is regulatedduring the cell cycle but also suggests that Hh signaling is linked tocell cycle control. In this work, we investigated Hh signaling duringthe cell cycle. We found that Hh signaling turns off in G2 phaseupon the phosphorylation and degradation of Gli1, turns on againduring the G1 and S phase of the next cell cycle, and participates inthe regulation of expression of G1/S transition regulators, such ascyclin E.Received 14 May 2018; Accepted 17 December 2018

TheMinistry of Education Key Laboratory of Cell Proliferation and Differentiation andthe State Key Laboratory of Membrane Biology, College of Life Sciences, PekingUniversity, Beijing 100871, China.

*Author for correspondence (zhangcm@pku.edu.cn)

C.Z., 0000-0003-1359-6475

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© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs220384. doi:10.1242/jcs.220384

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RESULTSExpression of Gli1 is downregulated at the G2 phaseTo investigate the coupling of cell cycle control with Hh signaling,we first examined the expression of Gli1, Smo and Sufu in severalcell lines. Western blotting analysis revealed their existence in thesecell lines (Fig. S1A). Since downstream members of the Hhsignaling pathway have strong effects on the activity of thispathway, we focused on the regulation of the Gli transcriptionfactors. When we expressed GFP-tagged exogenous Gli1 protein,we observed that the exogenous Gli1 proteins localized mostly inthe nucleus and modestly in the cytoplasm, while exogenous Gli2was predominantly localized in the nucleus (Fig. S1B), indicatingthat the Gli1 nuclear–cytoplasm transduction might be more active.Then, we treated cells with the Hh signaling agonist SAG and theantagonist cyclopamine, and analyzed the expression andlocalization of endogenous Gli proteins. We found that Gli1 washighly expressed and enriched in the nucleus upon Hh signaling

stimulation by SAG, whereas the expression and nuclearlocalization of Gli1 were reduced upon Hh signaling inhibitionupon treatment with cyclopamine (Fig. 1A,B). Through cell cyclesynchronization followed by western blotting analysis, we furtherrevealed that Gli1 expression was increased from the G1 to S phaseand decreased during the G2 and M phases, and this expression wasrestored in the next G1 phase (Fig. 1C–E). Furthermore, we foundthat Gli1 shuttled between the nucleus and the cytoplasm with morein the nucleus during the G1–S phases and more in the cytoplasm atthe G2 phase (Fig. 1D,E). To further verify the change of Gli1protein levels during the cell cycle, we synchronized NIH 3T3 cellsvia serum starvation for 48 h followed by western blotting analysis.We found that Gli1 protein levels were also downregulated atthe G2/M phase in NIH 3T3 cells (Fig. 1F). Taken together,these results show that the expression and localization of Gli1 isdependent on cell cycle control, and its downregulation occursduring the G2/M phases.

Fig. 1. Expression and dynamic localizationof Gli1 during cell cycle. (A) Expression of Gli1in cells treated with SAG or cyclopamine. MEFcells were treated with SAG, cyclopamine orDMSO as a control (Con). The cell lysates wereprocessed for electrophoresis on a 10% SDS-PAGE followed by western blotting analysis withspecific antibodies against Gli1 and α-tubulin.(B) Localization of Gli1 in cells subjected to SAGor cyclopamine treatment. Cells treated as in Awere processed for immunofluorescencelabeling with anti-Gli1 antibody and DAPI forDNA staining. (C) Expression of Gli1 in cellsthroughout the cell cycle. HeLa cells weresynchronized to G1/S phase by two courses ofthymidine treatment and release. The sampleswere collected at 0 h, 3 h, 6 h, 8 h, 9 h and 11 hand processed for western blotting analysisusing the indicated antibodies. Aurora A wasused as a G2/M phase indicator. (D) Localizationof Gli1 in cells throughout the cell cycle. HeLacells were treated as in C and processed forimmunofluorescence labeling with anti-Gli1antibody and DAPI for DNA staining.(E) Percentage of cells with more Gli1 in thenucleus than in the cytoplasm as shown inD. These data are mean±s.d. from threeindependent experiments with the nucleinumbers n>100. (F) Expression of Gli1 in cellsthroughout the cell cycle. NIH 3T3 cells werestarved for 48 h and released. Cells werecollected at 17, 19, 20, 21, 22 h after release andprocessed for western blotting analysis using theindicated antibodies. Note that, along with thecell cycle progress from the G2 to M phase,indicated by gradual expression of cyclin B1, andaccumulation of phosphorylated histone H3(pH3), Gli1 protein level gradually decreased.Numbers under western blot images in A, C andF are relative protein amount compared withloading control. Scale bars: 10 µm.

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Phosphorylation of Gli1 by Plk1 reduces its nuclear retentionand Hh signaling activityNext, we investigated how the Gli1 protein level is downregulatedduring the G2/M phase. Among the cell cycle control proteins, Plk1and Aurora A are two vital protein kinases that function during theG2/M phase and both take part in regulating primary ciliumdisassembly (Pugacheva et al., 2007; Wang et al., 2013). As the Hhpathway is transduced mainly in the primary cilium, we supposedthat these two kinases might be responsible for the downregulationof Hh signaling pathway during the G2 andM phase. To verify theireffects on Hh pathway activity, we treated cells with Bi2536, aninhibitor of Plk1, and MLN8237 (MLN), an inhibitor of Aurora A,and monitored changes in Hh pathway activity with a widely useddual-luciferase reporter system (Taipale et al., 2000). With thisreporter system, we showed that Hh pathway activity increasesunder the stimulation with the Hh ligand, the agonist SAG and upontreatment with exogenous Gli1 (Fig. S2). Interestingly, we foundthat, when the cells were treated with the Plk1 inhibitor Bi2536, Hhsignaling was also significantly increased whereas treatment withthe Aurora A inhibitor MLN had no effect (Fig. 2A). These resultssuggest that Plk1 but not Aurora A inhibits Hh signaling activity. Byusing immunofluorescence labelling, we revealed more nuclearretention of Gli1 in SAG- or BI2536-treated cells than in the controlcells (Fig. 2B). To better analyze the localization disparities, wetreated cells with Plk1 inhibitor or Hh signaling agonist thenseparated the nuclear and cytoplasmic fractions, followed bywestern blotting analysis to determine the Gli1 protein levels. Incells treated with Bi2536 and SAG, we found that levels of Gli1protein in the nucleus were significantly increased compared tothat of the control (Fig. 2C). Furthermore, we found that the Gli1bands in Bi2536- and SAG-treated samples were significantly

