Extended Data Figure 2: Depletion of RacGAP1 impairs myosin II equatorial accumulation and furrow ingression, but does not affect actin clearance from the poles at mid anaphase. | Nature

Extended Data Figure 2: Depletion of RacGAP1 impairs myosin II equatorial accumulation and furrow ingression, but does not affect actin clearance from the poles at mid anaphase.

From: Kinetochore-localized PP1–Sds22 couples chromosome segregation to polar relaxation

Extended Data Figure 2

a, Western blot showing RacGAP1 depletion in HeLa cells. b, Representative DIC stills from movies show HeLa cells at indicated times after the onset of anaphase. Images show control cells before and after furrow initiation and RacGAP1-depleted cells at mid anaphase, as all fail cytokinesis. Arrowheads point to blebs. c, d, Graphs show levels of cortical myosin II (c) and cortical actin (d) in the polar and equatorial regions of cells. Levels were measured in HeLa cells expressing myosin-II–GFP and Utrophin–Ruby treated with control siRNA (15 cells, 3 experiments) or siRacGAP1 siRNA (oligonucleotide no. 4, seen in a) (15 cells, 3 experiments). Data are shown as mean and s.d. e, f, Representative images and corresponding kymographs taken from 12 time-lapse movies of fly SOP cells, fluorescently labelled for both myosin II (Sqh–mCherry) and actin filaments (GMA), undergoing anaphase. Note that the same cells were used for the analysis in Extended Data Fig. 9a. Anaphase onset = 0 s. Asterisks mark the chromosomes. Kymographs of anaphase progression of the E–P perimeter section depicted in e. Note that actin and myosin II have distinct patterns of localization at the cortex during anaphase (also, see white arrowheads in e). g, Fly epithelial cell at mid anaphase immunostained for tubulin, DNA, p-myosin II and F-actin (phalloidin), representative of three cells. h, Fly epithelial cell at mid anaphase immunostained for p-moesin and DNA, representative of 15 cells. Scale bars in b, e, g, h, 5 µm. A two-tailed unpaired t-test was used to calculate statistical significance.

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