p73 regulates ependymal planar cell polarity by modulating actin and microtubule cytoskeleton

Fuertes-Alvarez, Sandra; Maeso-Alonso, Laura; Villoch-Fernandez, Javier; Wildung, Merit; Martin-Lopez, Marta; Marshall, Clayton; Villena-Cortes, Alberto J.; Diez-Prieto, Inmaculada; Pietenpol, Jennifer A.; Tissir, Fadel; Lizé, Muriel; Marques, Margarita M.; Marin, Maria C.

doi:10.1038/s41419-018-1205-6

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Published: 05 December 2018

p73 regulates ependymal planar cell polarity by modulating actin and microtubule cytoskeleton

Cell Death & Disease volume 9, Article number: 1183 (2018) Cite this article

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Abstract

Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.

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Introduction

The specific orientation of cells within the plane of the tissue, named planar cell polarity (PCP), is an essential feature of animal tissues¹. PCP signaling is required for polarized beating of motile cilia in a variety of tissues², including multiciliated ependymal cells (ECs), which carpet the wall of the lateral ventricles^3,4. ECs, which are established perinatally from radial glial cells (RGCs) in a multistep process, display two types of PCP, rotational (rPCP) and translational (tPCP). display two types of PCP, rotational (rPCP) and translational (tPCP), which are established perinatally from radial glial cells (RGCs) in a multistep process⁵. rPCP is defined by the unidirectional orientation of the motile cilia within the cell and is coordinated at tissue-level. tPCP initiates in RGCs when their primary cilium is asymmetrically displaced^3,6. Postnatally, immature multiciliated ECs displace their cilia clusters toward the anterior apical surface⁴.

PCP is regulated by asymmetric signaling through core and global regulatory modules. PCP-core module includes Frizzled (Fzd3, 6), Van Gogh-like (Vangl1/2), cadherin epidermal growth factor (EGF)-like laminin G-like seven-pass G-type receptor (Celsr1-3), Dishevelled (Dvl1-3), and Prickle (Pk1-4). In rodents, asymmetric localization of the PCP-core complexes is required for the rotational orientation of basal bodies (BBs) in multiciliated cells^{3,7,8,9,10,11,12}. There is an initial polarization of the apical microtubule (MT) cytoskeleton, which induces the asymmetric distribution of PCP-core complexes at apical junctions¹³. These, in turn, will communicate polarity information to ciliary BBs distributed within the sub-apical cytoskeleton networks^12,14. PCP-core signaling regulates the rotational, but not the translational, polarity in ECs¹⁵. In these cells, activation of the actin-binding protein non-muscle myosin-II (NMII) by the myosin light chain kinase (MLCK) is essential for tPCP establishment¹⁵. Thus, both actin and MT networks are key factors for setting up PCP in ECs.

The causal relationship between ciliogenesis and PCP signaling is not fully deciphered¹⁶. In this regard, the Trp73 gene is a key player in the organization of multiciliated cells^17,18,19 and in EC polarity^17,20. p73 belongs to the p53 family of transcription factors and generates functionally different TA and DNp73 isoforms²¹. Neither p73 mechanisms underlying PCP regulation nor the responsible p73-isoform, have been addressed.

In this work we analyzed the subcellular localization of PCP-regulatory proteins in ECs and the effect of Trp73 gene loss on their localization. p73 deficiency resulted in loss of PCP-core signaling complex asymmetry and lack of translational and rotational polarity in ECs. We demonstrate that p73 regulates PCP, at least in part, through TAp73-modulation of NMII activity via MLCK transcriptional regulation and regulation of MT dynamics signaling pathways.

Materials and methods

Animal care, genotyping, and isolation of wholemounts

All animal experiments were carried out in accordance with European (European Council Directive 2010/63/UE) and Spanish regulations (RD 53/2013) as well as institutional animal ethical guidelines. Mice heterozygous for Trp73 on a mixed background C57BL/6 × 129/svJae²² were backcrossed to C57BL/6, at least five times, to enrich for C57BL/6 background. Heterozygous animals were crossed to obtain the Trp73−/− mice (p73KO from now on). DNp73 and TAp73 mutant mice have been described before^23,31. Male and female mice, obtained from multiple litters, were used for the experiments. Genotyping of animals was performed by PCR analysis as described before^22,23,24.

For wholemount (WM) isolation, animals were euthanized and brains were dissected and placed in cold 0.1 M phosphate-buffered saline. Dissections of the lateral wall of the lateral ventricles were performed under the stereomicroscope as described before⁶.

Cell culture

Mouse wild type (WT) and p73KO-induced pluripotent stem cells (iPSCs) were cultured on feeder cells as described before²⁵. For transfection assays, iPSCs were subcultured once without feeders in plates coated with Matrigel (Corning, NY, USA) and then seeded (3.7 × 10⁴ cells/cm²) on coverslips (coated with Matrigel). Cells were transfected with the indicated plasmids using Lipofectamine™2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) and after 24 h were fixed with 3.7% paraformaldehyde (PFA) for 15 min at room temperature (RT). For MLCK recovery assays, p73KO-iPSCs were transfected with a constitutively active MLCK expression vector (pSLIK-CA-MLCK) (Addgene plasmid # 84647). After 24 h cells were treated with doxycycline (400 ng/mL) to induce MLCK expression and were analyzed 24 h later.

