MCF10A spindles exhibit high baseline twist that peaks in late metaphase and anaphase. (A) Schematic diagram of spindle twist quantification, using the method developed by Trupinic et al. (2022). Three-dimensional image stacks were rotated to view the spindle along the pole-to-pole axis. Optical flow was computed between successive frames, and flow vectors were converted to polar coordinates and averaged for each spindle (see Materials and methods). (B) Spindle helicity (average degrees rotated around the pole-to-pole axis per µm displacement along the pole-to-pole axis) at anaphase in three human epithelial (RPE1, MCF10A) or epithelial-like (U2OS) cell lines, calculated from GFP-α-tubulin or SiR-tubulin intensity. Negative values represent left-handed helicity, and positive values represent right-handed helicity. Black lines represent mean ± SD. n = 19, 27, 50, and 51 spindles pooled from N = 2, 4, 5, and 5 independent experiments for RPE1 GFP-tub, U2OS GFP-tub, MCF10A GFP-tub, and MCF10A SiR-tub, respectively. n.s. not significant, ****P = 1.50 × 10−9 (MCF10A GFP-tub) and P = 1.11 × 10−8 (MCF10A SiR-tub), one-sample t tests comparing each sample to a mean of 0. (C) Lattice light sheet images of the same MCF10A cell, labeled with SiR-tubulin, at four different timepoints (related to Video 1). The xy view (center) shows maximum intensity projections of the entire spindle region. The yz view (right) shows maximum intensity projections between 30% and 70% of the pole-to-pole axis for the same image volumes after rotating them by 90°. Colors indicate directions of Farnebäck optical flow vectors, according to the color legend shown in the top image. Scale bars = 3 µm. (D) Length (upper panel) and helicity (lower panel) over time of 12 MCF10A spindles, calculated from time-lapse lattice light sheet images. The center line and shaded region represent the mean and 95% confidence interval.
