Limits...
Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

Erasmus JC, Welsh NJ, Braga VM - Cell. Signal. (2015)

Bottom Line: In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process.However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions.Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, SW7 2AZ London, UK.

No MeSH data available.


DOCK180 stabilizes pre-formed junctions.Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H).A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B).C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation.C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates.D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1).E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p < 0.03; **, p < 0.0003; ***, p < 0.001; n.s., non-significant.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4508347&req=5

f0025: DOCK180 stabilizes pre-formed junctions.Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H).A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B).C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation.C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates.D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1).E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p < 0.03; **, p < 0.0003; ***, p < 0.001; n.s., non-significant.

Mentions: We investigated the above point further and tested whether DOCK180 and EGFR are both necessary for actin recruitment to cadherin clusters (Fig. 5), a process that is inhibited by dominant-negative Rac in keratinocytes [23]. Similarly to Rac inhibition, EGFR RNAi caused a partial, but significant reduction of cadherin-dependent actin recruitment (Fig. 5A–B). Strikingly, actin recruitment to clustered cadherin receptors was not perturbed by DOCK180 depletion (Fig. 5A–B). These results suggest that, following junction assembly, DOCK180 may regulate alternative Rac functions other than actin remodelling. In contrast, EGFR seems to be upstream of Rac in driving actin recruitment to junctions.


Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

Erasmus JC, Welsh NJ, Braga VM - Cell. Signal. (2015)

DOCK180 stabilizes pre-formed junctions.Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H).A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B).C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation.C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates.D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1).E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p < 0.03; **, p < 0.0003; ***, p < 0.001; n.s., non-significant.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508347&req=5

f0025: DOCK180 stabilizes pre-formed junctions.Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H).A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B).C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation.C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates.D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1).E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p < 0.03; **, p < 0.0003; ***, p < 0.001; n.s., non-significant.
Mentions: We investigated the above point further and tested whether DOCK180 and EGFR are both necessary for actin recruitment to cadherin clusters (Fig. 5), a process that is inhibited by dominant-negative Rac in keratinocytes [23]. Similarly to Rac inhibition, EGFR RNAi caused a partial, but significant reduction of cadherin-dependent actin recruitment (Fig. 5A–B). Strikingly, actin recruitment to clustered cadherin receptors was not perturbed by DOCK180 depletion (Fig. 5A–B). These results suggest that, following junction assembly, DOCK180 may regulate alternative Rac functions other than actin remodelling. In contrast, EGFR seems to be upstream of Rac in driving actin recruitment to junctions.

Bottom Line: In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process.However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions.Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, SW7 2AZ London, UK.

No MeSH data available.