3D nuclear organization of telomeres in normal and cancerous mammalian cells

Abstract

We present the high resolution 3D imaging and analyses of all the FISH-stained telomeres in mammalian nuclei. By using either confocal microscopy or 3D high-resolution microscopy measurements followed by an adequate 3D analysis that we have developed, we analyzed the space-filling properties of all the telomeres in the nucleus. The method was applied to normal, cancerous and c-myc disrupted cells. In normal cells we have found that telomeres occupy distinct and non-overlapping telomere territories (TTs), they localize in the middle of the nucleus during G0/G1 and S phases and assemble into a central telomeric disk (TD) during G2 phase. In tumors we have found that telomeres form aggregates and do not align in a telomere disk. 3D telomere order therefore forms an important tool for cancer research and possibly diagnostics. To analyze the data, a 3D image analysis program was developed. It segments the nucleus volume, counts the spots that are found and for each spot various parameters are calculated (volume, intensity and center of gravity). Then, the 3D distribution is determined by analyzing the shape of the volume occupying the telomeres. We have found that this volume can be described as a spheroid (i.e. an ellipsoid having two axes of equal length, a=b and a third different one c) and its variation from a perfect sphere is described by the ratio a/c. This parameter provides objective criteria for analyzing the telomeres distribution and forms the basis for the study and the conclusions mentioned above. Other future analysis may include measuring the anueploidy of the genome, using the number of telomeres together with an analysis of the DAPI stained chromosomes. Typical distribution of telomeres in a mouse Pre-B lymphocyte cell line is shown in the figures. Figure 1 shows the distribution of telomeres in a normal cell and figure 2 shows the distribution in a cell upon a transient activation of MycER with 4-hydroxy-tamoxifen and exhibit alterations of its telomere organization. A large aggregate is clearly observed. The 3D shape of the DAPI counter stain clearly indicates that we are not looking at a flat nucleus, where it would be trivial that the telomeres are assembled in a disk. The fixation technique used preserves the full 3D nature of the nucleus. We will describe the experimental and analysis methods and discuss about some of our results.

Figure 1	Figure 2