down-shifted compared with the control sample (Fig. 2C),indicating that Gli1 was phosphorylated in Bi2536- and SAG-untreated cells. Moreover, we synchronized cells at the G1/S or G2phase, and treated them with Bi2536 or MLN. We found thatBi2536 treatment could increase Hh signaling activity only in theG2 phase (Fig. 2D). Moreover, we revealed that Plk1 inhibition orexpression of the constitutively active mutant Plk1 T210D had noeffect on the nuclear localization of Gli2 (Fig. S3A,B). Collectively,these data demonstrate that the phosphorylation of Gli1 by Plk1reduces its nuclear retention and Hh signaling activity.

Gli1 colocalizes and interacts with Plk1 at the G2 phaseNext, we investigated whether Gli1 interacts with Plk1. Through co-immunoprecipitation (IP), we found that Plk1 interacted with Gli1via its polo-box domain (PBD), which serves as a phosphorylation-dependent binding site (Fig. 3A,B), and this interaction was muchweaker between Plk1 and Gli2 (Fig. S3C). Given that doublemutation of histidine 538 (H538) and lysine 540 (K540), which arelocated within the PBD, to alanine residues (2A, H538A/K540A)abolishes the interaction between Plk1 and its substrates (Barr et al.,2004), we investigated the interaction between Plk1-2A and Gli1and found no interaction between them (Fig. 3C). These findingsindicated that Gli1 might be a substrate of Plk1. By synchronizingGFP- or GFP–Gli1-expressing cells at the G1/S transition and Sphase by double-thymidine treatment and release, or at G2/Mtransition and M phase by treatment with S-trityl-L-cysteine(STLC), followed by IP, we found that the interaction betweenPlk1 and Gli1 was significantly increased during the G2/Mtransition and M phase (Fig. 3D), which is consistent with thetime window for Gli1 protein level decrease. Immunostaining alsorevealed the colocalization between Gli1 and Plk1. In cells treated

Fig. 2. Plk1 reduces Hh activity by impeding Gli1nuclear entry. (A) Gli-luciferase activity in NIH 3T3cells. Asynchronous NIH 3T3 cells treated with DMSO(Asy) or G0 phase NIH 3T3 cells (G0) serve as control,compared with asynchronous NIH 3T3 cells treatedwithBi2536, SAG and MLN8237. Data are mean±s.d.collected from three independent experiments.*P<0.05, **P<0.01 (Student’s t-test). (B) Localization ofGli1 in NIH 3T3 cells treated with DMSO as a control(Con), Bi2536 and SAG. The cells were fixed in 4%PFAfor 15 min and then permeabilized with PBS with 0.2%Triton X-100 for 30 s, followed by immunostaining withanti-Gli1 antibody. DNA was stained with DAPI. Scalebar: 10 µm. (C) Distribution of endogenous Gli1proteins in different cell fractions of the cells in B. Theproteins were analyzed by western blotting using ananti-Gli1 antibody. α-tubulin and lamin A/Cwere labeledto indicate the cytosol and nucleus fractions,respectively. Numbers under the images are relativeGli1 protein amount compared with lamin A/C. (D) Gli-luciferase activity in different phases of the cell cycle.HEK 293 cells were synchronized to G1/S or G2 phaseand treated with DMSO, as a control, Bi2536 orMLN8237. Data are mean±s.d. collected from threeindependent experiments. *P<0.05; NS, not significant(Student’s t-test).

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with Bi2536, Gli1 was still localized in nucleus even withexogenous expression of Plk1 (Fig. 3E). Taken together, theseresults indicate that Gli1 is a Plk1 substrate and that Plk1 is involvedin the downregulation of Gli1.

Gli1 is phosphorylated at S481 by Plk1We further tested whether Gli1 is phosphorylated by Plk1. Throughsequence analysis, we found that serine 481 in the Mus musculusGli1 molecule may be a Plk1 phosphorylation site, and it isconserved at serine 479 in human Gli1 (Fig. 4A). Then, we used aPhos-tag acrylamide gel and western blotting of SAG- and Bi2536-treated cells to analyze the modification of Gli1 proteins. We foundthat the Gli1 band was upshifted in the control and SAG treatmentsamples, but that this shift was inhibited upon the treatment with thePlk1-specific inhibitor Bi2536 (Fig. 4B), indicating that Plk1phosphorylates Gli1. To confirm this, we mutated serine 481 to analanine residue and performed an in vitro phosphorylation assay.

