ASPP2 Links the Apical Lateral Polarity Complex to the Regulation of YAP Activity in Epithelial Cells

<div><p>The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP <i>in vitro</i>, in epithelial cells, and <i>in vivo</i>, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.</p></div

ASPP2 scaffolds PP1 to dephosphorylate YAP.

<p>(<b>A</b>) The importance of the ASPP2/PP1 interaction in dephosphorylating YAP was tested by transfecting the indicated constructs in HEK293 cells. Exogenous YAP was subsequently immunoprecipitated using an anti-myc mouse monoclonal antibody (9E10) and SDS-Page/immunoblotting was performed using the indicated antibodies. The bar graph represents the ratio between pYAP S127 and total YAP protein levels (n = 3; *: p<0.05; n.s.: non-significant). Error bars indicate standard deviation. (<b>B</b>) Lysates obtained from HEK293 cells transfected with the indicated constructs were analysed by SDS-PAGE/immunoblotting on a Phos-Tag gel. (<b>C</b>) The ability of ASPP2 (Y869A/Y874A)-V5 to dephosphorylate YAP was tested in HEK293 cells to investigate the importance of the YAP/ASPP2 interaction in this process. Following transfection with the indicated constructs in HEK293 cells, Exogenous YAP was immunoprecipitated using an anti-myc mouse monoclonal antibody (9E10) and SDS-Page/immunoblotting was performed using the indicated antibodies. (<b>D</b>) The ability of ASPP2, iASPP and ASPP1-V5 to interact with and dephosphorylate YAP was tested in HEK293 cells. Following transfection with the indicated constructs in HEK293 cells, Exogenous YAP was immunoprecipitated using an anti-myc mouse monoclonal antibody (9E10) and SDS-Page/immunoblotting was performed using the indicated antibodies.</p

ASPP2 promotes the transcriptional activity of YAP.

<p>(<b>A</b>) Following transfection of Caco-2 cells with control or ASPP2 siRNA, the localisation of YAP and ASPP2 was analysed by immunostaining at low and high cell density. Scale bar: 20 µm. (<b>B</b>) The ability of wild type ASPP2 and ASPP2 (R<u>A</u>K<u>A</u>)-V5 to regulate TEAD-mediated transcription was analysed in a luciferase assay using the TEAD-luciferase reporter (8xGTIIC-luciferase) in Caco-2 cells. Values were obtained from three independent duplicate experiments and error bars indicate standard deviation (*: p<0.05). (<b>C</b>) YAP immunostaining of paraffin sections obtained from the colons of wild type and <i>ASPP2</i>^Δexon3 mice. Dashed white squares highlight magnified areas represented in the corresponding panels (C′-C′″). White arrowheads point to YAP positive nuclei in wild type crypts whereas yellow arrowheads point to nuclei devoid of YAP in <i>ASPP2</i>^Δexon3 crypts. Nuclei were counterstained with DAPI. Scale bars: 50 µm. (<b>D</b>) <i>CTGF</i> mRNA levels were quantified by qRT-PCR using RNA obtained from the colons of wild type (n = 3) and <i>ASPP2</i>^Δexon3 mice (n = 3). Error bars represent standard deviation (*: p<0.05). (<b>E</b>) Diagram representing the regulation of YAP by ASPP2 in epithelial cells. Once YAP is phosphorylated at serine 127, it can interact with ASPP2 at the apical lateral domain where ASPP2 induces its dephosphorylation via the recruitment of PP1. YAP is consequently able to relocalise to the nucleus where it can modulate TEAD transcriptional activity.</p

Modelling AVE migration.

<p>(A) Two-dimensional representation of force directions in the vertex model. At each vertex, tension forces act along the edges connecting neighbouring vertices, with unit direction vectors Tc (clockwise) and Ta (anti-clockwise). Pressure forces act normally at the vertex, bisecting the internal angle Φ, with unit direction vector P. (B) On the ellipsoid surface, forces act tangentially. To calculate the forces on a given vertex, its neighbours are projected onto the tangential plane. Unit direction vectors are then determined on this plane. (C) Each cell in the vertex model is 3-D, with associated height and volume. Forces act on the apical surface and depend on quantities such as surface area, edge lengths, height, and perimeter. (D) An initial cell configuration on the ellipsoid surface. Cells highlighted in green are the AVE. The polygon mesh represents the apical surfaces of cells of the VE. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001256#pbio.1001256.s005" target="_blank">Text S1</a> for further details. (E) Comparison of mean polygon number in the ExE-VE and Epi-VE early and late in simulation (roughly equivalent to “distal” and “anterior” embryos). As in wild-type embryos, there is a significant reduction in mean polygon number in the Epi-VE late in simulation as compared to early in simulation (Students <i>t</i> test, <i>p</i><0.001). (F) Frequencies of polygon numbers early and late in simulations. Late in simulations, there is a significant difference in the distribution in the Epi-VE as compared to the ExE-VE, with an increase in four-sided cells and a decrease in six-sided cells (Kolmogorov-Smirnov test, <i>p</i><0.001). There is no significant difference between the distribution in the ExE-VE and Epi-VE early in simulations. Early in simulations: <i>n</i> = 458 Epi-VE and 507 ExE-VE cells from five simulations. Late in simulations: <i>n</i> = 656 Epi-VE and 744 ExE-VE cells from five simulations.</p

Abnormal AVE migration and cellular geometry in mutants with disrupted PCP signalling.