HA-TAp73β-Saos-2-Tet-On cells²⁶, (inducible TAp73-Saos-2 cells from now on) kindly provided by Dr. Karen Vousden (Cancer Research UK Beatson Institute, Glasgow), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 100 U/mL penicillin–0.1 mg/mL streptomycin. To induce TAp73 expression, cells seeded on coverslips for 24 h (3 × 104 cells/cm²) were treated with 2.5 μg/mL of doxycycline for 48 h²⁷. Then, fixation was carried out with 3.7% PFA at RT during 15 min.

The human cell line HCT116 was cultured in RPMI supplemented with 10% FBS, 2 mM l-glutamine, and 100 U/mL penicillin–0.1 mg/mL streptomycin.

Luciferase assays

HCT116 cells were seeded in 24-well plates (3 × 105 cell/cm²) and transfected with 0.125 μg of the luciferase reporter pGL3-hMLCK(1Kb)-luc (kindly provided by Dr. Jerrold Turner, University of Chicago)²⁸, 0.0625 μg pRLNull renilla, and 0.6 or 0.8 μg of the indicated expression vectors. Transfection was performed using X-tremeGENE^TM HP DNA Transfection Reagent (Roche, Basel, Switzerland).

Saos-2 were transfected with empty pcDNA3.1 or containing p73 isoforms and a Firefly luciferase reporter containing the putative WT TAp73-binding sequence (WT, Table S2) as identified by chromatin immunoprecipitation (ChIP)-seq²⁹ or the same sequence lacking the predicted TAp73-binding motives (Mutated, Table S2). Transfection efficiency was controlled by co-transfecting a Renilla TK luciferase vector. Luciferase activity was always assayed 24 h after transfection using Dual luciferase assay. Firefly luciferase values were normalized to the corresponding Renilla luciferase levels.

Western blot analysis

Western blotting was performed as previously described³⁰ and is detailed in the Supplementary Experimental Procedures.

Immunostaining and image analysis

Immunostaining protocols are described in the Supplementary Experimental Procedures. WM samples were analyzed using the Zeiss LSM800 and Olympus FluoView FV10i confocal laser scanning microscopes. iPSCs and inducible-TAp73-Saos-2 samples were analyzed using a Zeiss LSM800 Confocal Laser Scanning Microscope and a Nikon Eclipse TE2000 Confocal Microscope. Confocal z-stack images were taken in all cases. Images were processed using ZEN blue software, FV10-ASW 2.1 viewer software, EZ-C1 software, and ImageJ software.

Morphometric analysis

For the quantification of BB displacement in RGCs (P1) and in immature ECs (P15 or P21), at least three independent mice from each genotype were considered, counting at least three non-overlapping fields (50 μm × 50 μm) of each animal. Image analysis was performed using ImageJ software, quantifying the displacement by measuring a vector from the cell center to the BB (P1) or to the patch centroid (P15/P21).

For the quantification of number and intensity of p-MLC2 membrane dots, five independent mice from each genotype were considered, counting six non-overlapping fields (1 cell/field; ±20 μm × 20 μm) of each animal. Single plane images were obtained from z-stacks. Image analysis was performed using ZEN blue software, analyzing number and intensity of p-MLC dots in membrane region.

For the quantification of BB docking, three independent adult mice from each genotype were considered, counting two non-overlapping fields of each animal (50 μm × 50 μm; ±40 cells per animal). Orthogonal view of z-stack image analysis was performed with ZEN blue software, counting the number of cells with BBs aligned with the plasma membrane and cells with BBs scattered within the cytoplasm.

For the quantification of rotational PCP, a combined staining of FGFR1 Oncogene Partner (FOP) and γ-tubulin was used to delineate cilia polarity³. The FOP signal is adjacent to that of γ-tubulin at the side opposite to the basal foot. A vector that represents the direction of the individual cilia beat was defined by the direction from FOP (blue) to γ-tubulin (red) dots, using ImageJ software and all data were transformed to positive angles. The angle of polarity formed by this vector with respect to a horizontal axis was drawn. For each cell, a mean rotational angle and the circular standard deviation (CSD) were calculated with Oriana software. Five independent mice from each genotype were considered, counting three non-overlapping fields of each animal (50 μm × 50 μm; ±21 cells per animal).

For the quantification of Vangl2 and Frizzled localization at the plasma membrane, confocal z-stack images were taken before and after transiently transfecting the indicated expression vectors into p73KO-iPSCs. Single-plane images were obtained from z-stacks. Gaussian Blur filter (σ = 10) was applied and then images were binarized using automatic threshold. The midline along the membrane was created using Skeletonize Option. This network was dilated (15 iterations) to create the plasma membrane selection. which was overlapped with Vangl2 and Frizzled3 staining and the mean fluorescence intensity was measured.

