Figure 7.
mTORC2–Rab35 activity is required for the adaptation to increased apical plasma membrane tension under hypo-osmotic stress. (A and B) mTORC2 activity under hypo-osmotic condition (150 mOsm/liter) were assessed by phosphorylation level of Akt Ser473 in EpH4 cells. N = 6 independent experiments. (C) Total cell lysates of wild-type EpH4 cells under iso- (Iso) and hypo-osmotic (Hypo) conditions, the latter treated with either DMSO or Torin-1 were resolved by SDS-PAGE and immunoblotted with indicated antibodies. (D) Quantification of C. N = 3 independent experiments; error bar, SD; **, P = 0.0019; ***, P = 0.0002; and ****, P < 0.0001 by one-way ANOVA with Tukey’s post-hoc test. (E) EpH4 cells expressing a GFP-based fluorogenic caspase-3 reporter, FlipGFP(casp3)-T2A-mCherry (Zhang et al., 2019), were treated with either iso- (Iso) or hypo-osmotic (Hypo) buffer. Cells treated with anti-Fas-antibody serve as the positive control for apoptosis. Scale bar, 10 µm. (F) Quantification of the rate of cells that undergo apoptosis under the conditions described in E. N = 3 independent experiments; error bar, SD; ***, P = 0.0003 and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (G) Wild-type EpH4 cells (WT), Rictor KD cells, and Rab35 KO cells were treated with hypo-osmotic buffer (30 mOsm/liter) for 30 min and stained with PI (red; nucleus of dead cells) and Calcein-AM (green; cytosol of live cells) to evaluate live and dead cells. Scale bar, 20 µm. (H) Quantification of the ratio of dead cells to total cells under the conditions described in G. N = 5 independent experiments; error bar, SD; ****, P < 0.0001 by one-way ANOVA with Tukey’s post-hoc test. (I) Scanning electron microscopy wild-type EpH4 cells under iso-osmotic buffer (Iso) and wild-type (WT), Rictor KD and Rab35 KO cells treated with hypo-osmotic buffer (30 mOsm/liter) for 10 min. Scale bar, 10 µm. (J) Quantification of the ratio of live cells to total cells in Rictor KD cells stably expressing mScarlet only or mScarlet-Rab35 Q67L and stained with Calcein-AM. Cells were treated with hypo-osmotic buffer (30 mOsm/liter) for 40 min. N = 5 independent experiments; error bar, SD; ****, P < 0.0001 by Student’s t test. This data is related to Videos 3 and 4. (K) Immunoblot of active, GTP-Rab35 purified by pull-down with the Rab-binding domain (RBD) of the Rab35 effector MICAL-3 in samples prepared from EpH4 cells stably expressing either wild-type or Q67L mScarlet-Rab35. Cells were maintained in iso-osmotic medium (-) and exposed to hypo-osmotic medium (Hypo, 150 mOsm/liter) for 2 min before lysis. (L) Quantification of K. N = 3 independent experiments; error bar, SD; P values indicated are from one-way ANOVA with Tukery’s post-hoc test. Data from Q67L was considered as outliers and excluded from comparison. (M) Localization of the GFP-tagged RBD of MICAL-3 in wild-type EpH4 cells stably expressing wild-type mScarlet-Rab35 treated with DMSO (Control) or 250 nM Torin-1 for 24 h. Scale bar, 5 µm. (N) Quantification of M. N = 3 independent experiments; error bar, SD; *, P = 0.0189 by Student’s t test. Source data are available for this figure: SourceData F7.

mTORC2–Rab35 activity is required for the adaptation to increased apical plasma membrane tension under hypo-osmotic stress. (A and B) mTORC2 activity under hypo-osmotic condition (150 mOsm/liter) were assessed by phosphorylation level of Akt Ser473 in EpH4 cells. N = 6 independent experiments. (C) Total cell lysates of wild-type EpH4 cells under iso- (Iso) and hypo-osmotic (Hypo) conditions, the latter treated with either DMSO or Torin-1 were resolved by SDS-PAGE and immunoblotted with indicated antibodies. (D) Quantification of C. N = 3 independent experiments; error bar, SD; **, P = 0.0019; ***, P = 0.0002; and ****, P < 0.0001 by one-way ANOVA with Tukey’s post-hoc test. (E) EpH4 cells expressing a GFP-based fluorogenic caspase-3 reporter, FlipGFP(casp3)-T2A-mCherry (Zhang et al., 2019), were treated with either iso- (Iso) or hypo-osmotic (Hypo) buffer. Cells treated with anti-Fas-antibody serve as the positive control for apoptosis. Scale bar, 10 µm. (F) Quantification of the rate of cells that undergo apoptosis under the conditions described in E. N = 3 independent experiments; error bar, SD; ***, P = 0.0003 and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (G) Wild-type EpH4 cells (WT), Rictor KD cells, and Rab35 KO cells were treated with hypo-osmotic buffer (30 mOsm/liter) for 30 min and stained with PI (red; nucleus of dead cells) and Calcein-AM (green; cytosol of live cells) to evaluate live and dead cells. Scale bar, 20 µm. (H) Quantification of the ratio of dead cells to total cells under the conditions described in G. N = 5 independent experiments; error bar, SD; ****, P < 0.0001 by one-way ANOVA with Tukey’s post-hoc test. (I) Scanning electron microscopy wild-type EpH4 cells under iso-osmotic buffer (Iso) and wild-type (WT), Rictor KD and Rab35 KO cells treated with hypo-osmotic buffer (30 mOsm/liter) for 10 min. Scale bar, 10 µm. (J) Quantification of the ratio of live cells to total cells in Rictor KD cells stably expressing mScarlet only or mScarlet-Rab35 Q67L and stained with Calcein-AM. Cells were treated with hypo-osmotic buffer (30 mOsm/liter) for 40 min. N = 5 independent experiments; error bar, SD; ****, P < 0.0001 by Student’s t test. This data is related to Videos 3 and 4. (K) Immunoblot of active, GTP-Rab35 purified by pull-down with the Rab-binding domain (RBD) of the Rab35 effector MICAL-3 in samples prepared from EpH4 cells stably expressing either wild-type or Q67L mScarlet-Rab35. Cells were maintained in iso-osmotic medium (-) and exposed to hypo-osmotic medium (Hypo, 150 mOsm/liter) for 2 min before lysis. (L) Quantification of K. N = 3 independent experiments; error bar, SD; P values indicated are from one-way ANOVA with Tukery’s post-hoc test. Data from Q67L was considered as outliers and excluded from comparison. (M) Localization of the GFP-tagged RBD of MICAL-3 in wild-type EpH4 cells stably expressing wild-type mScarlet-Rab35 treated with DMSO (Control) or 250 nM Torin-1 for 24 h. Scale bar, 5 µm. (N) Quantification of M. N = 3 independent experiments; error bar, SD; *, P = 0.0189 by Student’s t test. Source data are available for this figure: SourceData F7.

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