CT order is stably maintained during interphase
To detect or exclude large-scale CT movements, we produced criss-cross stripes of photobleached chromatin in HeLa cell nuclei with GFP-tagged histone H2B at different stages of interphase ()
. In the case of large-scale movements, the stripe pattern should become destroyed. In spite of translational and rotational nuclear movements, the stripe pattern was maintained until it became invisible after 1–2 h due to the replacement of bleached H2B-GFP by unbleached H2B-GFP ( C). This experiment provided evidence for the stability of large-scale CT arrangements during this period.
Figure 2. Stability of large-scale CT arrangements during interphase of HeLa cells studied by nuclear stripe photobleaching experiments. Cross-stripe (rows A and B) or mesh-like (row C) geometrical patterns bleached into HeLa cell nuclei with GFP-tagged H2B at (more ...)
To study the question of whether major changes of CT positions in interphase nuclei may occur during more extended time periods, we made use of a scratch-replication labeling protocol with Cy3-dUTP (Schermelleh et al., 2001
). The fluorescent nucleotides, which enter the cell during S-phase, are incorporated into newly synthesized DNA for roughly 1 h. Accordingly, each CT was represented only by a fraction of chromatin foci, called ~1-Mb chromatin domains (Cremer and Cremer, 2001
), that were replicated during the labeling period. After labeling, cells were grown for 5–8 additional cell cycles. During the second and subsequent post-labeling mitoses, Cy3-labeled and unlabeled chromatids were segregated, resulting in nuclei with a steadily decreasing number of labeled CTs. Clusters of domains were assigned to represent a single CT, and the 3-D coordinates of the common intensity gravity center were calculated to represent its nuclear location. It is possible that a larger signal cluster was occasionally assigned to one CT, although it represented several neighboring CTs. Small signal clusters, which consisted of few ~1-Mb chromatin domains, possibly resulted from sister chromatid exchanges, and therefore represented subchromosomal fragments. In spite of these limitations, the calculated 3-D coordinates served our purpose, namely to distinguish whether individual CTs in HeLa cell nuclei can move to remote nuclear locations during interphase. The 3-D intensity gravity center of the GFP-tagged nuclear chromatin served as the 3-D center of the nucleus (CN). 3-D CT-CN distances, as well as 3-D CT-CT distances were measured at different time points of the total observation period.
24 cells exhibiting nuclei with 1–4 labeled CTs () were chosen for the analysis of CT movements by time-lapse confocal microscopy with observation periods covering major parts up to a complete HeLa cell cycle of 18–20 h. Evaluation periods in represent the part of the total observation period used for a quantitative analysis. Light-optical nuclear image stacks were sampled at high resolution with intervals between 6 and 30 min. The simultaneous recording of H2B-GFP–tagged chromatin allowed us to monitor translational and rotational nuclear movements as well as changes in nuclear morphology, and to measure the increase of the nuclear volume during interphase. In 12 cells, the observation of a mitotic event preceded an evaluation period that covered G1 to mid S-phase. In 8 cells, a mitotic event resulting in two inconspicuous daughter nuclei was observed after the end of the evaluation period. In these cases, the end of telophase or the beginning of prophase was used as an indicator to estimate the interphase stages, where CT movements were analyzed (), taking into account the approximate length of each cell cycle stage determined for HeLa cells (see Materials and methods). In two cases (, nucleus 10 and 11), the total observation time encompassed two mitotic events. Two cells (, nucleus 15 and 16) did not undergo mitosis during an observation period of 10 h, but showed a nuclear volume increase typical for S-phase.
Nuclei included in the evaluation of CT-CT and CT-CN distances
For a typical example,
(A–D) shows the results obtained from nucleus 3m (), which was evaluated from late G1 to late G2 over a time period of 13 h. Optical image stacks from the Cy3 and GFP channels were simultaneously recorded every 15 min. 13.5 h after the start of observation, the cell went into mitosis, yielding two inconspicuous daughter cells. During the evaluation period, the nuclear volume increased from 1,200 μm3 to 1,780 μm3 (). From a total of 52 3-D image stacks which were recorded during this time, 27 time-image stacks with intervals of 30 min were used for a quantitative analysis of CT-CN and CT-CT distances. The results of these measurements are shown in . The differences between the maximum and minimum distances, Δ(Max-Min), were taken as a measure of motility. Absolute distances fluctuated and increased slightly over time for distantly located CTs ( C). This increase was likely a passive effect caused by the increase in nuclear volumes because it was no longer noticeable after correction for the nuclear diameter ( D; see Materials and methods). Accordingly, the corrected Δ(Max-Min) values were smaller than the absolute values (compare C with D). D demonstrates that the relative positions of CTs were stably maintained during the entire evaluation period.
Figure 3. Stability of large-scale CT arrangements studied in nuclei with Cy3-labeled CTs. (A–D) Confocal time-lapse series of a HeLa cell (Table I, nucleus 3m) with replication-labeled CTs and its daughters recorded for a total observation period of 18 (more ...)
