Fig. 5: Metaphase spindle length in H1299 cells depends on the pushing force generated by EB/HSET.

a Schematics of the two systems that allow endogenous EB light inactivation (π-EB1) and rapamycin-induced EB3 – HSET interaction. b Fluorescent images of π-EB1 H1299 cells with or without rapamycin treatment, light and dark blue-light activation. EB1 light inactivation induces spindle shortening, but only in cells lacking additional rapamycin activated EB/HSET system. At least five independent experiments with the same conditions independently for π-EB1 H1299 cells and wt H1299 cells were carried out. c Spindle length measured in H1299 and π-EB1 H1299 cells before (red points) and after (blue points) EB light deactivation with and without rapamycin. Gray lines connect single cell spindle length measurements before and after the blue light exposure. The boxes show mean, lower and upper quartiles. Whiskers represent ranges that fall within 1.5 times the interquartile range. Individual independent measurements are also shown. Number of measurements left to right: n = 11, 11, 26, 26, 14, 14, 14, 14. Significance was tested by Wilcoxon two-sided test, exact p values are shown. d Top data are simulations of the number of antiparallel tip/lattice interactions (red) and the area of the antiparallel lattice/lattice overlaps (blue) as the function of the pole-pole distance between asters. Points are simulations, lines are trendlines. Green is the difference between red and blue lines. e Illustration how a single minus-end directed motor and pushing microtubule tips separate microtubule asters and stabilize bipolar organization. See text for details. Source data for this figure are provided as a Source Data file.