To study the organization and structure of the genome in the nucleus, we took the approach of labelling only the telomeres and measuring their 3D organization as indicators for chromosomal distribution. After the 3D fluorescent measurements, the data were analyzed with a programme that was developed for this study. The programme finds all the telomeres in the nucleus; their size, intensity and shape; and determines the telomeric organization inside the volume of the nucleus. One crucial property that we analyzed was the distribution of the telomeres inside the nuclear volume. We first segmented the nucleus and found the centre of each telomere. We then found the smallest convex set of polygons that contains all the telomeres (Fig. 1). This was done by using the Quickhull algorithm [27]. In most cases, we found that the volume contained by the telomeres resembles either a sphere or a flattened sphere (disk). It can be described as an ellipsoid with two similar radii (a≈b) and a different third one (c; Fig. 2). Such a shape is called a spheroid. The level of flatness of the volume occupied by the telomeres can, therefore, be described by the ratio of the two radii that are different, a (or b) and c – a/c. The larger the ratio, the more oblate (or disk-like) is the shape of the volume occupied by the telomeres, while a/c≈1 means that the volume is spherical.
The optical resolution and signal-to-noise ratio are presented in Fig. 3. The images of two neighbouring telomeres that are 1200 nm and 400 nm apart, and the corresponding intensity along the line connecting the pair, indicates the smallest telomere distance that can still be unambiguously distinguished (approximately 200 nm).
It is expected that 80 telomeres will be observed in the interphase nucleus for normal mouse cells (92 for a normal somatic human cell), however, in our measurements we were usually able to identify approximately 40 separated telomere regions in each mouse cell (50 in human cells). Similar results have been described before [23, 28]. This is probably due to neighbouring telomeres that are closer than the optical resolution (see Fig. 3), but it does not affect the analysis of the telomere distribution in the nucleus as long as the hybridization efficiency is high. This was verified by two-dimensional measurements of all the telomeres in a metaphase spread (using the same probe), where at least 90% of the telomeres are unambiguously observed (Fig. 4).
We first described the major observation of primary BALB/c mouse B lymphocytes that were studied along the cell cycle. These studies were followed by the analysis of immortalized cells. The lymphocytes were sorted according to their DNA content for the determination of the G0/G1, S or G2/M phases (see Methods).
By analyzing cell-cycle sorted primary mouse lymphocytes we found that the 3D telomere organization changes during the cell cycle. Telomeres are widely distributed throughout the nucleus in the G0/G1 and S phases with a calculated a/c ratio of 0.9 ± 0.4, which means a spherical-like volume of distribution. However, during G2, telomeres are not observed throughout the whole nucleus. Their 3D organization changes, with all the telomeres assuming a central structure that we call the telomeric disk, which has never been reported before. In this ordered structure, all the telomeres align in the centre of the nucleus as cells progress into the late G2 phase. The a/c ratio they assume is 6.0 ± 2.0, which means a very flat disk (almost a coin shape).
Typical lymphocytes from different phases are shown in Fig. 5. The a/c ratio of these cells in the G0/G1, S and G2/M phases is 0.8, 0.8 and 6, respectively, and clearly shows the correlation of the a/c ratio with the telomere distribution and the organization of the telomeric disk that we found in the G2 phase. The elongation of the telomeres along the Z axis (the optical axis) relative to the XY plane has the same ratio as the point spread function of our system and results from the poorer optical resolution along the optical axis. However, this has a very small effect on the shape of the whole nucleus.
Similar results have been observed in primary human lymphocytes, primary human fibroblasts and in normal human epithelial tissue (see additional file for more data). This suggests that chromosomes assume a very precise order that pre-aligns them prior to the onset of mitosis. In order to ascertain that the telomeric disk was not the result of a distorted nucleus, our analysis programme compared the telomere distribution volume and shape with that of the 4'-6-Diamidino-2-phenylindole (DAPI) – stained nucleus, and verified that the nucleus itself still had a spherical-like volume. We rarely found distorted nuclei and excluded these cells from the analysis. The nucleus shown in G2 is not fully spherical. Such a shape is expected, because when the telomeres forms a disk, it pools the chromosomes and forces them to be closer to the disk, which results in an oblate shape as well.