(E–H) shows nucleus 4 (), which exemplifies the analysis of CT movements covering a period of 8 h from telophase into S-phase. From a total of 80 3-D image stacks recorded at intervals of 6 min, 25 image stacks with intervals of 12 min during early G1 and of 30 min from mid G1 to early S-phase were taken for a quantitative evaluation. (G and H) provides absolute and corrected CT-CN and CT-CT distance measurements. Δ(Max-Min) values measured during early G1 were higher than the values measured from mid G1 to early S. The Δ(Max-Min) values determined for nucleus 4 during mid G1 to early S were similar to the values noted for nucleus 3m from mid G1 to late G2 (). Accordingly, more extensive CT movements can occur during early G1.
summarizes the results from 3-D distance analyses of 24 cells and confirms the findings described above for the two example nuclei. Movements of labeled CTs were locally constrained during interphase. However, Δ(Max-Min) values, which express the variability of the measured CT-CN and CT-CT distances, were significantly larger during early G1 (ranging from 0.47 to 4.44 μm for corrected CT-CT distances; 7/19 (37%) of the values were >2 μm), compared with subsequent interphase stages (0.25–2.11 μm for corrected CT-CT distances; only 1/45 (2%) of these values were >2 μm). Notably, the mobility of individual CTs during early G1 varied largely (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200211103/DC1
). From mid G1 to late G2, absolute distances increased slightly over time, reflecting the increase in nuclear volume. During early G1, a significantly larger variability was noticed for both absolute and volume-corrected distances. Furthermore, in spite of the increase in nuclear volume, we observed occasional CT-CT and CT-CN distances that became even smaller during early G1 (). These findings indicate that the more pronounced CT movements during early G1 do not simply reflect the rapid increase of nuclear volume after telophase, but rather the movements of CTs to their final nuclear locations, which are then maintained within a range of 1 μm (maximum ~2 μm) from mid G1 to late G2. This corresponds to a radius of confinement of 0.5–1 μm.
Summary of CT-CN/CT-CT distance measurements
In a further experiment, a two-color scratch-replication labeling approach was performed. During the first S-phase, mother nuclei were labeled with Cy3-dUTP. During the next S-phase, daughter nuclei were labeled with Cy5-dUTP. Segregation of labeled and unlabeled chromatids during a post-labeling growth of cells for 4 d resulted in nuclei with a subset of CTs stained in different colors (
; Video 2). During observation periods of several hours neighboring, differently colored CTs moved repeatedly closer and further apart, confirming locally constrained CT movements in the order of 1 μm. We observed only very little color mixing, indicating the separation of the bulk DNA from different CTs. However, the sensitivity of this approach was not sufficient to exclude intermingling of a fraction of chromatin loops.
Figure 4. Stability of CT neighborhood during interphase of living HeLa cells. HeLa cells were replication-labeled during S-phase of two consecutive cell cycles (first cycle, Cy3-dUTP, false color red; second cycle, Cy5-dUTP, false color green). Frames (more ...)
Changes of chromosome neighborhoods occur during mitosis
To find out whether higher order chromatin arrangements change from one cell cycle to the next, we synchronized HeLa cells in early S-phase. 5–6 h after release from the block, when most of the cells were in late S-phase or had entered G2 (
, column I), we bleached the GFP-tagged chromatin, leaving a single, contiguous zone of unbleached chromatin at one nuclear pole (, column II). The cells were then followed by time-lapse confocal microscopy through mitosis into the next G1 phase. 34 cells yielded daughter nuclei with sufficient contrast of unbleached chromatin patches, reflecting the local decondensation of unbleached chromosome segments. 10 cases were excluded, because one or both daughter cells showed nuclei with morphological abnormalities. The remaining 24 cases were analyzed in detail.
Figure 5. Large-scale CT arrangements in HeLa cell nuclei change from one cell cycle to the next. A–C show examples of live-cell confocal image series from three HeLa cells (for movie sequences, see Video 3). After bleaching of GFP-labeled chromatin, except (more ...)
Until the onset of prophase, the area of unbleached nuclear chromatin retained its location and shape at the nuclear pole. At prophase, several unbleached chromosomal segments became visible within this zone ( C, column III). Mitotic rosettes were typically arranged perpendicular to the surface on which the cells grew. In 9 rosettes, unbleached chromosome segments clustered within a single area ( A, column IV), whereas in 13 rosettes, some or most of these segments were observed in distant locations (, column IV). Two rosettes could not be evaluated, as the light-optical serial sections did not cover the unbleached segments.