We found that the [32P]S481A mutant band was much weaker thanthe wild-type Gli1 band (Fig. 4C). We further performed in vivophosphorylation mass spectrometry (MS) by constructing a Gli1 2Rmutant, in which both asparagine 472 and cysteine 488 weremutated into arginine residues, to generate a cleavage site for trypsin(Fig. S4A). We confirmed that Gli1 2R showed no differences inlocalization and interaction with other proteins compared with wild-type Gli1 (Gli1 WT) (Fig. S4B,C). The MS results showed that Gli1was indeed phosphorylated at S481 in vivo (Fig. 4D). By expressingthe GFP-tagged phosphorylation-mimic mutant S481D and thenon-phosphorylatable mutant S481A, followed by microscopy andnucleus–cytoplasm separation, we found that, compared with Gli1WT, S481A showed increased nuclear localization, whereas S481Dtended to localize in the cytoplasm (Fig. 4E–G). Taken together,these data showed that Gli1 is phosphorylated at S481 by Plk1,and this phosphorylation status may be linked with the cellularlocalization of Gli1 proteins.

Fig. 3. Interaction and colocalization of Gli1 andPlk1. (A) Gli1 interacts with Plk1. First, HEK 293 cellswere transfected with either GFP or GFP–Gli1 for 48 h.Lysates of these cells were immunoprecipitated (IP)with a GFP antibody and analyzed by western blottingwith a Plk1 antibody. (B) Gli1 binds to the PBD domainof Plk1. First, HEK 293 cells were transfected withGFP–Gli1, and the cell lysates were sequentiallyincubated with GST or GST–Plk1-PBD (arrow) andglutathione–Sepharose beads. The beads were thenretrieved, and the bead-bound proteins were analyzedby western blotting with a GFP antibody. (C) Plk1-2A(H538A/K540A) does not bind Gli1. HEK 293 cellswere co-transfected with Myc–Gli1 and GFP, GFP–Plk1 or GFP–Plk1-2A, and the cell lysates wereprocessed for a co-IP assay using GFP and Mycantibodies. (D) Interaction of Gli1 with Plk1 at G2/Mphase. The HEK 293 cells were transfected with eitherGFP or GFP–Gli1 and synchronized to G1/S phase,through thymidine treatment, or G2/M phase, bytreatment with STLC. The cell lysates were processedfor IP using a GFP antibody followed by westernblotting analysis with a Plk1 antibody.(E) Colocalization of Gli1 and Plk1 in cells. HeLa cellswere fixed in 4% PFA for 15 min and thenpermeabilized with PBS containing 0.2% Triton X-100for 30 s, followed by immunostaining with anti-Gli1 and-Plk1 antibodies. Note that, in Plk1-expressing G2cells, most Gli1 proteins localized to the cytoplasm inthe control, and, after treatment with Bi2536, most ofthem localized in the nucleus. DNA was stained withDAPI. Scale bar: 10 µm.

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Phosphorylation of Gli1 at S481 enhances interaction of Gli1with Sufu, which promotes cytoplasmic retentionNext, we investigated why Plk1 affects Hh signaling activity. SinceSufu is a well-known antagonistic factor of Gli proteins, we firsttested whether Plk1 affects the interaction of Sufu and Gli1. Byexpressing Flag-tagged Sufu in cells, followed by immunostainingof endogenous Gli1, we found that nuclear localization of Gli1was significantly downregulated in Flag–Sufu-expressing cells(Fig. 5A). By co-expressing both Sufu and Gli1 and undertaking co-IP assays, we found that these proteins interacted with each other.Interestingly, we confirmed that the interaction between Sufu andGli2 was even stronger than the interaction between Sufu and Gli1,and, when GFP–Gli2 and Flag–Sufu were co-expressed in cells,Sufu could efficiently expel most Gli2 from nucleus (Fig. S5B).Then, we treated cells with the Plk1 inhibitor Bi2536 and performedco-IP assays. The results showed that, compared with the control,the Gli1 binding with both exogenous and endogenous Sufu wassignificantly reduced by the Plk1 inhibitor treatment (Fig. 5B,C).We further expressed Gli1 WT, S481A and S481D and performedco-IP assays. We found that the phosphorylation-mimicking mutantS481D as well as the wild-type Gli1 strongly bound endogenousSufu, whereas the non-phosphorylatable mutant S481A did not bind

(Fig. 5D). Immunostaining also revealed that S481D colocalizedwith Sufu (Fig. S5C). Taken together, these data demonstrate thatphosphorylation of Gli1 at S481 by Plk1 enhances its binding withSufu and mediates its cytoplasmic retention.

Plk1 facilitates Sufu-dependent nuclear export of Gli1We further explored how phosphorylation of Gli1 by Plk1 enhancesthe binding of Gli1 with Sufu and its cytoplasmic retention. Wetransiently expressed Flag–Sufu in cells, treated the cells with thePlk1 inhibitor and examined cells for Gli1 by immunostaining. Wefound that nuclear localized Gli1 was decreased in Flag–Sufu-expressing cells compared to non-Flag–Sufu-expressing cells.However, when the cells were treated with Bi2536, we observedmore nuclear localization of Gli1 in both the Flag–Sufu-expressingand non-Flag-Sufu-expressing cells (Fig. 6A). Statistical resultsshowed that the percentage of cells with nuclear Gli1 in Bi2536-treated cells was approximately threefold higher than that inuntreated control cells (Fig. 6B). These results were also confirmedby nucleus and cytoplasm separation followed by western blottinganalysis (Fig. 6C). More straightforwardly, we observed that Gli1vanished from the nucleus in cells expressing the constitutivelyactive Plk1 T210D mutant (Fig. 6D). We also investigated the

