Figure 2.
The midzone motors KIF4A and MKLP1 contribute to the anaphase spindle’s left-handed twist. (A) Schematic diagram of the midzone motors KIF4A (green), MKLP1 (blue), and Eg5 (purple) that cooperate to drive anaphase spindle elongation (gray “F” and arrows). (B) Schematic diagram of the experimental geometry of the in vitro microtubule bridge assay (see Materials and methods, not to scale). A fluorescently labeled microtubule was suspended between two beads of 2 µm diameter. A 0.51-µm diameter cargo bead was densely coated by multiple kinesins and brought onto the microtubule bridge by an optical trap (not shown) and bead motility was imaged using brightfield illumination. (C) Left: Example 3D trajectory of a kinesin-1 (K560)-coated cargo bead shows straight motility. Right: Histogram of the inverse of the helical pitch of K560-coated beads (−0.004 ± 0.112 µm−1, mean ± SD, n = 23 rotations). The left-handed helical motion was defined as negative pitch. (D) Left: Example 3D trajectory of a KIF4A-coated cargo bead shows left-handed helical motility. Right: Histogram of the inverse of the helical pitch of KIF4A-coated beads (−0.53 ± 0.16 µm−1, n = 14 rotations). (E) Helicity of anaphase spindles calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 64, 45, 46, and 36 spindles pooled from N = 6, 5, 7, and 6 independent experiments for siControl, siKIF4A, siMKLP1, and siMKLP+siKIF4A, respectively. n.s. not significant, *P = 0.042, one-way ANOVA with Tukey’s post-hoc test. (F) Confocal images of live MCF10A cells labeled with SiR-tubulin (see also Video 2). 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 (not visible in these high and low z-planes), manually assigned based on SiR-tubulin signal, are indicated by white circles. Scale bars = 3 µm.

The midzone motors KIF4A and MKLP1 contribute to the anaphase spindle’s left-handed twist. (A) Schematic diagram of the midzone motors KIF4A (green), MKLP1 (blue), and Eg5 (purple) that cooperate to drive anaphase spindle elongation (gray “F” and arrows). (B) Schematic diagram of the experimental geometry of the in vitro microtubule bridge assay (see Materials and methods, not to scale). A fluorescently labeled microtubule was suspended between two beads of 2 µm diameter. A 0.51-µm diameter cargo bead was densely coated by multiple kinesins and brought onto the microtubule bridge by an optical trap (not shown) and bead motility was imaged using brightfield illumination. (C) Left: Example 3D trajectory of a kinesin-1 (K560)-coated cargo bead shows straight motility. Right: Histogram of the inverse of the helical pitch of K560-coated beads (−0.004 ± 0.112 µm−1, mean ± SD, n = 23 rotations). The left-handed helical motion was defined as negative pitch. (D) Left: Example 3D trajectory of a KIF4A-coated cargo bead shows left-handed helical motility. Right: Histogram of the inverse of the helical pitch of KIF4A-coated beads (−0.53 ± 0.16 µm−1, n = 14 rotations). (E) Helicity of anaphase spindles calculated from SiR-tubulin intensity. Black lines represent mean ± SD. n = 64, 45, 46, and 36 spindles pooled from N = 6, 5, 7, and 6 independent experiments for siControl, siKIF4A, siMKLP1, and siMKLP+siKIF4A, respectively. n.s. not significant, *P = 0.042, one-way ANOVA with Tukey’s post-hoc test. (F) Confocal images of live MCF10A cells labeled with SiR-tubulin (see also Video 2). 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 (not visible in these high and low z-planes), manually assigned based on SiR-tubulin signal, are indicated by white circles. Scale bars = 3 µm.

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