In the 48 daughter cell nuclei, the degree of clustering of unbleached chromatin was scored on maximum intensity projections. 20 nuclei showed a nuclear subregion with a single cluster of unbleached chromatin patches ( A, column V and VI). These cases reflect the best restoration of CT order between the mother nucleus and its daughter nuclei. Nevertheless, the restoration was far from complete because patches of unbleached chromatin were separated by zones of bleached chromatin, suggesting that bleached chromosome segments had moved between unbleached chromosome segments. In 15 daughter cells, most unbleached chromatin patches were clustered, but some patches were located in a remote nuclear area ( B, columns V and VI). In 13 nuclei, patches of unbleached chromatin were distributed over the major part of the nucleus ( C, column VI). Controls excluded the possibility that fluorescent patches simply reflected areas of high chromatin density rather than unbleached chromosome segments. DNA staining of fixed daughter cells with propidium iodide (PI) showed no spatial correlation of intense PI signals with sites of strong GFP fluorescence. In nuclei, which were bleached completely and followed through mitosis, fluorescent patches were not detected (unpublished data). These experiments demonstrate that significant changes of CT order occur during mitosis.
In addition to the visual analysis, we developed a procedure to quantify the degree of nuclear clustering of unbleached chromatin. Pixel fractions with the 10 and 2% highest intensity values, respectively, were thresholded for a representation of the unbleached chromatin (
A). Distances between all possible pairs of pixels representing unbleached chromatin were determined and normalized to the size of the nucleus (relative distance d, see Materials and methods). Distance values were grouped into intervals of increasing relative distances. As a measure of the frequencies of pairs of pixels belonging to each interval, the radial autocorrelation function (RAC) was established (see Materials and methods and Fig. S1). (B and C) shows the average RAC calculated for both pixel fractions for the 24 mother nuclei and 48 daughter nuclei. Compared with the curve found for mother nuclei, the curve for daughter nuclei shows a decrease of smaller relative distances combined with an increase of larger relative distance values. For a nonparametrical, statistical test (see Materials and methods), median values were calculated from the mean relative distances <d> obtained for each of the 24 mother nuclei immediately after bleaching and before the onset of prophase, and for the 48 daughter nuclei. The comparison of the median values obtained for mother nuclei immediately after bleaching and before the onset of prophase showed a slight increase (4–7%). This increase was significant for the 2% highest intensity pixel fraction (P = 0.03) and suggests minor chromatin movements (). In contrast, the comparison of the median values from mother and daughter nuclei showed a marked (70–95%) and highly significant (P < 0.001) increase. Because daughter nuclei with scattered, unbleached chromatin patches might have contributed decisively to this significance level, we retested the 20 daughter nuclei showing a single cluster of unbleached chromatin patches. Still, a marked (45–60%) and highly significant difference (P < 0.001) was obtained. Our RAC analysis fully confirmed the conclusion derived from the visual inspection; the positions of unbleached chromosome segments studied in daughter nuclei differed significantly from the positions in the mother cell nuclei.
Figure 6. RAC of the distribution of unbleached chromatin in mother and daughter cell nuclei. (A) Projections of the mid-nuclear optical sections from a mother nucleus immediately after partial bleaching (I) and shortly before the onset of prophase (II), as well (more ...)
Mean relative distances of high intensity chromatin fractions in partially bleached nuclei
In conclusion, the experiments described in this section support Boveri's hypothesis that changes of chromosome neighborhoods occur when cells pass through mitosis. In the majority of cases (18 of 24), both daughter nuclei showed the same pattern of unbleached patches, suggesting that anaphase–telophase chromosome movements retain a certain level of symmetry. However, in six cases, scattering of unbleached chromatin patches was much more pronounced in one daughter nucleus than in the other (unpublished data). We suggest that different movements of unbleached sister chromatid segments during anaphase–telophase or in early G1 nuclei contributed to this apparent loss of symmetry.
Changes of CT arrangements during clonal growth
To study the variability of arrangements of specific CTs in two-cell clones from independent mitotic events, as well as between the two pairs of daughter nuclei in four-cell clones, we performed two-color 3-D FISH experiments on HeLa cell clones fixed at these stages. Painting probes for chromosomes #7 and #10 were chosen because these chromosome types were present as three free copies and not involved in translocations (unpublished data). Confocal serial sections were obtained from 12 two-cell clones and 10 four-cell clones, and projections were visually compared for translational and mirrorlike similarities of CT arrangements (for examples see and )
. Additionally, 3-D reconstructions from daughter nuclei were made, which could be freely rotated allowing the comparison of their CT arrangements from any angle ( B; Videos 4–7). In nuclei of two-cell clones, the CT arrangements showed an obvious (albeit far from perfect) symmetry in most clones, whereas different mitotic cells yielded daughter nuclei with largely different CT arrangements (). In four-cell clones, we could identify pairs of nuclei that showed a notable symmetry of their CT arrangements, whereas strong differences were noted between the two pairs (). In agreement with Boveri's findings ( B), CT arrangements in four-cell clones already differed largely.
Figure 7. CT #7 and #10 arrangements in nuclei of two-cell clones. Projections of confocal image stacks obtained after painting of chromosome #10 (visualized in red) and #7 (visualized in green). DNA counterstain, blue. A, B, and D represent daughter nuclei with (more ...)
Figure 8. CT #7 and #10 arrangements in a four-cell clone. (A) Projection of a confocal image stack through nuclei n1 to n4 after painting of chromosome #10 (visualized in red) and #7 (visualized in green). DNA counterstain, blue. (B) 3-D reconstructions of n1 (more ...)