Confinement spatially regulates RhoA/myosin-II dependent contractility. (a) Donor fluorescence lifetime of RhoA activity biosensor on 2D and in confinement, as measured by FLIM-FRET (n ≥ 25 cells from three independent experiments). (b) Heat map of RhoA activity biosensor in representative cells on 2D or in confinement, as imaged by FLIM-FRET (scale bars, 10 µm). DIC, differential interference contrast. (c) Schematic (left) depicting the front, perinuclear, and rear cell regions and spatial distribution of RhoA activity inside confined microchannels (right) as measured by FLIM-FRET (n ≥ 25 cells from three independent experiments). (d) Representative image (left) and quantification (right) of the average intensity of MIIA and MIIB across the cell length of confined cells, as visualized by MIIA-GFP and MIIB-mCherry (quantification from 16 cells). Scale bar, 10 µm. (e) Super-resolution images of the perinuclear actin and myosin-IIA of a representative HT-1080 cell (out of 10 cells analyzed) expressing myosin-IIA–GFP and stained for actin phalloidin (red) and Hoechst (blue) inside a confined channel. (i) XY plane, apical surface; (ii) orthogonal view, XZ plane. Arrows indicate regions of nuclear deformation. Scale bars, 5 µm. Data represent the mean ± SD. **, P < 0.01 relative to 2D; ##, P < 0.01 relative to confined front/rear.