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. 2021 May 3;56(9):1253–1267.e10. doi: 10.1016/j.devcel.2021.04.005

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© 2021 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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KIF4A depletion and EG5 inhibition does not impact the number or dynamic properties of midzone and astral microtubules

(A) Live-cell images of control RPE-1 cells expressing EB3-eGFP and H2B-mCherry showing schematically the definition of two astral regions, and the midzone region between H2B-labeled chromosomes (top) and the same cell with tracked EB3 spots (magenta circles) using TrackMate ImageJ tool (bottom).

(B) Live-cell images of RPE-1 cells expressing EB3-eGFP in control, KIF4A siRNA depletion and KIF4A siRNA depletion and 40-μM STLC treatment in early anaphase (top row) and late anaphase (bottom row). Images are maximum temporal projections of color-coded time points as shown on the scheme generated from a total of 1 min of early and late anaphase time frames.

(C) Live-cell images (top) and corresponding schemes (bottom) of RPE-1 cells in mid anaphase (3 min from the onset) and telophase (15 min from the onset) after KIF4A depletion and EG5 inhibition. Dashed lines indicate the cell borders.

(D) Graphs depicting change in the number of EB3 spots in astral regions (first), number of EB3 spots contacting the cortical area (second), number of EB3 spots per micron of midzone (third), and ratio of the number of EB3 spots in astral versus midzone arrays (fourth) in time in both control (n = 12 cells from two independent experiments) and KIF4A-depleted STLC-treated spindles (n = 6 cells from two independent experiments). Shaded areas represent the 95% confidence interval of the mean and thick lines represent the mean values for both indicated treatments.

(E) Whole spindle kymographs of EB3-GFP (gray) and H2B-mCherry-expressing (magenta) cells during 10 min of anaphase in the indicated treatments.

(F) Quantification of EB3 comet velocity (left) and EB3 comet track length (right) in the indicated treatments (n = 3 cells for KIF4A depletion from two independent experiments, other treatments are the same as in D; the number of comet traces is 80, 35, and 28 from left to right). Statistics: t test. Horizontal scale bars, 5 μm (in E, 1 μm). Vertical scale bar, 1 min. See also Figure S6.