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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 is recruited to E-cadherin complexes.A, Cell–cell contacts were initiated for 5 or 30 min and cells stained for endogenous E-cadherin or DOCK180. Bottom graphs show intensity profile of E-cadherin or DOCK180 by line scan at sites indicated by dashed arrows.B, E-cadherin and DOCK180 staining levels in the junctional area were quantified as a percentage of the total area in the image.C–D, DOCK180 recruitment was assessed to beads coated with anti-E-cadherin or BSA in normal keratinocytes (C) or cells treated with control (con) or EGFR RNAi (D). Beads were added onto cells for 30 min, washed and stained for endogenous DOCK180.E, GST–E-cadherin tail beads were incubated with keratinocyte lysates prepared after a time course of junction induction. Precipitates were probed with anti-DOCK180 antibody (D180); fusion proteins are shown by Comassie Blue (C.B.).F, Following depletion of EGFR, cells were induced to assemble contacts for 15 min and processed as described in E.
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f0020: DOCK180 is recruited to E-cadherin complexes.A, Cell–cell contacts were initiated for 5 or 30 min and cells stained for endogenous E-cadherin or DOCK180. Bottom graphs show intensity profile of E-cadherin or DOCK180 by line scan at sites indicated by dashed arrows.B, E-cadherin and DOCK180 staining levels in the junctional area were quantified as a percentage of the total area in the image.C–D, DOCK180 recruitment was assessed to beads coated with anti-E-cadherin or BSA in normal keratinocytes (C) or cells treated with control (con) or EGFR RNAi (D). Beads were added onto cells for 30 min, washed and stained for endogenous DOCK180.E, GST–E-cadherin tail beads were incubated with keratinocyte lysates prepared after a time course of junction induction. Precipitates were probed with anti-DOCK180 antibody (D180); fusion proteins are shown by Comassie Blue (C.B.).F, Following depletion of EGFR, cells were induced to assemble contacts for 15 min and processed as described in E.

Mentions: We tested whether DOCK180 is found at keratinocyte cell–cell contacts in a time course from early to more mature contacts. E-cadherin receptors are recruited to newly formed keratinocyte junctions as early as 5 min and by 30 min, junction assembly, cytoskeletal reorganization and cell polarization are mostly complete [26,27]. In the absence of cell adhesion, endogenous DOCK180 was found dispersed in the cytoplasm (Fig. 4A). Upon addition of calcium ions, a pool of DOCK180 was clearly recruited to the junctional region (Fig. 4A–B). This re-localization was significant and increased with time. This result is in contrast to the transient DOCK180 recruitment to MDCK new junctions (2.5 h), but absent at more established contacts (5 h) [7].


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

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

DOCK180 is recruited to E-cadherin complexes.A, Cell–cell contacts were initiated for 5 or 30 min and cells stained for endogenous E-cadherin or DOCK180. Bottom graphs show intensity profile of E-cadherin or DOCK180 by line scan at sites indicated by dashed arrows.B, E-cadherin and DOCK180 staining levels in the junctional area were quantified as a percentage of the total area in the image.C–D, DOCK180 recruitment was assessed to beads coated with anti-E-cadherin or BSA in normal keratinocytes (C) or cells treated with control (con) or EGFR RNAi (D). Beads were added onto cells for 30 min, washed and stained for endogenous DOCK180.E, GST–E-cadherin tail beads were incubated with keratinocyte lysates prepared after a time course of junction induction. Precipitates were probed with anti-DOCK180 antibody (D180); fusion proteins are shown by Comassie Blue (C.B.).F, Following depletion of EGFR, cells were induced to assemble contacts for 15 min and processed as described in E.
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f0020: DOCK180 is recruited to E-cadherin complexes.A, Cell–cell contacts were initiated for 5 or 30 min and cells stained for endogenous E-cadherin or DOCK180. Bottom graphs show intensity profile of E-cadherin or DOCK180 by line scan at sites indicated by dashed arrows.B, E-cadherin and DOCK180 staining levels in the junctional area were quantified as a percentage of the total area in the image.C–D, DOCK180 recruitment was assessed to beads coated with anti-E-cadherin or BSA in normal keratinocytes (C) or cells treated with control (con) or EGFR RNAi (D). Beads were added onto cells for 30 min, washed and stained for endogenous DOCK180.E, GST–E-cadherin tail beads were incubated with keratinocyte lysates prepared after a time course of junction induction. Precipitates were probed with anti-DOCK180 antibody (D180); fusion proteins are shown by Comassie Blue (C.B.).F, Following depletion of EGFR, cells were induced to assemble contacts for 15 min and processed as described in E.
Mentions: We tested whether DOCK180 is found at keratinocyte cell–cell contacts in a time course from early to more mature contacts. E-cadherin receptors are recruited to newly formed keratinocyte junctions as early as 5 min and by 30 min, junction assembly, cytoskeletal reorganization and cell polarization are mostly complete [26,27]. In the absence of cell adhesion, endogenous DOCK180 was found dispersed in the cytoplasm (Fig. 4A). Upon addition of calcium ions, a pool of DOCK180 was clearly recruited to the junctional region (Fig. 4A–B). This re-localization was significant and increased with time. This result is in contrast to the transient DOCK180 recruitment to MDCK new junctions (2.5 h), but absent at more established contacts (5 h) [7].

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.