The three-dimensional organization of telomeres in the nucleus of mammalian cells

Abstract

Background: The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells.

Results: We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed.

Conclusions: The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.

Figure 1

The distribution of the telomeres in the nucleus volume is found by fitting a convex set of polygons that contains all the telomeres. This volume usually looks like either a sphere or a disk and can be described as an ellipsoid.

Figure 2

In general, the ellipsoid's main axes along x'y'z' do not coincide with the microscope-slide plane and optical axes xyz. Our programme finds an ellipsoid that contains all the telomeres and the size of its main axes a,b,c. In most of the cases the x'y' axes of the ellipsoid are similar, i.e. a≈b. Therefore, the ratio a/c is a good measure of the flatness level of the ellipsoid and of the telomere organization inside the nucleus.

Figure 3

Demonstration of the signal-to-noise and spatial resolution of our measurements. The fluorescence intensity is bright (typical signal-to-noise ratio of 10:1). Two pairs of telomeres are shown, 1200 nm apart (top), which can be easily separated, and 400 nm apart (bottom). The inserts show the actual images.

Figure 4

Metaphase plate prepared from fetal liver cells directly isolated from day 10 old mouse embryos. Metaphase chromosomes and spreads were prepared as described [30] and hybridized with a PNA-telomeric probe that was Cy3 labelled. More than 90% of the telomeres are clearly observed.

Figure 5

The distribution of telomeres in the nucleus of three typical cells selected from the G0/G1 phase (upper row), S phase (middle row) and G2/M phase (lower row). Each telomere distribution is shown from a top view (the XY plane), along the optical axis Z (left column), from a side view (XZ plane) as observed along the Y axis (centre column) and as a 3D image of the telomeres in an open nucleus (right column). When shown from the top and side views, the telomeres are displayed on top of the projected image of the nucleus. This projection demonstrates the extent of the chromatin (and therefore chromosomes) and defines the volume and borderline of the nucleus.

Figure 6

BrdU-positive cells were live sorted and synchronized in the S phase. They were harvested from a culture at time intervals of 3.5–9 hours. The cells were then fixed for 3D analysis. For each time point we have measured: 1. the fraction of nuclei with a telomeric disk; 2. the fraction of cells in mitosis; and 3. the fraction of cells with interphase nuclei but without a telomeric disk. Ninety percent of the cells formed a telomeric disk 3.5 hours after BrdU incorporation and were therefore interpreted as cells in the late G2 phase (black line and circles). Cells entering mitosis (dashed line and squares) peaked at 7.5 hours (65%) and cells in G1 (dotted line and triangles) peaked after 8.5 hours (57%). The increase in the number of metaphases at 9.5 hours cannot be explained and probably lies within the limits of experimental errors.

Figure 7

Normal: A normal blood cell; RAJI: A Burkitt lymphoma cell line; PCT: A primary mouse plasmacytoma cell; HNSCC: A primary human head and neck squamous cell carcinoma (stage IV). The distribution of telomeres in cancer cells compared with a normal cell. Images are shown as explained in Fig. 5. Aggregates of telomeres are formed and the telomere disk that appears in the G2 phase is distorted.