RNA isolation and real-time quantitative reverse transcription-PCR analysis

Total RNA from cultured cells or dissected WMs was isolated using TRI reagent (Ambion, TX, USA). Kidney, bladder, and retina total RNA were isolated using RNeasy mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. cDNA was synthesized using up to 2 μg of total RNA and the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression was detected by real-time quantitative reverse transcription-PCR (qRT-PCR) in a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using FastStart Universal SYBR Green Master (Roche, Basel, Switzerland). qRT-PCR conditions were described before³¹. mRNA expression levels were expressed as 2^ΔCt (ΔCt = Ct internal reference − Ct gene), normalized to 18S mRNA expression. Primer sequences were as follows: 18S mouse gene³²: F 5′-AGTTCCAGCACATTTTGCGAG-3′ and R 5′-TCATCCTCCGTGAGTTCTCCA-3′; Mlck mouse gene (Primer Bank Database ID: 29650205a1): F 5′-TGGGGGACGTGAAACTGTTTG-3′ and R 5′-GGGGCAGAATGAAAGCTGG-3′; TAp73 mouse gene F 5′-GCACCTACTTTGACCTCCCC-3′ and R 5′-GCACTGCTGAGCAAATTGAAC-3′, and Eb3 mouse gene F 5′-ACTAGAACACGAGTACATCCACA-3′ and R 5′-CCATCATAGTTTGCGTCAAAGA-3′. 18S human gene: F 5′-GGCGCCCCCTCGATGCTCTTA-3′ and R 5′-GCTCGGGCCTGCTTTGAACAC-3′; MLCK human gene: F 5′-CACCGTCCATGAAAAGAAGAGTAG-3′ and R 5′-GAGAGGCCCTGCAGGAAGATGG-3′; TAp73 human gene: F 5′-GCACCTACTTCGACCTTCCC-3′ and R 5′-GTGCTGCTCAGCAGATTGAAC-3′; EB3 human gene: F 5′-GCCAATGATGCCTGAGATAACAG-3′ and R 5′-GCAGAGGGTTGTAATCCTTTCCA-3′.

Sequence analysis and ChIP

In silico prediction of putative p73-response elements within the mouse Mlck gene was performed using the open-access database for eukaryotic transcription factor-binding profiles JASPAR³³. Matrix models MA0861.1 and MA0525.1 were applied for the mouse Mlck gene and for the analysis of the ChIP-seq peak detected in the human MLCK gene, respectively. Only the sites with a score over 9.0 were selected for further in vitro analysis.

ChIP analyses were carried out as previously described²⁵ and are detailed in the Supplementary Experimental Procedures.

Gene ontology analysis

Gene ontology (GO) analysis was carried out using selected genes from GSE15780. DAVID Bioinformatics Resources 6.8^34,35 and PANTHER™ GO slim (version 13.1, released 2018-02-03)³⁶ were used for the analysis. The results were plotted using the ggplot2 library from R (3.5.1) Core Team (2014), a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.

Statistical analysis

Statistical analysis and Figure generation were performed with GraphPad Prism 7.04 software (Figs. 1–3, 6, 7) and Oriana 4 Software (Fig. 4). Data are expressed as means ± standard error (S.E.) of the mean. Differences were considered significant when p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). To test for normal distribution D’Agostino and Pearson tests were performed and non-parametric assumptions were made when necessary. In that case, the means of two experimental groups were compared with Mann-Whitney’s test; otherwise, unpaired two-tailed Student’s t-test was applied. In case of multiple comparisons, statistical differences were evaluated using either analysis of variance or Kruskal-Wallis test with Dunn’s multiple comparisons. To compare docking between WT and p73KO-ECs, a contingency analysis (Fisher’s exact test) was performed. Data in Fig. 4 are graphically represented using Oriana software (Kovach Computing Services, Anglesey, Wales) and statically compared by circular analysis (Watson’s U2 test).

**Fig. 1: p73 coordinates tPCP establishment in RGC and ECs through the regulation of polarized NMII activity.**

**Fig. 2: TAp73 transcriptionally regulates *Mlck* expression.**

**Fig. 3: p73 regulation of the actin cytoskeleton and NMII activation is essential for the formation of sub-apical actin lattices and BB docking in ECs.**

**Fig. 4: TAp73 regulates cilia organization and polarized junctional MT assembly required for rPCP, and is essential for the subcellular arrangement of the microtubule organizing protein, Fat4.**

**Fig. 5: p73 is essential for the polarized junctional assembly of PCP-core proteins and the asymmetric localization of their downstream signaling effectors, Dvl2 and Rac1, in ECs.**

**Fig. 6: p73 is necessary and sufficient for Vangl2 and Fzd3 membrane localization in vitro.**

**Fig. 7: MLCK activity is required, but not sufficient, for PCP-core protein membrane localization**

Results

p73 coordinates tPCP establishment through the regulation of polarized NMII activity

