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. 2018 Sep 3;9:3571. doi: 10.1038/s41467-018-06005-7

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Mitotic spindle is chiral. a STED image (single z-plane) of metaphase spindle in a live HeLa cell expressing EGFP-CENP-A and EGFP-centrin1 (both shown in magenta) (left and middle; middle panel shows traces of microtubule bundles superimposed on the image), and in a live U2OS cell expressing CENP-A-GFP (magenta) (right). Microtubules are labeled with SiR-tubulin (green). b Imaging scheme of a vertically oriented spindle. c Imaging plane of a vertical spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B (only PRC1-GFP is shown) (left); orthogonal plane of the same spindle (middle); arrows connecting starting and ending points of PRC1-GFP bundles traced upwards (right). Longer arrows roughly correspond to larger twist around the spindle axis (circle), colors show z-distance between starting and ending points, see color bar in g. d Schematic representation of the microtubule bundle helicity measurement. e Imaging scheme of a horizontally oriented spindle. f Horizontal spindle in a fixed HeLa cell expressing PRC1-GFP and mRFP-CENP-B, legend as in c. g Horizontal spindle in a fixed unlabeled HeLa cell immunostained for PRC1, with DNA stained by DAPI, legend as in c. Images in c left, and f, g middle are single planes; images in c middle, and f, g left are maximum intensity projections of five central planes. h Spindle helicity averaged over bundles for different conditions (vertical and horizontal spindles, fixed and live cells) and cell lines as indicated. Cell lines used were: HeLa cells expressing PRC1-GFP (1st, 2nd, 4th, and 5th bars), unlabeled HeLa cells immunostained for PRC1 (3rd bar), unlabeled U2OS cells immunostained for PRC1 (6th bar), U2OS cells expressing CENP-A-GFP, mCherry-α-tubulin, and photoactivatable (PA)-GFP-tubulin (7th bar). Data are representative of 4 independent experiments for unlabeled HeLa and U2OS cells immunostained for PRC1 and 3 independent experiments for all other conditions. Numbers represent the number of cells (top) and bundles (bottom). Data for individual cells are shown in Supplementary Fig. 1e. i Paper model of the spindle showing left-handed helicity of microtubule bundles and chirality of the whole spindle. Scale bars, 1 μm; error bars, s.e.m