Figure 9.
Figure 9. Filopodia mediated by EphB2 signaling required Myo1b motor activity and tyrosine phosphorylation of its tail. (A) YFP-EphB2-HCT116 cells stimulated or not with clustered ephrinB1-Fc were fixed and labeled with phalloidin and anti-Myo1b antibodies. Note the increase of protrusions labeled for EphB2, F-actin, and Myo1b after stimulation with clustered ephrinB1-Fc (see insets at higher magnification). Bars, 10 µM. (B) Mean number of protrusions observed per cell before and after stimulation of YFP-EphB2-Hek293T cells stimulated 10 min after ephrinB1-Fc. n = 44. (C and D) Membrane protrusions formed after 10 min of stimulation with clustered ephrinB1-Fc were quantified in YFP-EphB2-HCT116 cells transfected with control siRNA, Myo1b siRNA plus the empty Flag-HA plasmid (C and D), Myo1bsiRNA+Flag-HA-Myo1b5M (C and D), Myo1bsiRNA+Flag-HA-Myo1b5MR (C), or Myo1bsiRNA+Flag-HA-Myo1b5M4YF (D). Data are shown as the mean of the number of filopodia counted in three experiments. Error bars represent ± SEM. (C) n = 53 for control siRNA, 63 for Myo1b siRNA+Flag-HA, 75 for Myo1b siRNA+FlagHA-Myo1b-5M, and 58 for Myo1b siRNA+FlagHA-Myo1b-5MR. The probabilities of these data were analyzed with Paired Student’s t test. ***, P = 3 × 10−15 for Myo1b siRNA+Flag-HA and 1.4 × 10−12 for Myo1bsiRNA+FlagHA-Myo1b-5MR versus control siRNA-treated cells. (D) n = 59 for control siRNA, 58 for Myo1bsiRNA+FlagHA, 64 for Myo1b siRNA+FlagHA-Myo1b-5M, and 60 for Myo1b siRNA+Flag-HA-Myo1b-5M-4YF. ***, P = 2 × 10−20 for Myo1b siRNA+FlagHA and 2.5 × 10−13 for Myo1b siRNA+Flag-HA-Myo1b-5M-4YF–treated cells versus control siRNA-treated cells. (E) Sequence of fluorescent images of YFP-EphB2-Hek293T cells stimulated with clustered ephrinB1-Fc that illustrate filopodia formation growing from the cell edge and retraction fibers left behind after the retraction of the cell edge. Bars, 1 µm. The cell edge is marked by a yellow dashed line. (F) The increase of the number of protrusions after 10 min of stimulation with ephrinB1-Fc of YFP-EphB2-Hek293T cells transfected with control, Myo1b, or fascin siRNAs or treated with DMSO or 100 µM CK666 for 30 min at 37°C has been calculated from the total number of protrusions counted before and after stimulation and expressed as a percentage of the increase of the number of protrusions calculated in cells transfected with control siRNA. Data are shown as the mean of the number of protrusions counted for 44, 40, 32, and 39 control siRNA–, Myo1b siRNA–, fascin siRNA–, and CK666-treated cells, respectively. Error bars represent ± SEM. The probabilities of these data were analyzed with Paired Student’s t test: ***, P < 0.001 for protrusions formed in Myo1b siRNA–treated cells versus control siRNA–treated cells and CK666-treated cells versus DMSO-treated cells. (G) Filopodia growing from the cell edges (Fig. 8 A) after 10 min of stimulation with ephrinB1-Fc of YFP-EphB2-Hek293T cells transfected with control, Myo1b, or fascin siRNAs or treated with DMSO or CK666 were quantified and expressed as a percentage of the filopodia counted in cells transfected with control siRNA. Data are shown as the mean of the number of protrusions counted for 44, 40, 32, and 39 control siRNA–, Myo1b siRNA–, fascin siRNA–, and CK666-treated cells, respectively. Error bars represent ± SEM. The probabilities of these data were analyzed with Paired Student’s t test: ***, P < 0.001 for filopodia formed in Myo1b or fascin siRNA–treated cells versus control siRNA–treated cells and CK666-treated cells versus DMSO-treated cells.