To address tPCP in transforming RGCs³, we quantified primary cilia displacement (CD) from the cell center in WMs from P1 WT and p73KO lateral wall (LW) of the lateral ventricle. (Fig. 1a, white arrows). We found reduced CD in p73KO-RGCs. Thus, in the absence of p73, tPCP establishment at single-cell level is either delayed or defective. We next analyzed the effect of p73 deficiency on NMII activation and localization, both essential for tPCP establishment¹⁵. In WT-RGCs, activated NMII (p-MLC) was located in distinct puncta at the plasma membrane (Fig. 1b), colocalizing with the primary cilia BB (inserts, white arrows). At the same stage, p73KO-RGCs, with defective CD, have lower levels of p-MLC, especially at the plasma membrane (Fig. 1b), altogether confirming that NMII activation is associated with tPCP establishment.

In immature ECs, BBs packed tightly during migration and get arranged into clusters, termed “crescents”, coordinately displaced toward the anterior apical surface (Fig. 1c, arrows). p-MLC was closely associated to BB within crescents (Fig. 1c, dotted box, Fig. 1d, arrows), advocating the idea that NMII activation plays a fundamental role in BB polarized migration¹⁵. p73KO-ECs had less CD, crescents were never detected (Fig. 1a, c), and their BBs were scattered throughout the apical surface (Fig. 1c, solid arrows) with little p-MLC associated to them (Fig. 1d, green and red arrows and 1e, arrowheads). p-MLC also localize in puncta at membrane junctions, polarized in the same direction of the BB-cluster (Fig. 1c, dotted box), which are reduced in p73-deficient cells (Fig. 1e), highlighting the correlation between p73 expression and NMII activation and localization.

Analysis of immature ECs from TAp73KO brains, lacking only TAp73 isoforms³¹, revealed a significant decrease in BB-cluster displacement (Fig. 1f) in consonance with their low p-MLC levels (Fig. 1g). Moreover, CD in neighboring TAp73KO-ECs followed different directions, confirming the lack of tissular coordination (Fig. 1f, yellow arrows). Analysis of DNp73KO-ECs, lacking DNp73 isoforms²³, did not reveal defects in BB-cluster displacement and orientation, or NMII activation and localization (Supplementary 1a,b), indicating that TAp73, but not DNp73, plays a key role in tPCP establishment.

TAp73 could regulate tPCP, at least in part, by controlling NMII activity via the regulatory kinase MLCK¹⁵. A proximal p53 response element (p53RE) had been previously reported within the human MLCK promoter³⁷ (Fig. 2a). Moreover, ChIP-seq analysis of TAp73-transfected Saos-2 cells²⁹ revealed a TAp73 peak, which could correspond to a putative enhancer element (Fig. 2a, p73RE-enh). We performed luciferase and ChIP assays with the identified p53 and p73REs (Fig. 2b, c). Transcriptional analysis in HCT116 cells with the p53RE-MLCK-luc reporter²⁸ (Fig. 2b, left panel), and in Saos-2 cells with the p73RE-enh-MLCK-luc (right panel), demonstrated that TAp73 can significantly activate both reporters. Under the same conditions, DNp73 had a weak transactivating activity on the proximal p53RE but none over the enhancer. In inducible-TAp73-Saos-2 cells²⁶ TAp73 induction correlated with increased MLCK expression (Supplementary 2a) and ChIP assays revealed a significant TAp73-interaction with both sites (Fig. 2c). To demonstrate the relevance of this interaction in a physiological context, we searched for p73/p53REs (JASPAR Core Vertebrata Database) in the murine Mlck gene and predicted a strong p73RE within the proximal promoter (−1741 bp from Exon1*³⁸, Fig. 2a). ChIP assays showed a significant interaction of endogenous TAp73 with this site in WT, but not in p73KO brain tissue (Fig. 2d). p73-deficiency correlated with lower Mlck levels in brain (Fig. 2e and Supplementary 2b), in iPSCs²⁵ (Fig. 2f) and in retina and kidney, but not in bladder (Supplementary 2c–e). Moreover, TAp73 overexpression in p73KO-iPSCs significantly induced Mlck levels (Fig. 2g, h). These data, as a whole, uncover MLCK as a direct TAp73-transcriptional target.

MLCK influences cortical localization of NMII, which functions as a cortical organizer to concentrate E-cadherin to zonula adherens³⁹. Thus, p73-deficiency might affect the establishment of epithelial cell junctions. To address this, we used iPSCs, a model with epithelial-like cell junctions^25,40. In WT-iPSCs, p-MLC is strongly expressed at intercellular junctions localizing with F-actin (Fig. 3a, arrows). p73KO-iPSCs, with an impaired epithelial phenotype²⁵, had less and mostly cytoplasmic p-MLC (Fig. 3a and Supplementary 3a), not colocalizing with F-actin. TAp73 overexpression induced NMII phosphorylation (Supplementary 3b), restoring p-MLC levels and localization at plasma membrane in p73KO-iPSCs (Fig. 3b, arrows). In agreement, upon TAp73 induction, p-MLC increases and colocalizes with cortical actin bundles⁴¹ (Supplementary 3c, arrows), highlighting the functional interaction of TAp73, NMII activity, and the actin cytoskeleton.