Fig. 4. Plk1 phosphorylates Gli1 at S481. (A) S481 ofGli1 is conserved in mouse and human (S479). (B) Gli1 isphosphorylated upon Hh signaling activation, and thisphosphorylation can be inhibited by Plk1 inhibitortreatment. HEK 293 cells expressing Myc–Gli1 were firsttreated with Bi2536, SAG or DMSO, as a control (Con).Cell lysates were separated on a Phos-tag gel andanalyzed by western blotting with antibodies against Mycand GAPDH, as a loading control. Note that Bi2536treatment inhibits Gli1 phosphorylation. (C) The S481Amutation significantly reduces in vitro phosphorylation ofGli1 by Plk1. GFP, GFP–Gli1 and GFP-Gli1-S481Aproteins were expressed in HEK 293 cells and purified,and subjected to an in vitro Plk1 phosphorylation assay.Note that the phosphorylation of Gli1-S481A wassignificantly reduced (left). Coomassie Blue staining (CB)shows the protein loading (right). (D) Purified GFP–Glli1-2R protein was assessed by mass spectrometry,confirming that Gli1 S481 was phosphorylated by Plk1.(E) Localization of GFP, GFP–Gli1 (WT), GFP–Gli1-S481A or GFP-Gli1-S481D in HeLa cells. The cellsexpressing the GFP-tagged proteins were fixed andcounterstained with DAPI for DNA. Scale bar: 10 µm.Note that the non-phosphorylated mutant Gli1 S481Ashows complete nuclear localization, whereas thephosphorylation-mimic mutant S481D is largelycytoplasmic. (F) Statistics show the mean±s.d.percentage of cells with nuclear localization shown inE. The data were from three independent experiments,each with n>200. *P<0.05, **P<0.01, ***P<0.001(Student’s t-test). (G) Distribution of Gli1 WT and mutantsin HeLa cell fractions. The cells in E were processed fornuclear and cytoplasmic fractionation assays. The GFP-tagged proteins were analyzed by western blotting usingan anti-GFPantibody. α-tubulin and Lamin A/Cwere usedas indicators for the cytosol and nucleus, respectively.Numbers under western blot images are relative Gli1protein amount compared with Lamin A/C.

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localization of Gli2 under Plk1 inhibitor treatment, and found that,regardless of the presence or absence of Bi2536, the localization ofGli2 in Flag–Sufu-expressing and non-Flag–Sufu-expressing cellswas not changed (Fig. S6). Taken together, these results indicatethat phosphorylation of Gli1 by Plk1 promotes the nuclear export ofGli1 by enhancing formation of the Gli1–Sufu complex.

Phosphorylation of Gli1 by Plk1 reduces Hh signaling-regulated expression of cyclin E1To verify whether Hh signaling is negatively regulated by Plk1through phosphorylation of Gli1, we tested the expression status ofcyclin E1, a known transcriptional target of Gli1 and Hh signaling(Alvarez-Medina et al., 2009; Duman-Scheel et al., 2002). Wefound that the mRNA and protein levels of cyclin E1 were increasedwhen the cells were transfected with exogenous Gli1 (Fig. 7A,B),whereas the cyclin E1 protein level was significantly decreased

when the cells were transfected with exogenous Sufu (Fig. 7F). Weobserved that, while the mRNA and protein levels of cyclin E1 wereincreased in cells treated with the Plk1 inhibitor Bi2536 (Fig. 7C,D),this effect of Bi2536 treatment on cyclin E1 expression wasabolished in cells expressing siRNA targeting Gli1 (Fig. 7E;Fig. S7). These findings reveal the indispensable role of Gli1 inthe regulation of cyclin E1 expression with Bi2536 treatment.Furthermore, while exogenous Flag–Sufu in cells suppressedcyclin E1 expression, Bi2536 treatment could restore the cyclinE1 expression in these Flag–Sufu-expressing cells (Fig. 7F,G).Moreover, mutation of Gli1 into the Plk1 non-phosphorylatableform S481A, but not the phosphorylation-mimicking S481D form,could also enhance the mRNA and protein levels of cyclin E1(Fig. 7H,I). Taken together, these results demonstrate that Plk1negatively regulates Hh signaling by phosphorylating Gli1 topromote its nuclear exit.

Fig. 5. S481 phosphorylation enhances binding of Gli1 to Sufu. (A) Nuclear localization of endogenous Gli1 decreases in cells expressing Flag–Sufu. HeLacells were transfected with Flag–Sufu for 48 h, fixed in 4% PFA for 15 min and then permeabilized with PBS containing 0.2% Triton X-100 for 30 s, followed byimmunostaining with anti-Gli1 and Flag antibodies. DNA was stained with DAPI. Arrows highlight nuclear Gli1 in non-transfected cells. Scale bar: 10 µm.(B,C) Binding of Gli1 to Sufu, the reduction of this binding seen upon inhibition of Plk1. (B) HEK 293 cells were co-transfected with Flag–Sufu and either GFP orGFP-Gli1 and treated with or without Bi2536. The cell lysates were immunoprecipitated using a GFP antibody. Proteins co-immunoprecipitated with the GFPproteins were analyzed by western blotting with an antibody against Flag. (C) HEK 293 cells were transfected with either GFP or GFP–Gli1 alone and were thentreated with or without Bi2536. The cell lysates were immunoprecipitated using a GFP antibody. Proteins co-immunoprecipitated with the GFP proteins wereanalyzed by western blotting with an antibody against Sufu. The arrow indicates endogenous Sufu, and * indicates the heavy chain of IgG. (D) The ability to bindSufu differs between non-phosphorylatable and phosphorylation-mimic mutants of Gli1. First, HEK 293 cells were transfected with GFP, GFP–Gli1, GFP–Gli1-S481A or GFP–Gli1-S481D. The cell lysates were immunoprecipitated using a GFP antibody. Proteins co-immunoprecipitated with the GFP proteins wereanalyzed by western blotting with an antibody against Sufu. The arrow indicates endogenous Sufu, and * indicates the heavy chain of IgG. Note that the wild-typeand the phosphorylation-mimic mutant S481D form of Gli1 bind more Sufu than the non-phosphorylatable mutant S481A. Numbers under western blot images inB, C and D are relative protein amount compared with loading control.

