Characterizing the three‐dimensional organization of telomeres

Abstract

Background: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. Methods: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4 0 ,6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter r T , which indicated whether the telomeres were in a disk (r T ) 1) or not (r T % 1). We sorted mouse lymphocyte nuclei and measured r T . We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured r T at several instances.

The significance of telomeric aggregates in the interphase nuclei of tumor cells

“…This number is indicative of the distribution of telomeres throughout the entire nuclear space of primary lymphocytes, which is roughly spherical. Similarly, in S phase, the a/c ratio is small (1.5 AE 0.2; [Vermolen et al, 2005a]). The nuclear distribution of telomeres changes when cells enter into G2: telomeres align in the center of the nucleus and form a telomeric disk [Chuang et al, 2004].…”

“…Measurements of telomere positions in the 3D space of primary mouse lymphocyte nuclei have given a precise value to telomeres in this phase of the cell cycle. The a/c ratio indicates that telomeric positions in interphase nuclei is small in G0/G1 lymphocytes, and one usually measures values of 1.4 AE 0.1 [Vermolen et al, 2005a]. This number is indicative of the distribution of telomeres throughout the entire nuclear space of primary lymphocytes, which is roughly spherical.…”

“…The nuclear distribution of telomeres changes when cells enter into G2: telomeres align in the center of the nucleus and form a telomeric disk [Chuang et al, 2004]. At this time, the a/c ratio is large due to the organization of the telomeres in a disk-like volume, and the a/c ratio measurements usually are 14 AE 2 [Vermolen et al, 2005a]. Telomere dynamics in interphase nuclei of human osteosarcoma (U2OS), human cervical carcinoma (HeLa), and mouse MS5 cells has been carefully measured by live cell imaging approaches.…”

“…The most reliable one uses constrained iterative deconvolution [Schaefer et al, 2001;Vermolen et al, 2005b]. Telomere measurements are done with a special algorithm and software package that we developed, TeloView TM [Chuang et al, 2004;Vermolen et al, 2005a]. First, the position of each telomere is identified by using a threshold.…”

“…Since some of the aggregates represent fusions, dicentric chromosomes can form. These end-to-end fused chromosomes cannot appropriately separate during cell division, but will first generate anaphase bridges and then break apart, leaving one chromosome too short (with a terminal deletion) and the other one with [Vermolen et al, 2005a]. Each point represents the copy number of a telomere that is found in the nucleus.…”