To address the functional linkage between p73 and the actin cytoskeleton in vivo, we examined F-actin distribution in ECs. These cells displayed cortical F-actin staining³ (Fig. 3c, white arrow), a polarized and organized actin apical lattice between the BBs (orthogonal projections, white arrowheads), and a set of sub-apical actin filaments underneath the BBs (blue arrowheads). p-MLC puncta appeared to be associated to the actin lattices within the BBs, as well as with the cortical actin anchored to the plasma membrane (Fig. 3e, white arrowheads and arrows, respectively), suggesting that activated NMII have a key role orienting and keeping together the actin meshwork and, concomitantly, the BBs clusters. Consistent with this hypothesis, p73-deficient cells, with less p-MLC, lacked the organized apical actin lattices associated with BBs and the sub-apical actin filaments (Fig. 3d). Moreover, p-MLC puncta were not associated to the BB cluster or polarized at the plasma membrane (Fig. 3f, yellow arrowheads and arrows, respectively).

Thus, p73-deficiency results in blunted NMII activation leading to a failure in the actin-network assembly, implicated in BB apical migration, docking⁴², and spacing⁴³. In agreement, BBs were clearly identified and aligned with the plasma membrane in WT-ECs (Fig. 3g, g'), but p73KO-ECs (Fig. 3g) or TAp73KO-ECs (Fig. 3g′) had BBs scattered and deep within the cytoplasm, demonstrating TAp73 essential role in sub-apical cytoskeleton dynamics and BB docking.

TAp73 regulates the polarized junctional MT assembly promoting PCP-core complex asymmetric localization

p73 deficiency not only affected BB-cluster displacement, docking, and spacing, but also BB orientation (Fig. 4a). WT-ECs displayed well-organized and straight parallel bundles of cilia, while p73KO (Fig. 4b) and TAp73KO-ECs (Fig. 4b′) had disorganized cilia with a “disheveled” appearance (yellow arrows). Similar cilia alterations have been attributed to BBs’ rotational alignment defects^9,11. Thus, we measured rotational polarity of individual BBs by calculating an angle of polarity formed by a vector (marking the orientation of each BB) and the horizontal axis³ (Fig. 4c). Next, we determined the mean vector for each EC and calculated the CSD (Fig. 4d). This value was significantly larger in p73KO than in WT cells (p < 0.001), demonstrating p73 requirement for rPCP. Only few TAp73KO-ECs lacked cilia axoneme (Fig. 4b′, white arrows), but we did not detect ciliary axoneme defects in DNp73KO-ECs, which displayed well-organized and parallel bundles of cilia and well-oriented BBs (Fig. 4b″ and Supplementary 1b).

p73 regulation of actin dynamics, by itself, could not fully explain the generation of refined rPCP⁴⁰. Thus, we expanded our analysis to the MT cytoskeleton, since polarized MT dynamics is fundamental for rPCP^3,12,13. α-tubulin staining revealed a fine and polarized MT-meshwork (Fig. 4e, arrowhead), with similar disposition to the actin lattice within BB (Fig. 3c). In p73KO-ECs this meshwork was lax and disorganized (Fig. 4e, arrowhead). To address MT-growth orientation, we analyzed EB3, a plus-end MT protein, which marks newly synthesized MTs. In WT cells, a second MT network grew asymmetrically from the center of the cell toward the anterior region of the apical cell cortex contacting the plasma membrane (Fig. 4e′, arrow). These MT-anchoring points^3,12,44 were polarized at tissue-level (Fig. 4f, g, arrows). Interestingly, p73 deficiency resulted in lower and diffuse levels of EB3 staining around the scattered BBs, and a total absence of polarized MT-anchoring points at cell junctions (Fig. 4e–g), demonstrating TAp73 requirement for polarized MT network formation in developing ECs.

The protocadherins Fat (Fat4 in mammals⁴) and its ligand Dachsous (Ds) conform the global-PCP module, known to control the polarized junctional alignment and asymmetry of MT^13,45. Confocal images of WT-ECs revealed Fat4 polarized distribution toward the anterior apical cell surface (Fig. 4h, dashed arrows). Orthogonal planes revealed that Fat4 was closely associated with the BBs (white arrow). p73KO-ECs had weak Fat4 staining, scattered throughout the cytoplasm without association with the BBs, lacking any asymmetric distribution (Fig. 4h, i).

