NMM2 alignment induced by EphB2 stimulation requires Myo1b motor activity and tyrosine phosphorylation of its tail. (A) YFP-EphB2-Hek293T cells transfected with control siRNA were analyzed by live-cell imaging (Video 9). One frame before and one after 10 min of stimulation with clustered ephrinB1-Fc are shown. Bars, 5 µm. The yellow line outlines the cell periphery on the first frame before treatment. (B) YFP-EphB2-Hek293T cells, transfected with a plasmid encoding MRLC-RFP, were analyzed by live-cell imaging (Video 10) before and after stimulation with clustered ephrinB1-Fc. One frame before and one frame after 8 min of stimulation are shown. Bar, 5 µm. (C) YFP-EphB2-HCT116 cells were immunolabeled with anti-NMM2 antibodies and analyzed before or after 10 min of stimulation with clustered ephrinB1-Fc. Note the alignment of NMM2 after EphB2 stimulation. Bar, 6 µm. (D) YFP-EphB2-HCT116 cells transfected with Myo1b or control siRNAs were stimulated for 10 min or not with clustered ephrinB1-Fc and immunolabeled with anti-NMM2 antibodies, and the number of cells showing NMM2 alignment was quantified. Data are shown as the mean of three experiments (n = 1032 for control siRNA and 918 for Myo1b siRNA). Error bars represent ± SEM. (E and F) YFP-EphB2-HCT116 cells transfected with control siRNAs, Myo1b siRNA plus the empty Flag-HA plasmid (E and F), Myo1bsiRNA+Flag-HA-Myo1b5M (E and F), Myo1bsiRNA+Flag-HA-Myo1b5MR (E), or Myo1bsiRNA+Flag-HA-Myo1b5M4YF (F) were immunolabeled with anti-NMM2 antibodies and the number of cells showing NMM2 alignment was quantified. Data are shown as the mean of three experiments (E: n = 82 for control siRNA, 92 for Myo1b siRNA+Flag-HA, 74 for Myo1b siRNA+Flag-HA-Myo1b5M, and 74 for Myo1b siRNA+Flag-HA-Myo1b5MR; F: n = 70 for control siRNA, 57 for Myo1b siRNA+Flag-HA, 78 for Myo1b siRNA+Flag-HA-Myo1b5M, and 70 for Myo1b siRNA+Flag-HA-Myo1b5M4YF). Error bars represent ± SEM.