Figure 2.
NuMA acts at the time of nuclear formation, compacting the chromosome mass at mitotic exit. (A) Representative live images of observed nuclear phenotypes after 16-18 h of nocodazole and reversine treatment (spindle-less mitosis) in 96 h NuMA KO RPE1 cells stably expressing mCherry-H2B and EGFP-Lap2β. Arrow indicates a micronucleus. Scale bar, 5 µm. (B) Percentage of cells with different nuclear morphologies before and 16-18 h after nocodazole and reversine treatment (spindle-less mitosis). All cells, and not only those that went through mitosis, were analyzed. Plot shows mean ± SD n = 476 (control) and 820 (NuMA KO) cells from five and two independent experiments, respectively. Two-sided Fisher’s exact tests: ****, P < 0.0001; *, P < 0.04. (C) Experimental design as in Fig. 1 A, except that individual cell trajectories were followed by live imaging for the 16-18 h period in nocodazole (664 nM) and reversine (control 1 µM; NuMA KO 320 nM). Mother cell, cell before mitotic entry; daughter cell, cell after mitotic exit. (D) Representative time-lapse images of control and NuMA KO RPE1 cells stably expressing mCherry-H2B (magenta) and EGFP-Lap2β (green) that were followed live through spindle-less mitosis for 16-18 h, noting nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). White arrows indicate nuclear defects. Time in hours:minutes. Scale bar, 10 µm. See also Video 1 (control) and Video 2 (NuMA KO). (E) Percentage of cells with the indicated nuclear phenotypes in uninduced control and NuMA-KO cells treated with nocodazole and reversine just before mitotic entry (mother cells) into a spindle-less mitosis and just after mitotic exit (daughter cells). Mother cells include only cells that entered mitosis with a single, round nucleus. Plot shows mean ± SD n = 508 (control) and 308 (NuMA KO) cells from five and two independent experiments, respectively. Two-sided unpaired t test: ***, P < 0.001; *, P = 0.01. (F) Expansion of the chromosome mass from the time of nuclear envelope reformation (t = 0) in control and NuMA KO cells treated with nocodazole and reversine for a spindle-less mitosis. Plot shows mean ± SEM n = 5 cells in each condition. Two-sided Mann–Whitney test: **, P = 0.008 at 132 min. Fragm., fragmented; MN, micronuclei; Noc, nocodazole; and Rev, reversine.

NuMA acts at the time of nuclear formation, compacting the chromosome mass at mitotic exit. (A) Representative live images of observed nuclear phenotypes after 16-18 h of nocodazole and reversine treatment (spindle-less mitosis) in 96 h NuMA KO RPE1 cells stably expressing mCherry-H2B and EGFP-Lap2β. Arrow indicates a micronucleus. Scale bar, 5 µm. (B) Percentage of cells with different nuclear morphologies before and 16-18 h after nocodazole and reversine treatment (spindle-less mitosis). All cells, and not only those that went through mitosis, were analyzed. Plot shows mean ± SD n = 476 (control) and 820 (NuMA KO) cells from five and two independent experiments, respectively. Two-sided Fisher’s exact tests: ****, P < 0.0001; *, P < 0.04. (C) Experimental design as in Fig. 1 A, except that individual cell trajectories were followed by live imaging for the 16-18 h period in nocodazole (664 nM) and reversine (control 1 µM; NuMA KO 320 nM). Mother cell, cell before mitotic entry; daughter cell, cell after mitotic exit. (D) Representative time-lapse images of control and NuMA KO RPE1 cells stably expressing mCherry-H2B (magenta) and EGFP-Lap2β (green) that were followed live through spindle-less mitosis for 16-18 h, noting nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). White arrows indicate nuclear defects. Time in hours:minutes. Scale bar, 10 µm. See also Video 1 (control) and Video 2 (NuMA KO). (E) Percentage of cells with the indicated nuclear phenotypes in uninduced control and NuMA-KO cells treated with nocodazole and reversine just before mitotic entry (mother cells) into a spindle-less mitosis and just after mitotic exit (daughter cells). Mother cells include only cells that entered mitosis with a single, round nucleus. Plot shows mean ± SD n = 508 (control) and 308 (NuMA KO) cells from five and two independent experiments, respectively. Two-sided unpaired t test: ***, P < 0.001; *, P = 0.01. (F) Expansion of the chromosome mass from the time of nuclear envelope reformation (t = 0) in control and NuMA KO cells treated with nocodazole and reversine for a spindle-less mitosis. Plot shows mean ± SEM n = 5 cells in each condition. Two-sided Mann–Whitney test: **, P = 0.008 at 132 min. Fragm., fragmented; MN, micronuclei; Noc, nocodazole; and Rev, reversine.

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