Data from: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

Javadi, Arman; Deevi, Ravi K.; Evergren, Emma; Blondel-Tepaz, Elodie; Baillie, George S.; Scott, Mark G.H.; Campbell, Frederick Charles; Scott, Mark GH

doi:10.5061/dryad.ns5qs

Published August 17, 2017 | Version v1

Dataset Open

Data from: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

1. Queen's University Belfast
2. French National Centre for Scientific Research
3. University of Glasgow
4. Institut Cochin

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.

Notes

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.xlsx

Figure 1D Membrane β-Arrestin1 intensity

Data 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.xlsx

Figure 1F β-Arrestin1 membrane (ADU) in Caco-2 and Caco-2 ShPTEN cells after control or LPA treatment

Data 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.xlsx

Figure 1 supplement 2

Data 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 4

Data for Figure 1 Supplement 4 in Excel file. Membrane β-Arrestin1 ADU in PTEN+/+ and PTEN-/- HCT116 cells after LPA treatment. Experiments in triplicate

figure 1 supplement 4.xlsx

Figure 2B

Data for Figure 2B in Excel file. CDC42-GTP ADU in Caco-2 cells after β-Arrestin1 or NT SiRNA. Experiments in triplicate.

figure 2b.xlsx

Figure 2D

Data for Figure 2D in Excel file. CDC42-GTP (ADU) in Caco-2ShPTEN cells after ARHGAP10 or NT SiRNA transfections. Experiments in triplicate.

Figure 2d.xlsx

Figure 2 supplement 1

Data 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 2

Data 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 3

Data 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 4

Data 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 condition

Figure 2 supplement 5

Data 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.xlsx

Figure 2 supplement 6

Data 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 condition

Figure 2 supplement 7

Data 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 8

Data 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.xlsx

Figure 3B

Data for Figure 3B in Excel file. β-Arrestin1:ARHGAP binding ADU in Caco-2 or Caco-2 ShPTEN cells. Experiments in triplicate.

figure 3b.xlsx

Figure 3C

Data for Figure 3C in Excel file. β-Arrestin1:ARHGAP binding ADU in PTEN+/+ and PTEN-/- HCT116 cells

figure 3c.xlsx

Figure 3E

Data for Figure 3E in Excel file. β-Arrestin1:ARHGAP10 binding ADU in Caco-2 ShPTEN cells after transfection. Experiments in triplicate.

figure 3e.xlsx

Figure 3G

Data for Figure 3G in Excel file. β-Arrestin1:ARHGAP10 binding ADU in PTEN-/- HCT116 cells. Experiments in triplicate.

figure 3g.xlsx

Figure 4B

Data for Figure 4B in Excel file. Membrane β-Arrestin1 (ADU) in Caco-2ShPTEN cells after transfection. Experiments in triplicate.

figure 4b.xlsx

Figure 4C

Data for Figure 4C. Membrane ARHGAP10 ADU in Caco-2ShPTEN cells. Experiments in triplicate

figure 4c.xlsx

Figure 4E

Data for Figure 4E in Excel file. Membrane β-Arrestin1 (ADU) in PTEN-/-HCT116 cells after transfection. Experiments in triplicate.

figure 4e.xlsx

Figure 4F

Data for Figure 4F in Excel file. Membrane ARHGAP10 (ADU) in PTEN-/-HCT116 cells. Experiments in triplicate.

figure 4f.xlsx

Figure 4H

Data for Figure 4H in Excel file. Membrane β-Arrestin1:ARHGAP10 binding (ADU) in Caco-2 ShPTEN cells after transfection. Experiments in triplicate.

figure 4h.xlsx

Figure 4J

Data for Figure 4J in Excel file.Membrane β-Arrestin1:ARHGAP10 binding ADU in PTEN-/-HCT116 cells. Experiments in triplicate

figure 4J.xlsx

Figure 5B

Data 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.xlsx

Figure 5D

Data 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.xlsx

FIGURE 5F

Data 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 2

Data 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 percentage

figure 5 supplement 2.xlsx

Figure 6B

Data for Figure 6B in Excel file. β-Arrestin1:ARHGAP10 binding ADU in Caco-2 cells after treatment. Experiments in triplicate.

figure 6b.xlsx

Figure 6D

Data for Figure 6D in Excel file. CDC42-GTP ADU in Caco-2 cells after treatment. Experiments in triplicate.

figure 6d.xlsx

Figure 6F

Data for Figure 6F in Excel file. β-Arrestin1:ARHGAP10 binding ADU in PTEN+/+ HCT116 cells after treatment. Experiments in triplicate.

figure 6f.xlsx

Figure 6H

Data for Figure 6H in Excel file. CDC42-GTP (ADU) in PTEN+/+ HCT116 cells after treatment. Experiments in triplicate.

figure 6h.xlsx

Figure 6J

Data for Figure 6J in Excel file. Spindle angles in Caco-2 glands after treatment. Spindle angles calculated for 15 mitotic figures per treatment.

Figure 6L

Data for Figure 6L in Excel file. Caco-2 glands with single central lumens after treatment. Lumens assessed in 50 glands and expressed as a percentage

figure 6L.xlsx

Figure 7B

Data for Figure 7B in Excel file. Spindle angles in Organoids after treatment. Spindle angles calculated from 30 mitotic figures per treatment.

Figure 7b.xlsx

Figure 7C

Data 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

Funding provided by: National Science Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000001
Award Number: N/A

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Is cited by: 10.7554/elife.24578 (DOI)

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Data from: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

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