Fig. 5. Nocodazole inhibits the accumulation of E-cadherin at cell-cell contacts without affecting cadherin traffic to the cell surface. (A) Low concentrations of nocodazole disrupt the morphology of cadherin-based cell-cell contacts. MCF-7 monolayers were incubated with nocodazole (100 nM, 240 minutes) or DMSO carrier (Control) before fixation and immunostaining for E-cadherin. (B) Nocodazole reduces E-cadherin accumulation in newly forming cell-cell contacts. Cells were incubated with nocodazole (100 nM, Noc) or DMSO vehicle alone (Control) for 60 minutes beforehand, and throughout the experiments. Cell-cell contacts between confluent MCF-7 cells were disrupted by chelation of extracellular Ca²⁺ (0 minutes) then allowed to reform by addition of Ca²⁺. At varying times thereafter, samples were fixed and stained for E-cadherin. E-cadherin fluorescence intensity at cell-cell contacts (E-cadherin intensity) was measured by digital image analysis. Representative images of control and nocodazole-treated cells at 30 minutes are shown. (C) Nocodazole does not affect total or surface expression levels of E-cadherin. Surface expression of E-cadherin was measured using surface trypsin protection assays in control cells or cells pre-incubated with nocodazole (100 nM, 90 minutes). Cells were lysed immediately (WCL) or after trypsinisation in the presence (+Ca) or absence (-Ca) of extracellular Ca²⁺. Total cellular levels of E-cadherin (E-cad) were unaffected by nocodazole and all the cadherin remained accessible to surface trypsinisation in the absence of Ca²⁺ in both the control and drug-treated cells. (D) Nocodazole does not affect transport of E-cadherin to the cell surface. Cells were studied at baseline (BL) or trypsinised to strip E-cadherin from the cell surface (0'), then incubated for 2-4 hours before surface biotinylation. Biotinylated proteins were separated by SDS-PAGE and immunoblotted for E-cadherin or transferrin receptor (TfR), as a loading control. Cultures were incubated in the presence of nocodazole (100 nM, pre-incubated for 1 hour, then for the duration of the experiments) or with vehicle alone (C). Results are representative of three independent experiments.