<p>(A) Rosette density (number of rosettes divided by total VE cell number) at different wild-type stages (“pre-AVE”: before AVE induction, <i>n</i> = 9; “distal”: AVE at distal tip before migration, <i>n</i> = 5; “migrating”: AVE migrating, <i>n</i> = 5; and “anterior”: AVE finished proximal migration and moving laterally, <i>n</i> = 4) and in <i>ROSA26^Lyn-Celsr1</i> mutants (<i>n</i> = 7) with disrupted PCP signalling. There is a significant reduction in rosette density in <i>ROSA26^Lyn-Celsr1</i> mutants compared with “migrating” and “anterior” embryos. (A′) The same data as in (A), but depicted as mean number of rosettes per embryo (blue line), and mean number of VE cells per embryo (green bars) at the various stages. <i>ROSA26^Lyn-Celsr1</i> mutants have a comparable number of VE cells to stage matched “anterior” embryos, but show significantly fewer rosettes, leading to the reduced rosette density. (B, B′) En face and profile view of a representative “anterior” embryo, illustrating stereotypical ordered migration of AVE cells. The AVE is marked with a dotted line in (B′) and shows a single group of cells that does not extend more than half-way around the side of the embryo. (C, C′) En face and profile views of an equivalent stage <i>ROSA26^Lyn-Celsr1</i> mutant, showing abnormal AVE migration. AVE cells appear to have broken into several groups (outlined with dotted lines in (C′)) and spread much more broadly within the Epi-VE and even into the ExE-VE. Cell outlines in the embryos in (B) and (C) were visualised by staining for ZO-1 (magenta), and AVE cells by the expression of Hex-GFP (green). Nuclei are visualised with DAPI (dim grey). (D) Comparison of mean polygon number in the Epi-VE and ExE-VE of “anterior” embryos (<i>n</i> = 480 Epi-VE and 409 ExE-VE cells from three embryos) and equivalent stage <i>ROSA26^Lyn-Celsr1</i> mutants (<i>n</i> = 563 Epi-VE and 546 ExE-VE cells from four embryos). As in wild-type “anterior” embryos, the mean polygon number in the Epi-VE of <i>ROSA26^Lyn-Celsr1</i> mutants is significantly lower than that in the ExE-VE. (D′) The same polygon number data grouped according to the VE region. Though the mean polygon number in the ExE-VE is comparable for “anterior” and <i>ROSA26^Lyn-Celsr1</i> embryos, in the Epi-VE it is significantly lower in <i>ROSA26^Lyn-Celsr1</i> embryos, suggestive of increased disequilibrium in cell packing. The scale bar represents 50 µm. <i>p</i> values shown on the graphs were determined using Student's <i>t</i> test.</p

Quantitative characterisation of rosettes.

<p>(A) Rosette density (number of rosettes divided by total VE cell number) at different wild-type stages (“pre-AVE”: before AVE induction, <i>n</i> = 9; “distal”: AVE at distal tip before migration, <i>n</i> = 5; “migrating”: AVE migrating, <i>n</i> = 5; and “anterior”: AVE finished proximal migration and moving laterally, <i>n</i> = 4) and in the AVE arrest mutants <i>Nodal^Δ600/lacZ</i> (<i>n</i> = 5) and <i>Cripto</i>^−/− (<i>n</i> = 9). There is a significant increase in rosettes' density in “migrating” embryos as compared to “distal” embryos. The AVE arrest mutants <i>Nodal^Δ600/lacZ</i> and <i>Cripto</i>^−/− show significantly reduced rosette density compared to “migrating” and “anterior” embryos, suggestive of a direct link between rosettes and AVE migration. (A′) The same data as in (A), but depicted as mean number of rosettes per embryo (blue line), and mean number of VE cells per embryo (green bars) at the various stages. “Migrating” embryos have a comparable number of VE cells to “distal” embryos, but have significantly more rosettes, leading to an increase in rosette density. AVE arrest mutants have similar average VE cell numbers to stage matched “anterior” embryos, but show significantly fewer rosettes, leading to the reduced rosette density. (B) Polar plot showing distribution of rosettes in the VE of embryos. Migrating AVE cells were used to determine the anterior of embryos. Rosettes are localised predominantly to the Epi-VE. Within the Epi-VE, rosettes appear to be uniformly distributed with respect to the anterior-posterior axis (<i>n</i> = 39 rosettes from 7 embryos). <i>p</i> values shown on the graphs were determined using Student's <i>t</i> test.</p

The VE contains multi-cellular rosettes.

<p>(A) A ZO-1 stained embryo in which cells are coloured in to illustrate the presence of junctions where three, four, or five cells meet at a point. (B) Rosettes are formed by five or more cells meeting at a point. A variety of rosettes are shown, including two that share some cells (last panel).</p