Figure 1.
Chronological chart that summarizes a representative time-lapse experiment.
Cells were seeded in poly-D-lysine coated glass-bottom dishes and HU was added to the culture (150 µM). After 24 hours time-lapse image acquisition was started for this representative experiment. Six images (1.4 µm apart in the z-axis) were taken for both green fluorescence and phase contrast at 20 min intervals over 48 hours. Each nucleus was numbered (in parenthesis at time 0) by its position on the gridded field at the start time and is indicated by a circle in the figure. Cells with a micronucleus (µ) or buds (b) are noted in appropriate circles. Mitosis is marked as a rectangle, whose width corresponds to its duration. Tripolar or tetrapolar mitoses are drawn as three or four arrows emanating from the mitosis rectangles. As a result of multipolar mitosis, a multinucleated cell (surrounded by a larger oval) or a small nucleus appeared frequently. During the experiment, some cells moved out of the field (out) and some cells entered the field (in). Normal events are shown as dashed lines, whereas abnormal events are highlighted in bold lines. Ap, apoptosis; s, small nucleus; brg, chromatin bridge.
Figure 2.
Low concentrations of HU induced delay in cell cycle progression and accumulation of nuclear and mitotic abnormalities.
HeLa H2B-GFP cells were cultured in the absence or presence of 150 µM (A, B) or other indicated concentrations of HU (C, D). From representative time-lapse observations (Figs. 1, S1, S2, S3, S4), the length of interphase (from the completion of mitosis to the initiation of the next mitosis; A) or the length of the mitosis (prophase to telophase) was measured. A total of 135 non-treated and 72 HU-treated cells (A), and 158 non-treated and 73 HU-treated cells (B) were examined. Cells were fixed by PFA (C, D) and frequencies of mitotic cells, micronucleated cells, cells bearing nuclear buds and apoptotic cells were obtained by viewing triplicates of 1,000 cells for each sample (C). For D, frequencies of cells showing multipolar mitoses, cells bearing anaphase chromatin bridges or lagging chromatin were obtained by viewing triplicates of >150 mitotic anaphase/telophase cells for each sample. All error bars represent one standard deviation.
Figure 3.
Formation of micronuclei or nuclear buds after replication stress.
Micronuclei (indicated by arrows) were formed after formation of a chromosomal bridge between separating anaphase chromosomes (A), from a lagging chromatid that was located between anaphase chromosomes (B) or a chromatid located closer to the spindle pole (C). Both chromatin bridges and lagging chromatids can occur in a single cell (D). The nuclear bud (indicated by arrowheads) might form just after mitosis (C and D) or long after mitosis (E; 10 hours in this case). Buds can detach from the nucleus to generate micronuclei (F). Elapsed time (in hours:minutes) is shown above the images. The frequency of bridge- or lagging chromatid-generated buds or micronuclei after completion of mitosis was scored from more than 30 time-lapse movies and plotted in (G). The number of events observed within the first 72 hours after HU addition was noted.
Figure 4.
Multipolar mitosis is a frequent cause of micronuclei generation.
Representative time-lapse images for a tripolar mitosis that generated a chromatin bridge (A) or a lagging chromatid (B). By examining 349 normal and 57 multipolar mitoses, the frequency of chromatin bridge and/or lagging chromatid occurrence was scored and is summarized in (C). GFP, fluorescent channel; PC, phase contrast.
Figure 5.
Asymmetrical multipolar division generates hypoploid cells by partial genome elimination.
We followed cells produced from 39 tripolar or 16 tetrapolar mitoses and obtained the frequency of mononuclear and multinuclear cells (A). The mononuclear cells produced from the multipolar mitoses underwent apoptosis, whereas a portion (10/27) of the multinuclear cells reached the next mitoses (B). The distribution of genetic material among the multipolar mitoses can be asymmetric. Thus, in an extreme case, one pole might receive only a small amount of chromatin that resembled the lagging chromatid (C and D).
Figure 6.
Micronuclei-bearing cells frequently underwent apoptosis but not multipolar mitosis.
By examining more than 20 time-lapse movies, each of which captured more than 20 cells for over 48 hours, we followed the interphase cells bearing micronuclei and/or nuclear buds, and counted the frequency of cells that underwent apoptosis until they entered the next M phase (A) or the frequency of cells that underwent multipolar mitoses (B). The cells frequently underwent apoptosis if they contained micronuclei, buds, or especially both (A). However, the frequency of multipolar mitoses was similar between the cells with or without micronuclei or buds (B).
Figure 7.
The micronucleus was diminished or amplified after the cells passed through mitosis.
Two representative time-lapse images for the mitosis of micronucleus-bearing cells are shown in (A) and (B). In (A) the daughter cells did not bear a micronucleus (MN), whereas in (B) the daughter cells had many micronuclei. Among more than 30 time-lapse movies, we followed the cells with or without buds or micronuclei during mitoses, and recorded the frequencies of daughter cells with micronuclei or buds (C). The horizontal split of the bars between MN and buds indicates the fraction of daughter cells bearing both micronuclei and bud. In the graph, numbers of each event among the daughter cells were noted.
Figure 8.
The anaphase bridge-derived micronuclei had condensed chromatin and were devoid of nuclear lamin B.
(A) A series of time-lapse images for the H2B-GFP fluorescence shows that anaphase bridge-derived micronuclei (arrows) had condensed chromatin (c), whereas lagging chromatid-derived micronuclei had more relaxed chromatin (r; arrow head) and buds had normal condensation level (n) as the nucleus. (B, C) Among fixed wild-type HeLa cells, lamin B was detected by immunofluorescnce and DNA was counterstained with DAPI. The lagging chromatid-derived micronuclei seen in (B) (arrowhead) had both relaxed chromatin and lamin B (+), whereas the bridge-derived micronuclei had condensed chromatin that lacked lamin B (−) (see arrows in B and upper arrow in C). The lower arrow in (C) indicates a bud that was positive for lamin B stain. The frequencies of each of these cases were recorded and plotted in D and E. Data in (E) were obtained by scoring quadruplicates of more than 100 telophase cells for each sample.