Dynamitin is necessary to extend the microtubule cytoskeleton to the cell periphery and AJs and establish strong cell–cell adhesion. (A) Schematic model of a lentiviral shRNA vector used for the generation of dynamitin KD cells. CMV, cytomegalovirus; LTR, long terminal repeat. (B) Total protein extracts from wild-type (WT) and α–E-catenin^−/− (KO) cells transduced with β-galactosidase (β-gal; control) and dynamitin (Dyn; constructs 1 and 2) shRNA lentiviruses analyzed by blotting with antidynamitin (Dyn) and anti–β-actin antibodies. Numbers represent relative levels of dynamitin. (C and D) Dynamitin is necessary to localize microtubules to AJs. Wild-type keratinocytes transduced with β-galactosidase shRNA (β-galsh; C) or dynamitin shRNA-2 (Dynsh; D) were analyzed by immunostaining with anti–E-cadherin (E-cad) and anti–β-tubulin (β-tub) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in C‴ and D‴. Note the prominent localization of microtubules to AJs in β-galactosidase shRNA cells (arrows) but not in dynamitin KD cells. (E) Quantitation of junctional localization of microtubules in control (WT + β-gal shRNA) and dynamitin KD (WT + Dyn shRNA) cells. Quantitation was performed as described in Fig. 2 E. n = 50. (F) dynamitin KD cells display cell–cell adhesion defects. Wild-type and α–E-catenin^−/− keratinocytes expressing β-galactosidase shRNA or dynamitin shRNA-2 were allowed to aggregate for 1 h with and without Ca²⁺, and the total number of particles was counted. The degree of Ca²⁺-dependent cell aggregation (N_Ca+/N_Ca− percentage) was measured as a percentage of the decrease in the particle numbers in Ca²⁺-containing versus Ca²⁺-free conditions. Bars represent mean values; n = 3. The p-value was determined by t test. The error bars represent standard deviation. Bars: (C–C″ and D–D″) 28 μm; (C‴ and D‴) 11.2 μm.