Fig. 3: CENP-E promotes overlap bundle formation.

a Scheme of CENP-E-driven microtubule bundling. b Region around a kinetochore pair immunostained for CREST (magenta) and tubulin (white) in an untransfected HeLa cell, control (top) and after CENP-E depletion (bottom), merged channels and only tubulin. c Mean tubulin intensity at the bridging fiber center in control and CENP-E depleted cells, normalized to the cytoplasmatic anti-tubulin intensity; n = 78/10 bundles/cells (control) and 70/14 (CENP-E depletion), from 3 independent immunostainings. d Cross-section of a vertical prometaphase spindle immunostained for CREST (magenta) and CENP-E (cyan) in a HeLa-Kyoto cell expressing PRC1-GFP (white). e Line intensity plots of PRC1-GFP, CREST and CENP-E from the spindle shown in d, normalized to the corresponding maximum value. The line was drawn through the spindle cross-section center, with a thickness of 1/3 of the spindle cross-section. f Time-lapse images of a midplane of a vertical prometaphase spindle in a HeLa-Kyoto cell expressing PRC1-GFP (white), after addition of the CENP-E inhibitor GSK-923295. g Number of PRC1 segments (left) and normalized mean intensity of PRC1 segments (normalized to the intensity at t = 0, right) over time from squassh segmentation in control and GSK-923295-treated cells, as indicated. Colored surfaces around the central lines (mean) represent standard deviation, n = 9 cells from 9 independent experiments. h Horizontally oriented metaphase spindles immunostained for PRC1 (white) in a U2OS doxycycline-induced CENP-E wild-type (control, left) and T422A CENP-E mutant cell in which endogenous CENP-E was silenced (right). i Total PRC1 intensity on the spindle in cells expressing wild-type CENP-E (control, n = 59) and T422A CENP-E mutant (n = 66) from (h), from 5 immunostainings. j Horizontally oriented metaphase spindles immunostained for PRC1 (white) in an untransfected HeLa cell line (control, left) and after CENP-E overexpression (right). k Total PRC1 intensity on the spindle in control (n = 41) and CENP-E overexpression (n = 30), from 3 immunostainings. In (c, i, k), the black line shows the mean; the light and dark gray areas mark 95% confidence interval on the mean and standard deviation, respectively, and p values from a two-tailed t-test are given. All scale bars, 1 µm.