Figure 1.
Lamin B1-negative micronuclei and postmitotically-generated lamin B1 positive micronuclei were detected.
(A to D) COLO 320DM-GFP cells were fixed with PFA and the lamin B1 protein was detected by immunofluorescence. Representative confocal images of the DM-type micronucleus with lamin B1 (A; arrow) or without lamin B1 (B and C; arrow). The image of lamin B1 is shown in gray-scale, and it was shown in red in merged panels. The micronucleus in B is attached to the nucleus whereas the one in C is detached from the nucleus. In mitotic cells in telophase, the aggregated DMs were left behind the separating chromatids, and lamin B1 was detected at the rim (D; arrowheads). (E) Living COLO 320DM-GFP cells were stained with Hoechst 33342 and analyzed by time-lapse microscopy (E). DM aggregates were located separate from chromosomes at metaphase and anaphase, and generated the DM-type micronuclei after mitosis (arrowheads). Elapsed time (in hours:minutes:seconds) is shown in each images.
Figure 2.
The large cytoplasmic bleb entraps the nuclear bud/micronucleus that is devoid of lamin B1.
(A to D) COLO 320DM-GFP cells were fixed, and the lamin B1 protein was detected as in Figure 1. Representative confocal images of lamin-negative micronuclei (white arrows) connected to the nucleus by thin chromatin are shown. These micronuclei were inside the large cytoplasmic bleb. (E) The left two panels show the merged images of lamin B1 (red), DMs (green), DAPI (blue in left, gray in right) and DIC (gray in left). The rectangle region was enlarged in the right three panels showing the serial confocal images taken at 0.8 µm intervals in the Z-axis. The break in the lamin B1 envelope is indicated by a white arrow.
Figure 3.
Fresh serum induced the formation of large blebs and lamin B1-negative micronuclei.
(A) DIC images of living COLO 320DM-GFP cells are shown. The incubation time (min:sec) after the addition of fresh serum is shown in each panel. The arrows indicate the large blebs. (B) The cells were fixed with PFA and the frequency of the large blebs among the total cells was measured. (C) Cells cultured for two days in a medium containing 10% serum rarely showed the large bleb (-; “before stimulation”), whereas addition of fresh serum or treated serum at the final concentration of 10% induced extensive blebbing (+) after 10 min. (D) Cells were stimulated with fresh serum for the indicated time and fixed for detection of the lamin B protein. The frequency of the formation of the large bleb containing the micronuclei that were apart from the nuclei or the bud-shaped micronuclei that was connected to the nuclei among the total number of large blebs was calculated in 300 to 1,000 cells. The incidences among the counted blebs were noted in the graph. (E) In the same slides used in D, the frequencies of the micronuclei with/without DMs or with/without lamin B1 among the total cells were measured by examining 1,000 cells in three replicates. Error bars represent mean +/- SEM.
Figure 4.
Time-lapse microscopy indicated that nuclear budding is coupled to cytoplasmic blebbing, which generates lamin-negative micronuclei.
(A and B) The DNA in living COLO 320DM-GFP/lamin B1-mCherry cells was stained with Hoechst 33342 for time-lapse observation before and after fresh serum stimulation. The images were obtained at 3 min (A) or 5 min (B) intervals. The images corresponding to DM-GFP were omitted in A because the nuclear budding did not contain the DM-GFP signal. Elapsed time (in minutes:seconds) after the start of the experiment is shown in each image. White arrowheads indicate the cytoplasmic blebbing and nuclear budding.
Figure 5.
Induction of blebbing and budding by fresh serum was dependent on the cell line and the cell-to-substratum adherence.
(A) Several non-adherent cell lines were stimulated by fresh serum, and the generation of the large bleb was examined after 10 min. (B) HeLa cells were detached from the substratum by trypsin/EDTA and conditioned medium, and then stimulated by fresh serum. DIC images of the same microscopic field before (left) or after (right) 5 min of treatment with fresh serum are shown. The arrows indicate the large cytoplasmic bleb. (C) Several adherent cell lines were detached from the substratum as in B, stimulated with fresh serum for 3 or 10 min, and fixed. The frequency of the large bleb was scored and summarized as shown. (D and E) HeLa cells were treated with fresh serum for 10 min and fixed. Nuclear lamin B1 was detected by immunofluorescence. White arrows indicate the nuclear bud without lamin B1 that was inside the cytoplasmic large bleb. (F) The frequency of each micronuclei was assessed using the slide shown in D and E. To obtain the data in A, C and F, more than 500 cells were examined in each of three independent scoring processes at each point.
Figure 6.
CPT induced interphase micronucleation through blebbing and budding.
COLO 320DM-GFP cells were cultured in the presence of 5 µg/ml of CPT for the indicated time (A, E and F) or 3 hours (B to D) and fixed with PFA. Lamin B1 and DM-GFP (B), or lamin B1 and γH2AX (A, C to F) were simultaneously detected by immunofluorescence microscopy. (A) The frequencies of apoptotic cells and cells with large blebs among the total cells were scored and plotted. (B) The frequency of several kinds of micronuclei that were trapped in the large bleb among the cells with large blebs (85 non-treated and 246 CPT-treated) was scored and plotted. (C and D) Representative images of the large bleb trapping the lamin B-negative micronuclei. These micronuclei were either γH2AX-negative (C) or positive (D), while the neighboring nucleus was γH2AX-positive. (E) Time course of the appearance of γH2AX-positive cells. The intensities of the nuclear γH2AX-signal were classified from – to +++ according to a previous report [19]. (F) The lamin B-negative micronuclei were classified according to the distribution of the γH2AX-signal between the nucleus and the micronucleus. To obtain these data, more than 500 (A, E and F) or 1,000 (B) cells were examined in each of three independent scoring processes at each point.
Figure 7.
Cell cycle dependency and graphical summary of the blebbing and budding.
(A and B) BrdU (20 µg/ml) was added to the logarithmically growing culture of COLO 320DM-GFP cells. After 5 min, fresh serum was added at 10% concentration, and the cells were harvested 10 min later and fixed with PFA. The lamin B protein and BrdU were detected by immunofluorescence, and DMs were detected by FISH. Two representative cells showing blebbing and budding are shown. The cell cycle phase was determined by the nuclear distribution of BrdU [24]; the cell in A is non-S and the cell in B is at the early S phase. (C) The frequency of the large bleb-bearing cells among the cells at each cell cycle phase was scored, which was obtained by examining the number of cells shown in the denominator noted at each bar. (D) The frequency of the DM-positive or negative nuclear bud-bearing cells among the cells at each cell cycle phase is shown. (E) Model showing the generation of the different types of micronuclei as revealed by the findings of the study.