JCS02548 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig.S1. Src activity is required for Rap1 activation in response to the disruption of AJs.  FRT cells were either left untreated (Ctrl), treated with 4 mM EGTA for 30 minutes (EGTA) or pretreated with PP1 (10 mM) for 1 hour before EGTA treatment (PP1+EGTA). Active Rap1 levels were then measured by the pull-down assay. Notice that SFK inhibition by PP1 reduced the activation of Rap1 induced by EGTA treatment.

• Supplemental Figure 2 - Fig.S2. Rap1 activation promoted by the disassembly of AJs requires E-cadherin endocytosis. FRT cell monolayers kept overnight in serum-free medium were either left untreated (Ctrl), or treated with filipin (10 mg/ml) (Filipin) or Chlorpromazine (10 mg/ml) (CPZ) for 1 hour, to interfere with clathrin-independent and clathrin-dependent endocytosis, respectively, or EGTA (4 mM) for 30 minutes to disrupt cell-cell adhesion (EGTA), or pretreated with either filipin or Chlorpromazine for 30 minutes before EGTA treatment (Filipin+EGTA and CPZ+EGTA). (A) Cells were fixed, permeabilized, stained for E-cadherin and analyzed by immunofluorescence microscopy. Notice that the EGTA-induced endocytosis of E-cadherin (d) was inhibited by filipin (e) but not chlorpromazine (f). (B) Lysates from FRT cells treated as above were used to measure the GTP-loading of Rap1 by the pull-down assay. Notice that filipin pre-treatment effectively inhibited the EGTA-induced activation of Rap1, whereas CPZ pre-treatment was ineffective.

• Supplemental Figure 3 - Fig.S3. The recycling of endocytosed E-cadherin to the cell surface is required for the downregulation of Rap1 that occurs upon reformation of cell-cell contacts. Confluent FRT cells were treated with EGTA (4 mM) for 30 minutes, to disrupt cell-cell contacts and induce E-cadherin endocytosis. Treated cells were then either left in the presence of EGTA (EGTA), or allowed to reform adherens junctions by further incubation with fresh medium containing 1.8 mM Ca²⁺ for 30 minutes at 37°C either in the absence (EGTA/Ca²⁺) or presence of BAF (EGTA/BAF/Ca²⁺). (A) Cells were then fixed, permeabilized, stained for E-cadherin and analyzed by immunofluorescence microscopy.  Notice that upon Ca²⁺ restoration, BAF treatment reduced the reformation of cell-cell contacts by reducing recycling back to the cell surface of endocytosed E-cadherin. (B) Lysates from FRT cells treated as above were used to measure the GTP-loading of Rap1 by the pull-down assay. Notice that BAF prevented the downregulation of Rap1 activity induced by Ca²⁺ restoration.

• Supplemental Figure 4 - Fig.S4. The activation of Rap1 does not occur during the exocytosis of E-cadherin. Confluent FRT cells were either left untreated (Ctrl) or treated with NEM alone as a control (NEM), or pretreated with NEM, to inhibit endocytic trafficking, and then treated with EGTA (NEM/EGTA), or pretreated with EGTA, to induce E-cadherin endocytosis (EGTA), and then treated without (EGTA/Ca²⁺) or with NEM upon Ca²⁺ restoration (EGTA/NEM/Ca²⁺). (A) Cells were then fixed, permeabilized, stained for E-cadherin and analyzed by immunofluorescence microscopy.  Notice that NEM, prevented EGTA-induced E-cadherin internalization (d), but did not block the recycling of endocytosed E-cadherin to the cell surface and the reformation of cell-cell contacts upon Ca²⁺ restoration (f). (B) Lysates from FRT cells treated as above were used to measure the GTP-loading of Rap1 by the pull-down assay. Notice that the presence of NEM  prevented the activation of Rap1 induced by EGTA (NEM/EGTA), but did not affect the downregulation of Rap1 activity that occurred in EGTA-treated cells upon Ca²⁺ restoration (EGTA/NEM/Ca²⁺).

• Supplemental Figure 5 - Fig.S5. AJ disassembly induces a Rap1 activity-dependent assembly of paxillin-containing focal adhesions. FRT cells transiently transfected with EGFP-paxillin, as a marker of focal adhesions, were plated on FN-coated coverslips and grown to confluence for 12 hours. Living cells were then analyzed by time-lapse microscopy during disruption of cell-cell junctions by EGTA treatment in serum-free medium. (A) The two selected frames of the video sequence show the subcellular distribution of EGFP-paxillin before cell treatments (Ctrl), and after 30 minutes of EGTA treatment (EGTA). Notice the assembly of EGFP-paxillin-containing focal adhesions upon EGTA treatment. Phase contrast images of the EGFP-paxillin panels are also shown. QuickTime movies are available as supplementary material.

• Movie 1A
• Movie 1B -

  Supplementary Movies to Figs 8 and 14.

  Rap1 activity is required for the enhanced assembly of focal adhesions induced by the disruption of E-cadherin-mediated cell-cell junctions.

  Movie Legends

  Movie 1. (A) The subcellular distribution of EGFP-zyxin during the disruption of cell-cell junctions by EGTA treatment. Thirty video frames are shown. The time interval between frames is 1 minute. EGTA treatment was performed just before starting the video recording, and its effect on cell-cell junctions can be followed in phase contrast (B). Notice the assembly of EGFP-zyxin-containing focal adhesions during the disruption of cell-cell junctions.

• Movie 2A
• Movie 2B - Movie2. (A) The video sequence shows the subcellular distribution of EGFP-zyxin during the reformation of cell-cell junctions by Ca²⁺ restoration. Thirty video frames are shown. The time interval between frames is 1 minute. Ca²⁺ was restored just before starting the video recording, and its effect on cell-cell junctions can be followed in phase contrast (B). Notice the disassembly of EGFP-zyxin-containing focal adhesions during the reformation of cell-cell junctions.

• Movie 3A
• Movie 3B - Movie3. (A) The video sequence shows the subcellular distribution of EGFP-paxillin during the disruption of cell-cell junctions by EGTA treatment. Thirty video frames are shown. The time interval between frames is 1 minute. Cells were treated with EGTA just before starting the video recording, and its effect on cell-cell junctions can be followed in phase contrast (B). Notice the assembly of EGFP-paxillin-containing focal adhesions during the disruption of cell-cell junctions.

• Movie 4A
• Movie 4B - Movie4. (A) The video sequence shows the subcellular distribution of EGFP-zyxin during the disruption of cell-cell junctions in cells microinjected with either GST-Rap1GAPv (left cell) or with GST alone (right cell) 30 minutes before EGTA treatment. Thirty video frames are shown. The time interval between frames is 1 minute. Cells were treated with EGTA just before starting the video recording, and its effect on cell-cell junctions can be followed in phase contrast (B). Notice that the formation of EGFP-zyxin-containing focal adhesion is specifically and completely inhibited in the cell injected with Rap1GAPv.