Fig 1: Microtubule crosslinker PRC1 and nucleator augmin regulate spindle twist(A) First row, spindles in a non-transfected HeLa cell line stained with SiR-tubulin (first 2 spindles) and HeLa-Kyoto BAC cells expressing PRC1-GFP (last 3 spindles), after depletion of PRC1 and subunits of the augmin complex HAUS6 or HAUS8. Second row, images of spindles in hTERT-RPE1 cells expressing CENP-A-GFP and centrin1-GFP after perturbations of PRC1 and depletions of HAUS6 or HAUS8, as indicated. Color-coding for depth as in Figure 1B (see color bar), except SiR-tubulin is shown in non-transfected HeLa cells. In RPE1 spindles, gray represents SiR-tubulin, except in the cell with overexpressed PRC1 that shows PRC1-mCherry. Scale bars, 1 μm. Additional examples are given in Figures S4 and S5. Third row, the schemes showing the localization of PRC1 and augmin in the spindle. See also Video S8.(B) Spindle twist after perturbations of PRC1 and augmin in HeLa cells. Left, visual assessment of twist; right, twist calculated with the optical flow method; legend as in Figure 1D. The black dots denote the cell line/staining used for the corresponding treatment. On the right, the one-way ANOVA test showed a significant difference between group means (p = 8.06 × 10−5); numbers below the data show p values (Tukey’s HSD post hoc test); non-significant differences are not shown; the encircled dots represent cells shown on images. The experiments were performed on non-transfected HeLa cells (for the depletion of PRC1 and its control) and HeLa-Kyoto BAC cells expressing PRC1-GFP (for the depletion of HAUS6 and HAUS8 and their controls). Immunofluorescence after protein perturbations is shown in Figure S3A.(C) Spindle twist after perturbations of PRC1 and augmin in RPE1 cells expressing CENP-A-GFP and centrin1-GFP; legend as in (B). One-way ANOVA test showed a significant difference between group means (p = 1.72 × 10−9); immunofluorescence is shown in Figure S3B.