Actin promotes left-handed spindle twist at anaphase. (A) Confocal images of live MCF10A cells treated with 0.1% DMSO, 500 nM latrunculin A, or 25 µM blebbistatin labeled with SiR-tubulin (see also Video 3). Maximum intensity projections of a 2-µm thick low region (magenta) and a 2-µm thick high region (green) relative to the spindle midplane are overlaid. Dashed lines highlight individual microtubule bundles in each region. The helicity of each spindle is indicated in the top right. Positions of spindle poles are indicated by white circles. Scale bars = 3 µm. (B) Helicity of anaphase spindles calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 54, 43, 36, and 32 spindles pooled from N = 6, 5, 4, and 3 independent experiments for DMSO, LatA, blebbistatin, and Y27632, respectively. n.s. not significant, **P = 0.0039, one-way ANOVA with Tukey’s post-hoc test. (C) Confocal images of late anaphase MCF10A cells. 500 nM LatA and 25 µM blebbistatin treatment each inhibit cytokinetic furrow ingression. Brightfield images (upper row) represent a single z-plane and SiR-tubulin images (lower row) are maximum intensity projections of 10 µm z-stacks. Scale bars = 5 µm. (D) Helicity of anaphase RPE1 spindles, synchronized with RO-3306, calculated from GFP-tubulin intensity. The control and NuMA-KO+STLC conditions are the same cells included in a previous publication (Neahring et al., 2021), reanalyzed using the optical flow method. Black lines represent mean ± SD. n = 22, 18, and 7 spindles pooled from N = 4, 5, and 3 independent experiments for control, NuMA-KO+STLC, and NuMA-KO+STLC+LatA, respectively. **P = 9.99 × 10−4, ****P = 4.32 × 10−16 (control versus NuMA-KO+STLC) and ****P = 5.93 × 10−6 (control versus NuMA-KO+STLC+LatA), one-way ANOVA with Tukey’s post-hoc test.
