Reduction of PI(4,5)P2 by the activation of mTORC2–Rab35 axis is essential for the adaptation to hypo-osmotic stress. (A and B) Time-lapse imaging and quantification of EpH4 cells stably expressing GFP-Lifeact. Hypo-osmotic buffer (150 mOsm/liter) was added at time 0:00. Fluorescence intensities at apical membranes and basolateral membranes normalized to the value at t = 0 min and shown as the mean line. Scale bar, 10 µm. N = 15 independent experiments. (C and D) Time-lapse imaging and quantification of EpH4 cells stably expressing GFP-PH^PLCδ. hypo-osmotic buffer (150 mOsm/liter) was added at time 0:00. The decrease of GFP-PH^PLCδ signal in the apical membrane due to hypotonic stress was suppressed by the treatment with either 250 nM Torin-1 or 3 µM Ku-0063794 for 24 h. Scale bar, 10 µm. Fluorescence intensities normalized to the value at t = 0 min and shown as the mean line. N ≥ 3 independent experiments. (E) EpH4 cells expressing SS-GFP-Lys and Src-INPP5E-Scarlet were treated with DMSO (Control) or 250 nM Torin-1 for 24 h. Scale bar, 10 µm. (F and G) Representative images and quantification of Rictor KD cells expressing Src-GFP (control) vector or Src-INPP5E-GFP were treated with hypo-osmotic buffer (30 mOsm/liter) for 30 min and stained with PI (red; nucleus of dead cells) and NucBlue (total cell). N ≥ 8 independent experiments; error bar, SD; ****, P < 0.0001 by Student’s t test. (H) mTORC2 and Rab35 buffer plasma membrane tension by reducing the actin cortex and increasing transport of apical membrane components in order to protect against cell death by rupturing during hypo-osmotic challenge.