Figure 1B Membrane ARHGAP10 and β-Arrestin1 [ADU]). Experiments in triplicate.Data for Figure 1B in Excel file. Membrane ARHGAP10 and β-Arrestin1 [ADU]). Experiments in triplicate.Figure 1b.xlsxFigure 1D Membrane β-Arrestin1 intensityData for Fig 1D in Excel file. Membrane β-Arrestin1 intensity (AI) in Caco-2 and Caco-2 ShPTEN cell monolayers after control or LPA treatment. Experiments in triplicate. In each replicate, signal intensity was assessed in 10 cells (C-L). Mean ± sem values were plotted.Figure 1d.xlsxFigure 1F β-Arrestin1 membrane (ADU) in Caco-2 and Caco-2 ShPTEN cells after control or LPA treatmentData for Fig 1F in Excel file. β-Arrestin1 membrane (ADU) in Caco-2 and Caco-2 ShPTEN cells after control or LPA treatment. Experiments in triplicate.figure 1f.xlsxFigure 1 supplement 2Data for Fig 1 Supplement 2 in Excel file. Arbitrary intensity values for membrane β-Arrestin1 after LPA treatment in PTEN+/+ and PTEN-/- HCT116 cell monolayers. Confocal imaging conducted in 10 cells for each replicate (n=3) for each experimental condition.Figure 1 supplement 4Data for Figure 1 Supplement 4 in Excel file. Membrane β-Arrestin1 ADU in PTEN+/+ and PTEN-/- HCT116 cells after LPA treatment. Experiments in triplicatefigure 1 supplement 4.xlsxFigure 2BData for Figure 2B in Excel file. CDC42-GTP ADU in Caco-2 cells after β-Arrestin1 or NT SiRNA. Experiments in triplicate.figure 2b.xlsxFigure 2DData for Figure 2D in Excel file. CDC42-GTP (ADU) in Caco-2ShPTEN cells after ARHGAP10 or NT SiRNA transfections. Experiments in triplicate.Figure 2d.xlsxFigure 2 supplement 1Data for Figure 2 Supplement 1 in Excel file. CDC42-GTP ADU in PTEN+/+ cells after NT or β-Arrestin SiRNA transfection. Experiments conducted in triplicate.Figure 2 supplement 2Data for Figure 2 Supplement 2 in Excel file. CDC42-GTP ADU in PTEN-/- HCT116 cells after NT or ARHGAP SiRNA transfections. Experiments conducted in triplicate.Figure 2 supplement 3Data for Figure 2 Supplement 3 in Excel file. CDC42-GTP intensity (AI) in Caco-2 glands. Intensity was assessed in 10 randomly selected glands for each replicate (n=3) for each experimental condition.Figure 2 supplement 4Data for Figure 2 supplement 4 in Excel file. Spindle angles in Caco-2 cells after NT or β-Arrestin1 SiRNA knock down. Spindle angles of 15 mitotic figures assessed for each experimental conditionFigure 2 supplement 5Data for Figure 2 supplement 5 in Excel file. Caco-2 glands with single lumens after transfection. Assays in 50 glands per replicate (N=3). Results expressed as a percentage.figure 2 supplement 5.xlsxFigure 2 supplement 6Data for Figure 2 supplement 6 in Excel file. CDC42-GTP AI in Caco-2 ShPTEN glands. Assays in 10 glands for each replicate (n=3) in each experimental conditionFigure 2 supplement 7Data for Figure 2 Supplement 7 in Excel file. Spindle angles in Caco-2 ShPTEN cells after NT or ARHGAP10 SiRNA transfection. Assays in 15 glands for each experimental condition.Figure 2 supplement 8Data for Figure 2 supplement 8 in Excel file. Caco-2ShPTEN glands with single central lumens after transfection. 50 glands counted per replicate (n=3). Those with single lumens expressed as a percentage.figure 2 supplement 8.xlsxFigure 3BData for Figure 3B in Excel file. β-Arrestin1:ARHGAP binding ADU in Caco-2 or Caco-2 ShPTEN cells. Experiments in triplicate.figure 3b.xlsxFigure 3CData for Figure 3C in Excel file. β-Arrestin1:ARHGAP binding ADU in PTEN+/+ and PTEN-/- HCT116 cellsfigure 3c.xlsxFigure 3EData for Figure 3E in Excel file. β-Arrestin1:ARHGAP10 binding ADU in Caco-2 ShPTEN cells after transfection. Experiments in triplicate.figure 3e.xlsxFigure 3GData for Figure 3G in Excel