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DISCUSSIONHh signaling regulates not only cell metabolism, individualdevelopment and tumorigenesis but also cell proliferation,although the underlying mechanisms remain unclear and evencontroversial (Agathocleous et al., 2007; Lum and Beachy, 2004;Neumann, 2005; Phua et al., 2017; Wang et al., 2010). Hh signalingfunctions mainly through the primary cilium in mammals (Goetzand Anderson, 2010). The primary cilium shows a dynamicbehavior in a cell cycle regulation-dependent manner, withdisassembly or resorption during the mitotic entry and reassemblyafter mitosis (Plotnikova et al., 2008; Pugacheva et al., 2007; Zhanget al., 2017, 2015). All these findings indicate a link between cellcycle control and Hh signaling, and that both cell cycle control andHh signaling might precisely regulate each other. In this work, westudied the relationship between cell cycle control and Hh signaling.We demonstrated that one of the key cell cycle regulators, Plk1kinase, negatively regulates Hh signaling by phosphorylating andinhibiting the transcriptional activity of Gli1.Gli1 is the main effector of Hh signaling activation and plays crucial

roles in the expression of the downstream target genes of this signalingpathway by serving as a transcriptional activator. The members of theGli protein family can act as both transcriptional activators andrepressors of Hh signaling (Hui andAngers, 2011). Of theseGli familymembers, Gli1 actively regulates the G1-S transition during neuralprogenitor proliferation in Drosophila (Alvarez-Medina et al., 2009)and the S phase checkpoint in tumor cells, promoting tumorprogression and resistance to chemotherapy (Tripathi et al., 2014). Inthis work, we found that Gli1 activity is negatively regulated by Plk1kinase, a putative target gene product modulated by Gli1 (Lee et al.,

2010). Thus, our findings very likely reveal a feedback mechanism ofHh signaling for the G1-S phase transition, in which Gli1 regulatesexpression of its target genes, including Plk1, which in turn limits theactivity of Gli1 to prevent overexpression of its downstream genes.

Plk1 kinase regulates many aspects of the cell cycle, especiallythe centrosomal cycle, mitosis and cytokinesis (Wang et al., 2014).Knockdown of Plk1 leads to G2/M arrest in cells, and this mighthave a negative effect on Hh signaling activity (Evangelista et al.,2008). Moreover, Plk1 has crucial roles in the regulation of ciliaryresorption before mitotic entry (Wang et al., 2013; Zitouni et al.,2014). In this work, by controlling the duration of Plk1 inhibition(Bi2536, 2–3 h), we further show that Plk1 phosphorylation of Gli1also promotes cell cycle progression into the G2/M phase by turningoff the transcriptional activity of Gli1, in addition to the previousfinding that Plk1 promotes DNA replication by phosphorylatingHbo1 to regulate pre-replicative complex (pre-RC) formation andDNA replication licensing (Wu and Liu, 2008).

Based on our findings, we propose a working model of theregulation of Hh signaling by Plk1 during the cell cycle (Fig. 7J).During mitotic exit and the G1 phase, Gli1 proteins areprogressively expressed along with Hh signaling activation andmove into the nucleus to function as transcriptional activators toinduce target gene expression. These target gene products mayinclude the regulators for the G1/S phase transition and DNAreplication, such as cyclin E and Plk1, which then phosphorylateand inhibit Gli1 as a feedback regulation. Along with theprogression of the cell cycle from G1 into S and G2/M phases,Plk1 accumulates, which, on the one hand, promotes cell cycleprogression and, on the other hand, phosphorylates and inhibits Gli1

Fig. 6. Plk1 kinase activity facilitates Sufu-dependent Gli1nuclear exit. (A) Exogenous Sufu expression induces nuclearexit of Gli1. HeLa cells transiently expressing Flag–Sufu weretreated with Bi2536 or STLC, as a control, and immunostainedwith Flag and Gli1 antibodies. Note that cells expressing Flag–Sufu have less nuclear Gli1 (yellow arrow in the control) than non-Flag-expressing cells (yellowarrowheads in the control), and that,after treatment with Bi2536, both cells expressing Flag–Sufu(white arrows) and non-Flag-Sufu (white arrowheads) have moreGli1 in the nucleus than in the cytoplasm. DNA was stained withDAPI. Scale bar: 10 µm. (B) Mean±s.d. percentage of cells withmore Gli1 in the nucleus than in the cytoplasm for cells as shownin A. Data were collected from three independent experimentswith n>100. ***P<0.001 (Student’s t-test). (C) Distribution of Gli1in between the cytoplasmic and nuclear fractions of cellstransfected with Flag or Flag–Sufu and treated with or withoutBi2536. α-tubulin and Lamin A/C were used to indicate the cytosoland nucleus fractions, respectively. Numbers under western blotimages are relative Gli1 protein amount compared with Lamin A/C. (D) GFP–Plk1-T210D induces nuclear exit of Gli1. HeLa cellstransiently expressing GFP–Plk1-T210D were immunostainedwith Gli1 antibody. DNAwas stained with DAPI. Scale bar: 10 µm.Note that GFP–Plk1-T210D-expressing cells (green) possessless nuclear Gli1 (red in dashed white line-enclosed nuclearareas) than non-GFP-Plk1-T210D-expressing cells (red indashed yellow line-enclosed nuclear areas).

