Figure 3.

Increasing Membrane Tension Abrogates Rear Caveolae Formation and Forward Translocation

(A) A2780 cell in 3D CDM imaged before and during hypo-osmotic shock and after recovery in isotonic media.

(B) Rear mCherry-caveolin-1 intensity before and during osmotic shock, and after recovery in isotonic medium. N=12 cells, 3 repeats, bars = SEM.

(C) Top left: cells as in (A) imaged every 30 s immediately after media change; top right: average normalized mCherry-caveolin-1 intensity in 10% rear mask (white inset in images) and rest of cell regions over time following osmotic to isotonic switch (N=17 cells, 3 repeats, bars = SEM, fitted quadratic curves showed in red and blue); bottom: montage of zoomed, 90° rotated rear region (highlighted by yellow box) imaged every 30 s post- osmotic-isotonic switch showing mCherry-caveolin-1 accumulation.

(D) Left: A2780 cell in CDM expressing GFP-membrane (left), Ezrin wild type (WT, center) or Ezrin constitutively active (CA, right); center: pairwise normalized peak Ezrin intensity within front and rear regions (N>32 cells/condition, 3 repeats); right: average rear forward movement over 5 min (N > 39, 3 repeats).

(E) Left: cells in CDM stained for F-actin (phalloidin) and caveolin-1, MIPs shown; center: rear/nuclear peak caveolin-1 intensity ratio (N>11 cells/condition, 3 repeats); right: paired trap force measurements at the front and rear of cells expressing CFP Ezrin-CA on durotactic gradients (N=10 cells, 3 repeats).

^∗∗p < 0.01; ^∗∗∗∗p < 0.0001; ns, not significant. See also Figure S3 and Video S5.