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

Abstract

Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics. ' 2008 International Society for Advancement of Cytometry Key termstelomere; TRF1; TRF2; nuclear organization; chromatin dynamics; CLEM; live cell imaging TELOMERES are the natural ends of linear chromosomes. A mammalian telomere consists of a double stranded array of simple TTAGGG repeats ending in a single stranded overhang that folds back to form a T-loop structure (1). In combination with sufficient telomere repeats, a complex of indirect and direct telomere binding proteins, dubbed shelterin, assures proper telomere structure and function. The specificity of shelterin for telomeric DNA is due to the recognition of TTAGGG repeats by three of its components: the homodimers telomeric repeat binding factor 1 and 2 (TRF1 and TRF2) that bind the duplex part of telomeres, and protection of telomeres 1 that binds to the single stranded TTAGGG repeats present at the three-overhang and in the D loop of the T-loop configuration (2-5).The nucleus has a meticulous organization with functional relevance to the cell assuring proper gene expression, replication and genome stability (6-8). Telomeres are considered to play an important role in spatial genome organization. Whereas yeast telomeres are known to form few large silencing clusters at the heterochromatic nuclear periphery (9,10), there is no consensus on how telomeres are organized in the mammalian nucleus. From an architectural point of view, human telomeres have been proposed to be anchored to a nuclear scaffold, thereby possibly providing struc-

Three-Dimensional Telomere Dynamics in Follicular Thyroid Cancer

“…Of the expected 160 telomeres of mouse chromosomes (four signals per chromosome), 3D telomere FISH and subsequent quantitative analysis using TeloView detects on average 40 separated telomeres in whole interphase nuclei of cultured cells with complete hybridization efficiency as shown in 2D FISH metaphase spreads (30,31). This number is equivalent to previously published findings (33)(34)(35) Table 2). Thus, we considered nuclear segments in a 5 lm thick section of thyroid tissue having more than 30 telomeric signals as potentially being aneuploid and nuclear segments having more than 40 signals as potentially being near tetraploid or tetraploid.…”

Light exposure and cell viability in fluorescence microscopy

“…laser scanning) microscopy by nonuniform illumination, where excitation light was adapted to the local concentration of fluorophores (Hoebe et al, 2007, de Vos et al, 2009). In particular, by using an electronic feedback system, illumination was reduced in regions of high fluorescence as well as in the background between fluorophores.…”

3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost' nuclei and very short telomeres

“…39 There is growing evidence that the integrity of nuclear chromosome territories is essential for successful mitosis. 40,41 With that in mind, it will be hard to imagine that trinuclear LMP1-positive RS cells (as shown in Figure 5) composed of a first nucleus with a few short telomeres, a second telomerefree 'ghost' nucleus and a third micronucleus with several midsized telomeres or aggregates, will be able to get through the next round of successful karyokinesis and thus may represent true end-stage tumor cells. Thus, our findings in ND ¼ not done (see Table 2).…”