Polarized MTs are required for targeting PCP proteins to the proper asymmetric membrane domains^12,46. PCP-core molecular complexes formed by Vangl2, Celsr1, and Pk2 displayed asymmetric distribution in WT-ECs (Fig. 5a–c), with Vangl2 exhibiting the characteristic zigzag pattern³ indicative of tissular coordination of polarity. This asymmetric distribution was completely lost in p73KO-ECs (Fig. 5a–c) and Vangl2 had cytoplasmic localization (Fig. 5a, a′) suggesting a defect in Vangl2-vesicle trafficking.

PCP-core proteins transduce the polarity signals through the adaptor protein Dvl2^9,15 and its effector Rac1⁴⁷. Orthogonal projections demonstrated that Dvl2 was located at the base of the individual BBs (Fig. 5d, white arrows) clustered at the anterior cell surface, establishing a polarized Dvl2-distribution. As expected, Rac1 was localized on top of the BBs (Fig. 5e, white arrows) at the base of the axoneme (Fig. 5f, white arrows). In p73 absence, Dvl2 did not associate with BBs (Fig. 5d, yellow arrows), and Rac1 was found scattered throughout the disheveled axonemes (Fig. 5e, f, yellow arrow), all in consonance with a lack of the polarized anchoring structures required to scaffold and orient the PCP signaling machinery.

TAp73 induces PCP-core protein membrane localization and modulates MT dynamics

TAp73 acts as a transcriptional integrator of multiciliogenesis^18,19 and regulator of EC differentiation¹⁷. Therefore, it was possible that like in airway epithelial cells¹⁶, the defects observed in p73KO-ECs were a consequence of their defective differentiation and ciliogenesis impairment. To rule this out, we analyzed PCP-core protein localization in a ciliogenesis-independent model, the iPSCs. In WT-iPSCs, Vangl2, Celsr1, and Fzd3 localized at the membrane, with intense staining at the lateral junctions (Fig. 6a, white arrows). Prickle2 also had cortical localization, close to Celsr1 (Fig. 6a, yellow arrows). Lack of p73 resulted in cytoplasmic Vangl2, which had lost most of its colocalization with Celsr1, confirming p73 requirement for Vangl2 membrane localization. In p73KO-iPSCs Fzd3 had faint and diffuse cytoplasmic staining (Fig. 6a, arrowheads), while little Prickle2 localized at the cell cortex associated with Celsr1 (Fig. 6a, yellow arrowheads). To test whether p73 was sufficient for PCP-core protein membrane localization, we expressed TAp73 or DNp73 in p73KO-iPSCs and analyzed the restoration of Vangl2 and Fzd3 at the plasma membrane and Celsr1 colocalization. Confocal microscopy (Fig. 6b, c, e) and image quantification (Fig. 6d) revealed that TAp73, but not DNp73, could rescue the p73KO phenotype and demonstrated that TAp73 is necessary and sufficient for membrane localization of PCP-core proteins, independently of its function as multiciliogenesis regulator.

MLCK is required for the establishment of epithelial cell junctions, which are necessary for polarized transport. Moreover, in some systems MLCK mediates intracellular trafficking over MTs⁴⁸. Therefore, we asked whether MLCK could be involved in PCP-core protein membrane transport. We treated iPSCs with the MLCK inhibitor ML7¹⁵ and confirmed that upon treatment p-MLC went down, disappearing from intercellular junctions (Fig. 7a, a′). Concomitantly, Vangl2 membrane localization decreased (Fig. 7b, b′), partially phenocopying p73KO-iPSC. Furthermore, overexpression of constitutively active MLCK (CA-MLCK⁴⁹) in p73KO-iPSCs resulted in an enhancement of p-MLC levels (Fig. 7c) and a partial restoration of PCP-core proteins membrane localization (Fig. 7d), indicating that MLCK/NMII signaling pathway is required for PCP-core proteins membrane delivery, but other factors are also involved.

To determine if a defect in MT dynamics was causing the observed p73KO-iPSCs defects, we asked whether Fat4, which promotes the formation of polarized MT networks, could rescue the phenotype, similarly to TAp73 overexpression^13,50. p73KO cells displayed a loosely formed MT meshwork (Fig. 8a, white arrowheads); however, upon Fat4 or TAp73 expression, the MT cytoskeleton formed a tight cortical network at the proximity of the cell periphery (where PCP-core proteins were localized), resembling WT phenotype (Fig. 8a, yellow arrowheads). Concurrently, Vangl2 and Fzd3 levels at the plasma membrane significantly increased, approaching those of WT-iPSCs (Fig. 8a′). Thus, Fat4 was sufficient to rescue the MT disposition and restore PCP-core proteins membrane localization in p73KO-iPSCs, confirming that defective MT dynamics were an underlying cause of p73KO phenotype.

