(A) Identification of centrosomes in an extract supplemented with purified HeLa cell centrosomes. (B) Montage showing that centrosomes duplicate and promote the division of cell-like compartments. SiR-Tubulin fluorescence is shown in red. (C) Montage showing SiR-tubulin fluorescence (red), NLS-mCherry fluorescence (green), and centrosome positions (cyan) (top), and heat maps of the mitotic sources (bottom), as a function of cycle number from a typical experiment. The coloring of the heat map denotes the time at which each pixel entered mitosis relative to the earliest pixels in that cycle. See also Figure 4—video 1. (D) The number of centrosomes and adventitiously-produced nuclei per cell cycle for the experiment shown in (C). (E) Suppression of nucleus formation by aphidicolin. (F) The fraction of the mitotic sources associated with nuclei/centrosomes (orange), edges (red), centrosomes (cyan) or others (blue) as a function of cycle number. The data points are mean fractions from 18 experiments where the extracts cycled at least 10 times, and the shaded regions show the ± SEM. (G, H) The distribution of distances from mitotic sources to the nearest centrosome (G, cyan), from 19 cycles, 11 experiments, and 494 sources, and from mitotic sources to the nearest nucleus (H, orange), from 149 cycles, 21 experiments, and 3993 sources. Only those cycles with fewer than 100 centrosomes or nuclei were included in G or H, respectively. The expected random distributions (mock distribution) of distances are shown in gray. The insets show probabilities (p-values) of obtaining the observed number of mitotic sources (or more than the observed number) that are close (<100 µm) to centrosomes (G) or nuclei for the individual analyzed cycles. p-values were calculated by bootstrapping with 105 randomized source positions for each individual cycle. (I) Acceleration of the cell cycle (measured as Δcycle times) relative to the slowest 15% of the cytoplasm. These Δcycle times were calculated for the first cell cycle after the appearance of centrosomes. Data are from 17 experiments and include 74 centrosome-, five nucleus-, 103 edge-associated sources and 167 other sources. (J) Acceleration of the cell cycle (measured as Δcycle times) relative to the slowest 15% of the cytoplasm, calculated for the first cell cycle after the appearance of nuclei (for those experiments where nuclei appeared). Data are from 19 experiments and include 369 centrosome-, 28 nucleus-, 132 edge-associated and 123 other sources. (K) Montage showing SiR-tubulin (red), NLS-mCherry (green), and centrosomes (cyan) for a centrosome-supplemented extract treated with 15 µM aphidicolin. (L) The fraction of the mitotic sources associated with centrosomes (cyan), edges (red) or others (blue) as a function of cycle number for aphidicolin-treated (15 to 60 µM), centrosome-supplemented extracts. From 11 experiments where the extracts cycled at least 10 times. (M) The Δcycle times for the three classes of mitotic sources. Data are from 14 experiments and include 82 centrosome-, 121 edge-associated sources and 169 other sources.