Filopodia mediated by EphB2 signaling required Myo1b motor activity and tyrosine phosphorylation of its tail. (A) YFP-EphB2-HCT116 cells stimulated or not with clustered ephrinB1-Fc were fixed and labeled with phalloidin and anti-Myo1b antibodies. Note the increase of protrusions labeled for EphB2, F-actin, and Myo1b after stimulation with clustered ephrinB1-Fc (see insets at higher magnification). Bars, 10 µM. (B) Mean number of protrusions observed per cell before and after stimulation of YFP-EphB2-Hek293T cells stimulated 10 min after ephrinB1-Fc. n = 44. (C and D) Membrane protrusions formed after 10 min of stimulation with clustered ephrinB1-Fc were quantified in YFP-EphB2-HCT116 cells transfected with control siRNA, Myo1b siRNA plus the empty Flag-HA plasmid (C and D), Myo1bsiRNA+Flag-HA-Myo1b5M (C and D), Myo1bsiRNA+Flag-HA-Myo1b5MR (C), or Myo1bsiRNA+Flag-HA-Myo1b5M4YF (D). Data are shown as the mean of the number of filopodia counted in three experiments. Error bars represent ± SEM. (C) n = 53 for control siRNA, 63 for Myo1b siRNA+Flag-HA, 75 for Myo1b siRNA+FlagHA-Myo1b-5M, and 58 for Myo1b siRNA+FlagHA-Myo1b-5MR. The probabilities of these data were analyzed with Paired Student’s t test. ***, P = 3 × 10−15 for Myo1b siRNA+Flag-HA and 1.4 × 10−12 for Myo1bsiRNA+FlagHA-Myo1b-5MR versus control siRNA-treated cells. (D) n = 59 for control siRNA, 58 for Myo1bsiRNA+FlagHA, 64 for Myo1b siRNA+FlagHA-Myo1b-5M, and 60 for Myo1b siRNA+Flag-HA-Myo1b-5M-4YF. ***, P = 2 × 10−20 for Myo1b siRNA+FlagHA and 2.5 × 10−13 for Myo1b siRNA+Flag-HA-Myo1b-5M-4YF–treated cells versus control siRNA-treated cells. (E) Sequence of fluorescent images of YFP-EphB2-Hek293T cells stimulated with clustered ephrinB1-Fc that illustrate filopodia formation growing from the cell edge and retraction fibers left behind after the retraction of the cell edge. Bars, 1 µm. The cell edge is marked by a yellow dashed line. (F) The increase of the number of protrusions after 10 min of stimulation with ephrinB1-Fc of YFP-EphB2-Hek293T cells transfected with control, Myo1b, or fascin siRNAs or treated with DMSO or 100 µM CK666 for 30 min at 37°C has been calculated from the total number of protrusions counted before and after stimulation and expressed as a percentage of the increase of the number of protrusions calculated in cells transfected with control siRNA. Data are shown as the mean of the number of protrusions counted for 44, 40, 32, and 39 control siRNA–, Myo1b siRNA–, fascin siRNA–, and CK666-treated cells, respectively. Error bars represent ± SEM. The probabilities of these data were analyzed with Paired Student’s t test: ***, P < 0.001 for protrusions formed in Myo1b siRNA–treated cells versus control siRNA–treated cells and CK666-treated cells versus DMSO-treated cells. (G) Filopodia growing from the cell edges (Fig. 8 A) after 10 min of stimulation with ephrinB1-Fc of YFP-EphB2-Hek293T cells transfected with control, Myo1b, or fascin siRNAs or treated with DMSO or CK666 were quantified and expressed as a percentage of the filopodia counted in cells transfected with control siRNA. Data are shown as the mean of the number of protrusions counted for 44, 40, 32, and 39 control siRNA–, Myo1b siRNA–, fascin siRNA–, and CK666-treated cells, respectively. Error bars represent ± SEM. The probabilities of these data were analyzed with Paired Student’s t test: ***, P < 0.001 for filopodia formed in Myo1b or fascin siRNA–treated cells versus control siRNA–treated cells and CK666-treated cells versus DMSO-treated cells.

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