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by enhancing the nuclear export of Gli1 and the interaction of Gli1with Sufu, which sequesters Gli1 in the cytoplasm, leading to theswitch-off of Hh signaling. With a unique structure of PBD domain,

Plk1 normally requires a priming phosphorylation in its substratesfor it to bind to them. Unfortunately, so far we do not know thispriming kinase. Furthermore, we show that it is possible that Plk1

Fig. 7. Hh signaling activation enhances cyclin E1 expression, whereas inhibition of Plk1 kinase activity reduces cyclin E1 expression. (A) ExogenousGFP–Gli1 increases the mRNA level of cyclin E1. RT-qPCR analysis of the cyclin E1 mRNA level in HEK 293 cells transfected with GFP or GFP–Gli1. Data werenormalized to GAPDH and are shown as the mean±s.d. from three independent experiments. **P<0.01 (Student’s t-test). (B) Exogenous GFP–Gli1enhances cyclin E1 protein expression. HEK 293 cells expressing GFPor GFP–Gli1 were subjected towestern blotting analysis with antibodies against cyclin E1,GFP and GAPDH, as the loading control. (C) Bi2536 treatment enhances the cyclin E1 mRNA level. RT-qPCR analysis of cyclin E1 mRNA level in HEK 293 cellstreated with STLC (control) or Bi2536. Data were normalized to GAPDH and are shown as the mean±s.d. from three independent experiments. *P<0.05(Student’s t-test). (D) Bi2536 treatment enhances cyclin E1 protein expression. HEK 293 cells were treated with STLC (control) or Bi2536, and subjected towestern blotting analysis with antibodies against cyclin E1 and GAPDH, as a loading control. (E) Gli1 siRNA (RNAi) abolished the effect of Bi2536 treatment oncyclin E1 expression. HEK 293 cells were transfected with RNA control or Gli1 siRNA and then treated with STLC (control) or Bi2536, respectively. Cells weresubjected to western blotting analysis with antibodies against cyclin E1 and GAPDH, as the loading control. The siRNA knockdown efficiency and loadingcontrol (GAPDH) are shown on the left. (F) Exogenous Flag–Sufu reduces cyclin E1 expression. HEK 293 cells expressing exogenous Flag or Flag–Sufu weresubjected to western blotting with antibodies against cyclin E1, Flag and α-Tubulin, as a loading control. (G) Bi2536 treatment restored the cyclin E1 expression inFlag–Sufu-expressing cells. HEK 293 cells expressing Flag–Sufu were treated with either STLC (control) or Bi2536 followed by western blotting analysiswith antibodies against cyclin E1, Flag and α-Tubulin, as a loading control. (H) The non-phosphorylatable Gli1 mutant Gli1-S481A enhances the cyclin E1 mRNAlevel, while the phosphorylation mimic mutant Gli1-S481D decreases it. RT-qPCR analyses of cyclin E1 mRNA levels in HEK 293 cells transfected with GFP–Gli1-WT, GFP–Gli1-S481A or GFP–Gli1-S481D. Data were normalized to GAPDH and are shown as the mean±s.d. from three independent experiments.*P<0.05, **P<0.01 (Student’s t-test). (I) The non-phosphorylatable Gli1 mutant Gli1-S481A enhances the cyclin E1 protein level, while phosphorylation mimicmutant Gli1-S481D decreases it. HEK 293 cells expressing GFP–Gli1-WT, GFP–Gli1-S481A or GFP–Gli1-S481Dwere subject to western blotting analysis usingantibodies against cyclin E1, GFP and GAPDH, as a loading control. Numbers under western blot images in B, D–G and I are relative protein amountcompared with loading control. (J) Working model of the relationship between Hh signaling and the cell cycle regulation by Plk1. When the cell enters G1 phase,Gli1 proteins are progressively expressed alongwithHh signaling activation, and are imported to the nucleus to function as transcription factors regulating expressionof the target genes, such as cyclin E and Plk1. Plk1, in turn, phosphorylates and inhibits Gli1 as a feedback regulation. Along with cell cycle progression from the G1to the S andG2/M phases, Plk1 accumulates and phosphorylates and inhibits Gli1 by enhancing nuclear export and binding of Gli1 with Sufu, which sequesters Gli1in the cytoplasm. These changes switch off Hh signaling and promote the cell cycle progression of the G2 and M phases at multiple levels.

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could also phosphorylate Gli2 (data not shown), but thephosphorylation sites and functions are still unknown. In general,our findings provide an insight into the crosstalk between Hhsignaling pathway and cell cycle control, and may have importantimplications in the understanding of the link between cell signalingand cell proliferation.

MATERIALS AND METHODSMolecular cloning and RNA interferenceHuman Plk1 was cloned from a HeLa cell cDNA library. Mouse Gli1 wassubcloned from the Flag–mGli1 plasmid, a kind gift from Dr Qing Zhang(Nanjing University, China). cDNAs encoding Plk1, Plk1-PBD, Plk1-PBD-2A and Gli1 WT, S481A and S481D were subcloned into the pEGFP-C2(Clontech, 6083-1), pCMV-Myc (Clontech, 635689) or pET-28a (Novagen,69864) vectors. The GFP–mGli2 and Flag–hSufu plasmids were kindgifts from Dr Yun Zhao (Institute of Biochemistry and Cell Biology, SIBS,CAS, Shanghai, China). The siRNA sequence targeting Gli1 was 5′-CCAGGAAUUUGACUCCCAA-3′. Three additional siRNAs targetingGli1were used in this project: siRNA1: 5′-CCGAGUA-UCCAGGAUACAATT-3′, siRNA2: 5′-CCGAAGGACAGGUAUGUAATT-3′, siRNA3: 5′-CUU-CCCACCUACUGAUACUTT-3′.

