Skip to main content
. 2021 Mar 3;22(4):e51400. doi: 10.15252/embr.202051400

Figure EV5. Kinetochore–microtubule attachment stability is not absolutely required for bipolar microtubule organization.

Figure EV5

  1. Ndc80‐9D‐expressing oocytes do not exhibit increased collapse in spindle shape. Microtubules (EGFP‐Map4) and chromosomes (H2B‐mCherry) were visualized in Zp3‐Cre Ndc80f/f oocytes injected with Ndc80‐WT, Ndc80‐9D, or Ndc80‐9A. The aspect ratio (length/width) of 3D‐reconstructed spindles was measured over time (n = 18, 18, and 18 oocytes, respectively, from three independent experiments; identical to the dataset shown in Fig 4).
  2. Experimental pipeline for the quantification of microtubule organization. Oocytes were imaged with the microtubule plus‐end marker EB3‐3mEGFP (green) and the chromosome marker H2B‐mCherry (magenta) at 5 h after NEBD. The comets of EB3‐labeled microtubule plus‐ends were detected following image processing for peak enhancement and background subtraction. The comets were tracked to visualize their movements. The spindle was divided into 21 equal regions between the two spindle poles along the spindle axis, and divisions 4–6 and 16–18 were defined as polar regions. In the polar regions, comets moving toward the inside of the spindle were categorized as inward comets (magenta lines), and the others as outward comets (green lines). Inward comets were predominant in the polar regions of the spindle, which represents bipolar microtubule organization.
  3. EB3 dynamics are largely unaffected by kinetochore–microtubule stability. Zp3‐Cre Ndc80f/f oocytes expressing Ndc80‐WT, Ndc80‐9D, or Ndc80‐9A were analyzed as in (B).
  4. EB3 dynamics are not significantly affected. Total number of comets going inwards and outwards in the polar regions of the spindle (n = 14, 13, 16 oocytes from at least two independent experiments) is shown. Welch’s t‐test was performed. Boxes show 25–75 percentiles, and whiskers encompass data points within 1.5 times the interquartile range.

Data information: Scale bars, 10 μm. See also Movie EV9.

Source data are available online for this figure.