Single–Drosophila embryo extract recapitulates repeated nuclear divisions and distribution of nuclei in space. (A) Schematic of the embryo extraction procedure. (B) Sequence of fluorescence microscopy images of metaphase spindles in four consecutive division cycles in embryo extract, with Jupiter-GFP–labeled microtubules and Histone 2av–mRFP-labeled DNA. Dark round areas are yolk spheres. Bar, 10 µm. (C) Cycle time as a function of the cycle number for undiluted or buffer-diluted extract at 25°C. Each data point represents one experiment. In vivo data (Foe and Alberts, 1983; Foe et al., 1993) are shown in gray for comparison. (D) Plot of the metaphase spindle length for division cycles 7–9 of spindles in extract. Data points are in gray, black dots are the mean, error bars represent SD, and the number of measured spindles is shown in brackets (eight experiments). (E and F) Time course of the quantified spindle elongation (pole-to-pole distance) and DNA separation (chromosomes or nuclei) during nuclear division in extract (E) and example images (F). Bar, 5 µm. Solid and dotted lines are the mean and SD, respectively, of 15 (red) and 11 (green) observed divisions in different experiments. Anaphase onset is time 0. At the telophase–interphase transition, duplicated centrosomes of each daughter nucleus start separating (E [dashed bold] and F [bottom]), forming new poles, whereas nuclear separation levels off (red). The horizontal dashed line indicates the interphase mean nuclear diameter. (G) Time course of microtubule aster size. Error bars represent SD of astral microtubule lengths (n ≥ 28, representative out of more than three repeats). (H) Image sequence (inverted gray values) of microtubules illustrating the cycle of aster size growth and shrinkage. Chromosomes and nuclei are schematically overlaid (red). Time is shown in minutes/seconds. Bar, 5 µm.