Cell culture, synchronization and transfectionHeLa (ATCC, CCL-2), HEK 293T (ATCC, ACS-4500), HEK 293 (ATCC,CRL-1573), murine embryonic fibroblast (MEF; ATCC, SCRC-1008) andNIH 3T3 (ATCC, CRL-1658) cells were cultured at 37°C and 5% CO2 inDMEM (Gibco) with 10% FBS (HyClone Laboratories, Inc.). Resting (G0phase) cells were obtained by serum starvation. G1/S phase synchronizationwas achieved by double-thymidine (Sigma-Aldrich) treatment. Briefly, thecells were treated with 2.5 mM thymidine for 18 h twice with a 9 h internalrelease. The cells were harvested (500 g for 5 min) at the indicated time pointafter they were released to fresh medium. G2 phase cells were obtained with a6 h release after the double thymidine treatment. G2/M phase cells wereobtained by adding 100 ng/ml STLC (Sigma-Aldrich) for 15 h after releasefrom the thymidine block. Bi2536 (Axon Medchem) (100 nM) was added tothe medium for 2–3 h before harvest to manipulate the kinase activity of Plk1.Transient cDNA transfections were carried out on cells using MegaTranstransfection reagent (Origene) according to the manufacturer’s instructions,and siRNA transfectionwas performedwith Lipofectamine 2000 (Invitrogen)according to the manufacturer’s instructions.

Gli-luciferase reporter assayNIH 3T3 cells were pre-plated into 24-well plates. Then, the cells weretransfected with a Gli-Luc reporter and Renilla luciferase pRL-SV40 (kindgifts from Dr Yun Zhao, Institute of Biochemistry and Cell Biology,Shanghai, China). We treated the cells with SAG (3 µM, 12 h), Bi2536(100 nM, 2–3 h), MLN (250 nM, 2–3 h) or starved the cells to simulate theirtransition to G0 phase by using medium containing only 0.5% FBS for 24 h,the next day. Then, the cells were collected for luminometric detection usingdual luciferase reagents (Promega). Transfection efficiency was normalizedvia the Renilla luciferase activity.

Immunofluorescence microscopyThe cells were fixed in 4% PFA for 15 min and then permeabilized withPBS/0.2% Triton X-100 for 30 s, followed by immunostaining with theindicated primary [rabbit anti-Gli1 (1:100, ab49314, Abcam), rabbit anti-Gli2 (1:100, ab7195, Abcam), mouse anti-Plk1 (1:200, 06-813, EMDMillipore), mouse anti-γ-tubulin (1:200, T6557, Sigma-Aldrich) and mouseanti-Flag (1:200, PM020, MBL International)] and secondary antibodies[Alexa Fluor 488-conjugated donkey anti-mouse-IgG, Alexa Fluor 594-conjugated donkey anti-rabbit-IgG, Alexa Fluor 488-conjugated donkeyanti-rabbit-IgG or Alexa Fluor 594-conjugated donkey anti-mouse-IgG(Invitrogen)]. DNA was stained with DAPI (Sigma-Aldrich). Images wereanalyzed under a 63×/1.4 NA oil objective of a microscope (Axiovert 200M;Carl Zeiss) and captured with a charge-coupled device (CCD) camera(MRM; Carl Zeiss) and Axiovert image acquisition software.

Immunoprecipitation, GST fusion protein pulldown assay andwestern blottingNIH 3T3 or HEK 293 cells transfected with indicated plasmids were lysedin cell lysis buffer (20 mM Tris-HCl pH 8.0, 150 mMNaCl, 2 mM EGTA,0.5 mM EDTA, 0.5% NP-40, 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF,and 500× protease inhibitor cocktail; Calbiochem) for 15 min on ice. Thelysates were centrifuged at 9000 g for 15 min, and the supernatants wereincubated with the rabbit anti-GFP polyclonal antibody-coated protein A–Sepharose beads for 2 h at 4°C. The rabbit anti-GFP polyclonal antibodieswere raised against GFP protein in rabbit in our laboratory. Then, the beadswere washed three times with lysis buffer and suspended in Laemmlisample buffer before resolution on SDS-PAGE gels. For GST pulldownassays, cell lysates were incubated with 5 µg of soluble GST or GST-fusedproteins bound to 15 µl glutathione–Sepharose beads for 2 h at 4°C. Then,the beads were washed three times with lysis buffer and suspended inLaemmli sample buffer before resolution on SDS-PAGE gels.

