Figure 3.
NuMA’s coiled-coil is required for the formation of a single nucleus and modulates its mobility in the nucleus. (A) Schematic representations of FL NuMA and truncations NuMA-Bonsai and NuMA-NC, with amino acid numbers indicated. (B) Representative immunofluorescence images of nuclear morphologies observed in uninduced NuMA KO RPE1 cells (control), NuMA KO cells without exogenous constructs (–, two examples), and NuMA KO cells transiently expressing the rescue constructs NuMA-FL-EGFP or NuMA-Bonsai-EGFP. Cells were stained for tubulin (not shown), DNA (Hoechst), GFP, and NuMA. Scale bar, 5 µm. (C) Percentage of cells with different nuclear morphologies observed in experiment from B. n = 38 (control), 35 (NuMA KO), 21 (NuMA KO + NuMA-FL-EGFP), and 18 (NuMA KO + NuMA-Bonsai-EGFP) cells. These rescues were repeated one more time, with similar results (not shown). Two-sided Fisher’s exact test: ***, P < 0.0006; *, P = 0.04; ns, nonsignificant. (D and E) FRAP of NuMA-FL-EGFP, NuMA-Bonsai-EGFP, and NuMA-NC-EGFP in the nucleus of uninduced NuMA KO RPE1 cells. GFP intensity was measured in the bleached area (red circle) and in a nonbleached area (blue circle) to account for photobleaching. Scale bar, 10 µm. Time in minutes:seconds; 0:00 indicates the time of bleaching. n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP) cells. (F) Distribution of the fast and slow recovery halftimes (t1/2) of the different GFP-tagged NuMA proteins during FRAP. Plot shows mean ± SEM n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP) cells. Two-sided Mann–Whitney test: ***, P < 0.001; **, P = 0.001; ns, nonsignificant. (G) Time-lapse images showing the nucleus (dashed white line) of an uninduced NuMA KO RPE1 cell highly overexpressing NuMA-FL-EGFP and forming cable-like structures (left) and FRAP of the same cell (right). NuMA was bleached in the indicated red circle at 0:00, and only minimal GFP intensity recovered by 39 s. Time in minutes:seconds. Scale bar, 5 µm. See also Video 3. Fragm., fragmented; and MN, micronuclei.

NuMA’s coiled-coil is required for the formation of a single nucleus and modulates its mobility in the nucleus. (A) Schematic representations of FL NuMA and truncations NuMA-Bonsai and NuMA-NC, with amino acid numbers indicated. (B) Representative immunofluorescence images of nuclear morphologies observed in uninduced NuMA KO RPE1 cells (control), NuMA KO cells without exogenous constructs (–, two examples), and NuMA KO cells transiently expressing the rescue constructs NuMA-FL-EGFP or NuMA-Bonsai-EGFP. Cells were stained for tubulin (not shown), DNA (Hoechst), GFP, and NuMA. Scale bar, 5 µm. (C) Percentage of cells with different nuclear morphologies observed in experiment from B. n = 38 (control), 35 (NuMA KO), 21 (NuMA KO + NuMA-FL-EGFP), and 18 (NuMA KO + NuMA-Bonsai-EGFP) cells. These rescues were repeated one more time, with similar results (not shown). Two-sided Fisher’s exact test: ***, P < 0.0006; *, P = 0.04; ns, nonsignificant. (D and E) FRAP of NuMA-FL-EGFP, NuMA-Bonsai-EGFP, and NuMA-NC-EGFP in the nucleus of uninduced NuMA KO RPE1 cells. GFP intensity was measured in the bleached area (red circle) and in a nonbleached area (blue circle) to account for photobleaching. Scale bar, 10 µm. Time in minutes:seconds; 0:00 indicates the time of bleaching. n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP) cells. (F) Distribution of the fast and slow recovery halftimes (t1/2) of the different GFP-tagged NuMA proteins during FRAP. Plot shows mean ± SEM n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP) cells. Two-sided Mann–Whitney test: ***, P < 0.001; **, P = 0.001; ns, nonsignificant. (G) Time-lapse images showing the nucleus (dashed white line) of an uninduced NuMA KO RPE1 cell highly overexpressing NuMA-FL-EGFP and forming cable-like structures (left) and FRAP of the same cell (right). NuMA was bleached in the indicated red circle at 0:00, and only minimal GFP intensity recovered by 39 s. Time in minutes:seconds. Scale bar, 5 µm. See also Video 3. Fragm., fragmented; and MN, micronuclei.

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