file. β-Arrestin1:ARHGAP10 binding ADU in PTEN-/- HCT116 cells. Experiments in triplicate.figure 3g.xlsxFigure 4BData for Figure 4B in Excel file. Membrane β-Arrestin1 (ADU) in Caco-2ShPTEN cells after transfection. Experiments in triplicate.figure 4b.xlsxFigure 4CData for Figure 4C. Membrane ARHGAP10 ADU in Caco-2ShPTEN cells. Experiments in triplicatefigure 4c.xlsxFigure 4EData for Figure 4E in Excel file. Membrane β-Arrestin1 (ADU) in PTEN-/-HCT116 cells after transfection. Experiments in triplicate.figure 4e.xlsxFigure 4FData for Figure 4F in Excel file. Membrane ARHGAP10 (ADU) in PTEN-/-HCT116 cells. Experiments in triplicate.figure 4f.xlsxFigure 4HData for Figure 4H in Excel file. Membrane β-Arrestin1:ARHGAP10 binding (ADU) in Caco-2 ShPTEN cells after transfection. Experiments in triplicate.figure 4h.xlsxFigure 4JData for Figure 4J in Excel file.Membrane β-Arrestin1:ARHGAP10 binding ADU in PTEN-/-HCT116 cells. Experiments in triplicatefigure 4J.xlsxFigure 5BData for Figure 5B in Excel file. Membrane β-Arrestin1 intensity (AI) in Caco-2 ShPTEN glands after transfection. Intensity assessed in 10 glands for each replicate (n=3) for each transfection.figure 5b.xlsxFigure 5DData for Figure 5D in Excel file. Membrane ARHGAP10 intensity (AI) in Caco-2 ShPTEN glands after transfection. Intensity assessed in 10 glands for each replicate (n=3) for each transfection.figure 5d.xlsxFIGURE 5FData for Figure 5F in Excel file. Spindle angle in Caco-2 ShPTEN glands after transfection with EV or C2 or MCBR3 constructs. Spinlde angles of 15 mitotic figures per transfection.Figure 5 Supplement 2Data for Figure 5 Supplement 2 in Excel file. Caco-2 ShPTEN glands with single central lumens. Lumens counted in 50 glands and edpressed as a percentagefigure 5 supplement 2.xlsxFigure 6BData for Figure 6B in Excel file. β-Arrestin1:ARHGAP10 binding ADU in Caco-2 cells after treatment. Experiments in triplicate.figure 6b.xlsxFigure 6DData for Figure 6D in Excel file. CDC42-GTP ADU in Caco-2 cells after treatment. Experiments in triplicate.figure 6d.xlsxFigure 6FData for Figure 6F in Excel file. β-Arrestin1:ARHGAP10 binding ADU in PTEN+/+ HCT116 cells after treatment. Experiments in triplicate.figure 6f.xlsxFigure 6HData for Figure 6H in Excel file. CDC42-GTP (ADU) in PTEN+/+ HCT116 cells after treatment. Experiments in triplicate.figure 6h.xlsxFigure 6JData for Figure 6J in Excel file. Spindle angles in Caco-2 glands after treatment. Spindle angles calculated for 15 mitotic figures per treatment.Figure 6LData for Figure 6L in Excel file. Caco-2 glands with single central lumens after treatment. Lumens assessed in 50 glands and expressed as a percentagefigure 6L.xlsxFigure 7BData for Figure 7B in Excel file. Spindle angles in Organoids after treatment. Spindle angles calculated from 30 mitotic figures per treatment.Figure 7b.xlsxFigure 7CData for Figure 7C in Excel file. Organoids with single central lumen after treatment. In each replicate (n=3), in each experimental condition 10 organoids were studied and the number with single lumens were expressed as a percentage (%).Figure 7c.xlsx

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signalling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein β-Arrestin1. Because β-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42-dependent morphogenic processes through a β-Arrestin1-ARHGAP21 complex. Here we show that PTEN knockdown (KD) impairs β-Arrestin1 membrane localization, β-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN-deficiency were phenocopied by β-Arrestin1 KD or inhibition of β-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of β-Arrestin1, ARHGAP21 and Cdc42.