**Fig. 8: TAp73 modulate MT dynamics and actin cytoskeleton.**

MTs play a central role in the Golgi complex integrity and agents that alter MT distribution induce Golgi fragmentation^51,52. As expected, cis-Golgi fragmentation was detected in p73KO-iPSCs (Fig. 8b). Next, we analyzed MT cytoskeleton and Vangl2 localization in inducible-TAp73-Saos-2 cells. Untreated cells, with very low levels of p73 (Fig. 8c), displayed a lax MT disposition and diffuse cytoplasmic Vangl2 staining (Fig. 8c–e). Upon TAp73 induction, changes in Vangl2 expression pattern, concurrently with changes in MTs disposition, were observed. These adopted a radial structure that radiated from a juxtanuclear cellular compartment where Vangl2 was enriched (Fig. 8d, arrowheads). This subcellular compartment accumulated acetylated-tubulin, which colocalized with Vangl2 (Fig. 8e, arrowheads) and Golgi marker GM130 (Fig. 8f). Therefore, it was consistent with Golgi-derived MTs, reported to preferentially accumulate acetylated-tubulin⁵³ and indispensable for cell polarity⁵⁴. Upon TAp73 induction we detected an increase on EB3 levels (Fig. 8g), confirmed by RT-PCR in inducible-TAp73-Saos-2 cells and p73KO-iPSCs (Supplementary Fig. 4a, b), all demonstrating that p73 could induce changes in MT dynamics. These changes were accompanied by a dramatic alteration in actin cytoskeleton and peripheral stress fiber architecture, readouts of MLCK enhancement⁵⁵.

To determine whether TAp73 could induce transcriptional programs involved in these processes, we analyzed previously published TAp73β-ChIP-seq/RNA-seq data from Saos-2 cells (GSE15780)²⁹. We used differentially expressed genes (DEGs) upon TAp73β expression that contained TAp73 peaks either at the promoter and/or at regulatory regions (within 25 kb). GO analysis identified terms like “Regulation of MT based processes”, “Regulation of MT polymerization”, “Post-Golgi vesicle–mediated transport”, and “Regulation of Actin cytoskeleton organization” as highly enriched biological processes (Fig. 8h and Supplementary 4c,d). These DEGs included key regulators of Golgi organization and MT dynamics like AKAP9 (AKAP 450), PDE4DIP, GOLGA1, or CLIP1 also required for cell polarity^56,57. Among actin cytoskeleton regulators, we confirmed MYLK (MLCK) as a TAp73β-DEG. These results support that TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and MT dynamics and Golgi organization.

Discussion

Polarity establishment relies on polarized actin and MT cytoskeletal rearrangements, but knowledge of the signals that organize these processes remains incomplete⁴³. This work demonstrates an essential role of TAp73 in PCP through the regulation of both, actin and MT-polarized network assembly, providing a model to the mechanism of planar polarization regulated by TAp73 (Fig. 8h).

We show that TAp73, but not DNp73, is necessary for the efficient tPCP establishment in RGCs, suggesting that the initial mechanoreceptors signaling, which instruct primary CD, cannot work properly in the absence of p73. While TAp73KO-ECs show PCP- and BB-docking defects, cilia axoneme alterations are milder than in the total p73KO mice, suggesting that DNp73 could have a compensatory role. However, consistent with the lack of DNp73 expression in ECs²³, we did not detect any cilia or PCP defects in DNp73KO-ECs. These data suggest that compensatory and redundant ciliary programs are induced in the absence of TAp73 when DNp73 is present, but not in total p73 deficiency. Nevertheless, these signaling programs do not compensate for PCP establishment in the absence of TAp73, uncoupling ciliogenesis, and PCP signaling in ECs.

Actin remodeling and the actin-based motor protein NMII are essential to establish tPCP¹⁵. Our results support a model in which NMII activation and membrane localization correlates with anterior migration of the BB crescent. Mechanistically, we identify TAp73 isoform as an upstream modulator of NMII through the transcriptional regulation of MLCK in vivo and in vitro. This directly links p73 with actin microfilament dynamics, essential for proper cilia docking and spacing, and for the establishment of global coordination of cilia polarity^43,58.

PCP regulation in epithelial cells relies on the PCP-core and the Fat/Ds modules to generate morphological asymmetries⁵⁹. PCP-core complexes transduce polarity signals through the adaptor protein Dvl2^9,15 and its effectors aPKC, Rac1, and RhoA, leading to reorganization of the actin cytoskeleton⁹. We show that Rac1 is localized at the base of the axoneme in a region corresponding to the ciliary pocket, a proposed interface with the actin cytoskeleton and a platform for vesicle trafficking⁶⁰. These molecular complexes are not established in the absence of p73. We report for the first time in ECs⁴, the expression of the Fat/Ds-module components^13,45 at the base of the cilia closely associated with the BBs, supporting its role in ciliary functions. Interestingly, p73-deficient cells displayed a defective global-PCP signaling that could be underlying, at least in part, the lack of polarized MT-junctional structures in p73KO cells.

Polarized MTs are an integral component of early steps in cell polarity driving asymmetric accumulation of PCP-core proteins^45,61. The cytoplasmic distribution of Vangl2 in p73-deficient ECs suggests a possible defect in Vangl2-vesicle trafficking and points toward a possible role of p73 in MT dynamics. Moreover, disassembling of MTs by nocodazole treatment of brain ventricles impairs the trafficking and distribution of PCP-core proteins in ECs³, phenocopying the observed p73KO phenotype. Lack of p73 affected both sets of MT described in ECs. Such defects could impair PCP-core proteins traffic to the plasma membrane at the MT-anchoring points and the assembly of PCP-asymmetric complexes.

