Extended Data Figure 3: Actin clearance from the poles is independent of centrosomes and astral microtubules. | Nature

Extended Data Figure 3: Actin clearance from the poles is independent of centrosomes and astral microtubules.

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

Extended Data Figure 3

a, SOP cell imaged at metaphase and anaphase (left) (representative of three cells imaged precisely in this way), together with a kymograph of the cross-section (yellow box). Cnn indicates centrosomin. Lifeact–GFP was used to label F-actin. b, c, Fly epithelial cells were fixed and immunostained for centrosomin, tubulin and DNA. Green arrowheads indicate the presence of centrosomes in control cells. Representative images are shown (b), together with a quantification of relative centrosomin levels at the centrosome (c) for 25 cells from three control animals and for 26 cells from three AslmecD animals. A two-tailed unpaired t-test indicated that there was a significant difference in centrosomal centrosomin levels in the two cases. d, Scheme of SOP cells dividing in different orientations. A–P axis = 0° (left). Rosette plots indicate spindle axis angle measured at the onset of anaphase for 34 control cells from three animals and for 23 AslmecD cells from three animals. e, Time-lapse stills of SOP cells expressing GMA to label F-actin taken at early and mid anaphase (ana) in control (representative of 12 cells) and AslmecD (representative of 16 cells) mutant backgrounds (as shown in Fig. 1e, f), together with plot profiles (right) denoting the relative actin levels across the cell. Asterisks mark the chromosomes. f, Images show representative STLC-treated RPE-1 cells treated with or without 20 nM nocodazole (Noco) and/or RO3306 (15 cells were analysed for each condition), fixed and stained for p-ERM proteins, DNA and tubulin. g, Images in top panel show representative Mad2-depleted S2 cells treated with 25 µM colchicine and stained for F-actin (phalloidin, red in merged image), anillin (green in merged image) and Hoechst 33258 (blue in merged image), from a population of 13 cells. Similarly, the bottom panel shows images of S2 cells (representative of 13 cells) treated with colchicine, and 20 µM RO3306 to induce forced exit from mitosis, and stained for F-actin (phalloidin, red in merged image) and p-moesin (p-ERM antibody, green in merged image) and Hoechst 33258 (blue in merged image). h, i, Ratio of proximal/distal levels of cortical F-actin (h) and p-moesin (i) (refers to g). Mean is labelled in red. j, S2 cells, expressing either H2B–GFP/anillin–Cherry or Lifeact–GFP/H2B–Cherry, were imaged during mitotic exit. Representative stills and the corresponding kymographs are shown in j (equivalent to phenotype I in k). Dashed lines were used to generate the kymographs. Top panel, n = 68 cells, three experiments; bottom panel, n = 24 cells, one experiment. k, Phenotypic quantification of anillin–Cherry-expressing S2 cells treated with colchicine and forced to exit mitosis through either Mad2 depletion (as depicted in j, top panel) or through treatment with RO3306. Bar graphs depict mean and s.d. Phenotype I: DNA and cortex are polarized. Phenotype II: neither DNA nor cortex is polarized. Phenotype III: DNA is polarized but cortex is not. Mad2 RNAi, n = 68 cells, three experiments. RO3306, n = 121 cells, two experiments. In a, b, e, f, g and j, scale bars, 5 µm.

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