Figure 4: Height elevation of rounding mitotic MDCK cells confined by narrowly spaced micropillars depends on actomyosin cortex and hydrostatic pressure.

(a) Confocal fluorescence microscopy of MDCK cells expressing actin-mCherry (red) and H2B-eGFP (green) grown on DiD-labelled micropillar arrays (blue). Micropillars had an average distance of ≈6.8 μm and length of ≈8.2 μm. Upon entering mitosis, rounding MDCK cells travel up the micropillars until they protrude above the micropillar apices. After division, daughter cells move back between the micropillars. (b) Confocal microscopy of MDCK cells expressing actin-mCherry and H2B-eGFP grown on the micropillar array in the presence of 2 μM latrunculin A. Perturbed mitotic cells cannot travel up the micropillars and remain in space confined by micropillars. Scale bars, 6 μm. (c) Perturbations were tested using a micropillar array with ≈6.8 μm interpillar distance. Maximum heights from the substrate to the top of metaphase cells were recorded using confocal fluorescence microscopy (see Methods). The blue dashed lines give the average maximum height of non-perturbed metaphase cells on micropillar substrates and Petri dishes as references. Perturbations are categorized into those affecting the actomyosin (pink background), microtubule spindle (green background) and transporters generating a hydrostatic pressure of the mitotic cell (yellow background). Perturbants were added 20 min before cells entered prophase except for EIPA and ouabain, which were added during prophase because they inhibit the G2/M transition. Each dot or triangle represents one experimentally characterized cell. For each condition, the bar denotes the average. Perturbants are described in Supplementary Table 1.