We demonstrate that TAp73 is necessary and sufficient for membrane localization of PCP-core proteins independently of multiciliogenesis. Moreover, MLCK/NMII signaling pathway is necessary for PCP protein plasma membrane delivery, but not sufficient to completely rescue p73KO phenotype. Since the reciprocal interaction between MTs and the actin cytoskeleton is necessary for intracellular transport⁶², it is possible that similar functional interactions are triggered by TAp73 in this system.

We found that p73-deficient ECs displayed lower levels of EB3, a TIP+-binding protein required to modulate MT dynamics and the Golgi apparatus organization⁶³. Interestingly, EB1/3 disruption is known to perturb non-centrosomal MT organization⁵⁷. Congruently, TAp73 overexpression induces enhanced EB3 staining and dramatic changes in the dynamics of non-centrosomal Golgi-derived MTs, indispensable for post-Golgi trafficking and cell polarity⁵⁴.

GO analysis of DEGs containing TAp73 peaks showed enrichment in Golgi organization and MT dynamics, as well as actin cytoskeleton organization GO-terms, confirming TAp73 role as modulator of actin and MT dynamics signaling programs. However, further studies will be required to unravel how p73, directly or indirectly, regulates these modules.

Altogether, our data demonstrate that TAp73 not only regulates ECs development and ciliogenesis but also is an essential regulator of PCP establishment. This novel p73 function as an epithelial architect is not only fundamental during development but could also play a relevant role in TAp73 tumor suppression.

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Acknowledgements

This work was supported by Grants SAF2015-71381-R from Spanish Ministerio de Economía y Competitividad co-financed by FEDER funds (to M.C.M.) and LE021P17 from Junta de Castilla y Leon, and from the Queen Elisabeth Medical Foundation to F.T. J.V.-F. and S.F.-A. are holders of predoctoral fellowships from the Junta de Castilla y León. L.M.-A. is supported by a predoctoral scholarship from the Asociación Española contra el Cáncer (AECC). F.T. is a Research Director of the FNRS. M.W. and M.L. are funded by the Deutsche Forschungsgemeinschaft (DFG) under grant number LI 2405/2.

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Authors and Affiliations

Instituto de Biomedicina (IBIOMED) and Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, 24071, León, Spain
Sandra Fuertes-Alvarez, Laura Maeso-Alonso, Javier Villoch-Fernandez, Marta Martin-Lopez, Alberto J. Villena-Cortes & Maria C. Marin
Molecular and Experimental Pneumology Group, Clinic for Cardiology and Pneumology, University Medical Center, 37077, Göttingen, Germany
Merit Wildung & Muriel Lizé
Institute of Molecular Oncology, Clinic for Cardiology and Pneumology, Department of Pneumology, University Medical Center Göttingen, Göttingen, Germany
Merit Wildung & Muriel Lizé
Department of Biochemistry and Vanderbilt-Ingram Cancer Center, Vanderbilt University and Vanderbilt University Medical Center, Nashville, TN, 37232, USA
Clayton Marshall & Jennifer A. Pietenpol
Departamento de Medicina, Cirugía y Anatomía Veterinaria, Universidad de León, Campus de Vegazana, 24071, León, Spain
Inmaculada Diez-Prieto
Developmental Neurobiology, Institute of Neuroscience, Universite Catholique de Louvain, Avenue E. Mounier, 73, Box B1.73.16, B1200, Brussels, Belgium
Fadel Tissir
Instituto de Desarrollo Ganadero (INDEGSAL) and Departamento de Producción Animal, Universidad de León, Campus de Vegazana, 24071, León, Spain
Margarita M. Marques

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Sandra Fuertes-Alvarez
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Laura Maeso-Alonso
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41419_2018_1205_MOESM3_ESM.pdf

Supplementary Figure 1. Lack of DNp73 has no effect on PCP establishment or pMLC localization or rotational PCP Basal Bodies organization

Supplementary Figure 2. p73 expression correlates with MLCK levels

Supplementary Figure 3. TAp73 overexpression induces NMII activation

41419_2018_1205_MOESM6_ESM.pdf

Supplementary Figure 4. TAp73 overexpression results in changes in “Golgi and MT organization” signaling pathways as well as Actin dynamics

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Fuertes-Alvarez, S., Maeso-Alonso, L., Villoch-Fernandez, J. et al. p73 regulates ependymal planar cell polarity by modulating actin and microtubule cytoskeleton. Cell Death Dis 9, 1183 (2018). https://doi.org/10.1038/s41419-018-1205-6

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Received: 26 October 2018
Accepted: 02 November 2018
Published: 05 December 2018
DOI: https://doi.org/10.1038/s41419-018-1205-6

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