Visualizing telomere dynamics in living mammalian cells using PNA probes

Abstract

Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A¯uorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to speci®cally associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital¯uorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed signi®cant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

“…Occasionally, telomeres have been found to travel large distances within the nuclear volume and show (coincidental?) collisions (23). Our results confirm these previous findings.…”

“…Without CLEM, confocal imaging was limited to a time span of not more than 60 min, which was also experienced by other research groups (23). Because of the small fraction of the entire image occupied by (telomere) spots, the overall illumination with CLEM is low; for a typical interphase cell the light dose was roughly reduced by a factor 5.…”

“…These functions are not mutually exclusive and could very well be of importance in cycling cells. Transient telomere collisions, observed by Molenaar et al, in osteosarcoma cells (U2OS), were situated in a context of recombination because the cell type relies on the ALT mechanism for telomere maintenance (23). ECV-cells are telomerase positive cells (54).…”

“…Therefore there is no proof that these variations in telomere distribution are significant in the living cell. In a pilot study telomeres were visualized in living osteosarcoma cells with PNA probes during a period of 2 h to demonstrate a constrained diffusive motion for most telomeres, and an increased mobility of some telomeres (23). This pivotal study was a great step forward although it made use of tags with unknown effects on cellular health and photobleaching and phototoxicity (photodamage) were serious limiting factors.…”

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

“…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. Long ranging as well as short movements were observed over a time period of 20 min [Molenaar et al, 2003]. Telomere dynamics has also been observed in interphase nuclei of human keratinocytes [Ermler et al, 2004].…”