To further study the phase transition timing along the cell cycle we used the synchronous bromodeoxyuridine (BrdU) sorting method. The cell population was pulse-labelled with BrdU in the S phase and flow sorted. Cells were placed back into culture and sub-populations harvested at 3.5, 4, 5, 6, 7, 8, 8.5, 9 and 10 hours after labelling and sorting. The cells were then fixed for 3D analysis. A minimum of 20 cells from each of these sub-populations were measured, analyzed and divided into the following three categories: 1) nuclei with a telomeric disk; 2) cells in mitosis; 3) cells in interphase without telomeric disk and mitotic figures (evaluated as G1 cells). The cell fractions as a function of time are shown in Fig. 6. Most cells (90%) form a telomeric disk 3.5 hours after BrdU incorporation. These cells are, therefore, interpreted as cells in the G2 phase. The fraction of metaphase cells peaks at 7.5 hours (65%) and the cell fraction of interphase cells that does not have a telomeric disk (and is interpreted as being in the G1 phase) peaks at 8.5 hours (57%).
These results reveal that the telomeric disk is formed in the late G2 phase. As cells progress from G2 to M, chromosomes organize into metaphases and, therefore, the number of cells in interphase with a telomeric disk decreases. Because there is no other state of transition between telomeric disk and mitosis, we conclude that the telomeric disk is the 3D telomeric organization assumed in late G2. Thus, it is also the final stage of the interphase nucleus that permits the organization of the genetic material prior to its entry into the M phase and prior to chromosome segregation. Cells in late G2 with a telomeric disk have additional characteristic features: i) they exhibit a larger overall nuclear volume than their G1 or S phase counterparts (this increase in size was also confirmed by fluorescent activated cell sorter [FACS] analysis); and ii) they begin to show signs of early re-organization of the chromatin into partially condensed areas (as visualized using the DAPI stained image).
At the end of the M phase, we observe cells that enter into the G1 conformation of telomeres, with a wide spatial distribution of telomeres throughout a smaller nucleus.
In conclusion, this data indicates that the telomeric disk is a novel structure within the interphase nucleus in late G2 that has not been previously described. Its existence points to the fundamental importance of ordered nuclear organization at the end of G2. The telomeric disk probably assures the proper organization of chromosomes prior to mitosis and their organized segregation during mitosis. Together with information that has been previously published on telomeric dynamics [26, 28], it is tempting to speculate that telomeres take an active part in the process of chromosome organization into a unique structure, the telomeric disk, during G2. This alignment of telomeres and chromosomes would facilitate the proper subsequent organization of the chromosomes into an equatorial plane during cell division. This process may be driven by the telomeres themselves (that are free of the nuclear matrix) or through the nuclear matrix. The telomeric disk may also allow for a late G2 checkpoint.
Further work on the subject can also be performed in vivo, as has been shown by Molenaar et al. [26]. In such a way the full dynamic process can be observed, which is complementary to the single time-points that are shown in our work.
We have continued to observe the distribution of telomeres in cancer cells. Typical 3D images constructed from normal nuclei and from a Burkitt lymphoma cell line (Raji), as well as from primary mouse plasmacytoma (PCT) and primary human head and neck squamous cell carcinoma (HNSCC) stage IV (Fig. 7), show that telomeres form aggregates and thus a partially altered telomeric disk. Such telomeric aggregates are characterized by both a larger volume and larger integrated intensity than their normal non-overlapping and non-aggregated counterparts. They are not observed in normal cells. Similar results for altered telomeric organization have also been found in human neuroblastoma and colon carcinoma tumor cell lines.
In line with these concepts, oncogenic activation remodels this nuclear order and sets the stage for genomic instability as we have recently measured for conditional c-Myc deregulation. We have found that deregulated expression of c-Myc alters the 3D nuclear organization of chromosomes and telomeres, and makes genomic rearrangements topologically feasible (Chuang et al., in preparation).