(A) Total internal reflection fluorescence (TIRF) microscopy of hTERT-RPE-1 under confinement stably expressing LifeAct-mCherry. Representative images of n = 11 cells. Time = 0 s at anaphase onset, as determined in phase contrast images of the same cell. Dashed yellow line indicates position of kymograph shown in (B), whereby yellow arrow indicates onset of lateral cortex displacement toward equator. (C-F) Laser microsurgery in live hTERT-RPE-1 cells grown in confinement microchambers, at different stages and orientations as illustrated in the schematics. Cells stably expressing LifeAct-mCherry were cut at their bottom actin cortex along a 5 μm linear path using a pulsed 915 nm laser as indicated by the dashed lines. Red indicates cutting perpendicular to the cell equator circumference, blue indicates parallel cutting orientation. (G) Quantification of cortex tension based on the initial cortical outward movement within 0.9 s after laser cutting perpendicular to the cell equator, for different mitotic stages as indicated. Dots represent individual cells, bars indicate median (metaphase: n = 9, pre-ingression: n = 10, early ingression: n = 15, late ingression: n = 14). (H) Cortical tension quantification for laser cutting parallel to the cell equator (metaphase: n = 8, pre-ingression: n = 9, early ingression: n = 15, late ingression: n = 4). (I) Laser microsurgery as in (C-F), but combined with fluorescence polarization microscopy. Dots represent mean normalized emission ratio values in individual cells measured adjacent to the cutting region before and after laser cutting (metaphase: n = 8, early-ingression: n = 12, late ingression: n = 8; **p<0.0021, n.s. >0.05 by MannWhitney-U test). (J) The increase in initial outward movement in perpendicular cutting experiments (G) correlates with an increase in normalized emission ratio (I). Dots represent mean, bars represent s.e.m. (normalized emission ratio, metaphase = 8, early ingression = 12, late ingression = 8; Initial outward movement, metaphase = 9, early ingression = 15, late ingression = 14). Scale bars = 10 µm.