α–E-catenin negatively regulates dynactin-mediated intracellular traffic in an AJ-independent manner. (A) Quantitation of lysosome movements in wild-type (WT) and α–E-catenin^−/− (KO) keratinocytes. The total length of lysosome movements within 5 min was determined using Imaris software analysis of the time-lapse videos. Bars represent the percentage of the vesicles that moved over the indicated distances; n = 315 for wild type and n = 342 for α–E-catenin^−/−. Note the prominent increase in lysosome motility in α–E-catenin^−/− cells. (B and C) Expression of exogenous α–E-catenin in α–E-catenin^−/− cells. Keratinocytes were transduced with retroviruses expressing the HBT tag (HBT) or HBT-tagged α–E-catenin (HBT–α-cat) and analyzed by blotting (B) and immunostaining (C) with streptavidin or anti–α-catenin (α-cat), anti–β-actin, and anti–E-cadherin (E-cad) antibodies. (D) Reexpression of α–E-catenin in α–E-catenin^−/− cells rescues lysosome motility defects. Quantitation of lysosome movements in α–E-catenin^−/− keratinocytes expressing HBT (KO + HBT) or HBT-α–E-catenin (KO + HBT–α-cat). n = 354 for KO + HBT and n = 318 for KO + HBT–α-cat. (E) Disruption of AJs in keratinocytes cultured in low-calcium media. Immunofluorescence staining of wild-type and α–E-catenin^−/− cells incubated in normal or low-calcium (LowCa) media with anti–E-cadherin (green) antibodies and phalloidin (actin; red). (F and G) Quantitation of lysosome movements in wild-type (F) and α–E-catenin^−/− (G) keratinocytes incubated in normal or low-calcium media. n = 319 for WT, n = 326 for WT + LowCa, n = 307 for KO, and n = 347 for KO + LowCa. Bars: (C) 25 μm; (E) 33 μm.