After separation on SDS-PAGE gels, the proteins were transferred tonitrocellulose membranes that were then blocked in TTBS (20 mM Tris-HCl, pH 7.5, 500 mMNaCl and 0.1%Tween 20) containing 2% nonfat milkat room temperature for 1 h. Then, they were probed with primary antibodiesdiluted in TTBS containing 2% nonfat milk at 4°C overnight. Themembranes were washed three times with TTBS before they were incubatedwith HRP-conjugated secondary antibody at room temperature for 1 h.After that, the membranes were washed three times with TTBS again.The membranes were developed for visualization by enhancedchemiluminescence (Sigma-Aldrich) and X-ray film. The followingprimary antibodies were used for immunoblotting: rabbit anti-Gli1 (1:500,V812, Cell Signaling Technology), rabbit anti-Smo (1:500, ab38686,Abcam), rabbit anti-cyclin B1 (1:1000, sc-25764, Santa CruzBiotechnology, Inc.), rabbit anti-cyclin E1 (1:1000, A0112, ABclonalTechnology), rabbit anti-Lamin A/C (1:1000, raised in our laboratory withLamin A/C protein), mouse anti-Plk1 (1:1000, 06-813, EMD Millipore),mouse anti-Sufu (1:1000, sc-137014, Santa Cruz Biotechnology, Inc.),mouse anti-α-Tubulin (1:5000, T6074, Sigma-Aldrich), mouse anti-GFP(1:5000, M048-3, MBL International), mouse anti-Flag (1:1000, PM020,MBL International), mouse anti-Myc (1:1000, M4439-100UL, Sigma-Aldrich), mouse anti-H3pS10 (1:1000, ab47297, Abcam) and mouse anti-GAPDH (1:5000, 60004-1-lg, Proteintech). The western blot results fordistinct proteins came from the same samples processed simultaneously inseparate gels. All animal experiments were performed according toapproved guidelines.

In vitrokinaseassayandphospho-peptide identification bymassspectrometryGFP–Gli1-WT or GFP–Gli1-481A proteins were purified from HEK 239Tcells by immunoprecipitation. Beads coated with equal amounts of bothproteins were combined with Plk1 kinase (PV3501, Life Technologies),50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM EGTA, 5 mM DTT,100 mM ATP and 1 µCi γ-[32P]ATP (10 mCi/ml, 6000 Ci/mmol; GEHealthcare) for 30 min at 30°C. Loading buffer was added to stop thereaction. SDS-PAGE samples underwent electrophoresis. The gel wasexposed to X-ray film for 6 h or overnight at 4°C. For mass spectrometry,GFP–Gli1-2R was purified from HEK 293T cells by immunoprecipitationand was electrophoresed by SDS-PAGE. The gel was stained withCoomassie Brilliant Blue to visualize the protein bands. The GFP–Gli1-2R bands were sliced and subjected to MS analysis (Zhang et al., 2015).

Nucleocytoplasmic separationNIH 3T3 or HEK 293T cells were washed by low-permeability buffer A(20 mM HEPES-K pH 7.8, 5 mM KAc, 0.5 mMMgCl2 and 0.5 mM DTT)and treated with the same buffer A at 2 ml/dish for 10 min on ice. Then, thecells were scraped from the dishes and homogenized using a homogenizermore than 20 times. Samples were then centrifuged at 1000 g for 5 min. Thesupernatants were taken as cytosol segments, and the sediments were takenas nuclei. The cytosol segments were centrifuged at 20,000 g for 30 min,and the supernatants were mixed with Laemmli sample buffer beforeresolution on SDS-PAGE gels. The nuclei were washed three times with

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PBS and centrifuged at 1500 g for 5 min each time. The nuclei were lysedwith high permeability buffer B (10 ml buffer A and 0.234 g NaCl) at∼1.5×108 nuclei/ml for 9 min at 4°C. The lysates were centrifuged at12,000 g for 30 min, and the supernatants were mixed with Laemmli samplebuffer before separation on SDS-PAGE gels.

RT-qPCR analysisTotal RNA was extracted and 1 µg RNA was reverse transcribed usingPrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa). QuantitativePCR (qPCR) was performed with a FastStart Universal SYBRGreenMaster(Rox) kit (Roche) on a Roche Light Cycler 96 machine using a SYBRGreenqPCR template. The synthesis of cyclin E1 was detected using sense 5′-GGAGTTCTCGGCTCGCTCC-3′ and antisense 5′-CGTCCTGTCGATT-TTGGCC-3′ primers. Cyclin E1 gene levels were normalized to GAPDHlevels. The qPCR results were analyzed by Roche LightCycler 96 software.

AcknowledgementsWe thank Drs Qing Zhang (Nanjing University, China) and Yun Zhao (Institute ofBiochemistry and Cell Biology, SIBS, CAS, Shanghai, China) for reagents. We aregrateful to other members of the laboratory for critical reading of this manuscript andDrs Hongxia Lv, Liying Du, and Dong Cao (Peking University, China) for technicalsupport, and Dr John Olson (Peking University, China) for language editing.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: C.Z.; Methodology: T. Zhang, G.X., M.J., T. Zhuang, S.Z., B.Z.,G.W., C.Z.; Validation: T. Zhang, G.X., M.J., T. Zhuang, S.Z., B.Z.; Formal analysis:T. Zhang, G.X., M.J., T. Zhuang, S.Z., B.Z., G.W., Q.J., C.Z.; Investigation: G.X.,M.J., T. Zhuang, S.Z., B.Z., G.W.; Resources: T. Zhang, Q.J., C.Z.; Data curation:T. Zhang, G.X., M.J., T. Zhuang, S.Z., B.Z., G.W.; Writing - original draft: T. Zhang;Writing - review & editing: Q.J., C.Z.; Visualization: T. Zhang, G.X., M.J., T. Zhuang,S.Z., B.Z., G.W., Q.J., C.Z.; Supervision: Q.J., C.Z.; Project administration: Q.J.,C.Z.; Funding acquisition: B.Z., Q.J., C.Z.

FundingThis work was supported by funds from the Ministry of Science and Technology ofthe People’s Republic of China (2016YFA0100501 and 2016YFA0500201) and theNational Natural Science Foundation of China (NSFC) (31520103906, 31430051,31571386 and 91854204).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.220384.supplemental

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