FIGURE 9:

KIF20B depletion delays and dysregulates abscission. (A, B) Example wide-field time-lapse imaging of abscission in a HeLa cell labeled with SiR-tubulin at 20× (A) and 63× (B) (arrowhead a1: abscission 1, arrowhead a2: abscission 2; filled arrow: midbody flanks, open arrow: midbody remnant). Images captured every 7.5 min (A) and every 15 min (B). Insets in B show the intact midbody and the first abscission. (C) Frequency distribution of time from anaphase to first abscission shows a similar median time (67.5 min, arrows, p_M-W = 0.13) and similar distribution shape (p_K-S = 0.6) for siLUC (black bars) and siKIF-treated cells (white bars) imaged and scored using SiR-tubulin. n = 44 siLUC cells and 41 siKIF20B cells from four experiments. (D) The median time between first and second abscissions (arrows, 7.5 min, p_M-W = 0.09) imaged with SiR-tubulin was not different in KIF20B-depleted cells (siKIF) and controls (siLUC). The distributions were not significantly different between siLUC- and sKIF-treated cells (p_K-S = 0.9). The sample is the same as in C. (E) Representative selected planes from time-lapse z-stack images of abscission using differential interference contrast (DIC) microscopy, captured every 7.5 min (arrowhead with “a” signifies abscission). Top, in this siLUC-treated cell, abscission was observed 67.5 min after anaphase onset. Bottom, in this siKIF-treated cell, abscission was observed 90 min after anaphase onset. The second abscission event was not discernible by DIC imaging. (F) Increased median time (67.5 vs. 82.5 min) and altered distribution of time to abscission after KIF20B depletion, shown in a frequency graph of time from anaphase to first abscission discerned using DIC time-lapse microscopy in siKIF- (white bars) compared with siLUC-treated cells (black bars). For distribution, *p_K-S = 0.04; for medians, *p_M-W = 0.04. n = 25 siLUC cells and 30 siKIF cells from four experiments. K-S: Kolmogorov-Smirnov test; M-W: Mann-Whitney U test. Scale bars, 10 µm.