High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

Reymann, Jürgen; Baddeley, David; Gunkel, Manuel; Lemmer, Paul; Stadter, Werner; Jegou, Thibaud; Rippe, Karsten; Cremer, Christoph; Birk, Udo

doi:10.1007/s10577-008-1238-2

Cited by 68 publications

(62 citation statements)

References 40 publications

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“…Because of the diffraction-limited resolution of the optical system used here, the true dimensions of the array are smaller. This is inferred from comparitive measurements of array sizes by spatially modulated illumination microscopy (Reymann et al, 2008). On the other hand the chromatin density of the lacO arrays appears to be above average.…”

Section: Characterization Of U2os Cell Lines With Integrated Laco Arraysmentioning

confidence: 89%

Dynamics of Telomeres and Promyelocytic Leukemia Nuclear Bodies in a Telomerase-negative Human Cell Line

Jegou

Chung²,

Heuvelman³

et al. 2009

MBoC

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121

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Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism.This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs. INTRODUCTIONTelomeres are specialized chromatin structures at the end of linear chromosomes, in which repetitive DNA sequences (5Ј-TTAGGG-3Ј in vertebrates) associate into a nucleoprotein complex (de Lange et al., 2006). This complex-the "telosome"-protects the chromosome ends from degradation and genomic rearrangement and includes the "shelterin" proteins TRF1, TRF2, and POT1 that directly recognize the TTAGGG repeat sequence (de Lange, 2005;Bertuch and Lundblad, 2006;Croy and Wuttke, 2006;Blasco, 2007). Because of incomplete DNA synthesis at the chromosome ends, 50 -200 base pairs of telomeric DNA are lost during each replication cycle (Harley et al., 1990;Martens et al., 2000). After ϳ60 -80 cell divisions telomere repeats are shortened from a typical initial length of 10 -15 kb in human cells to ϳ5 kb and below, which triggers cell senescence or apoptosis (Harley et al., 1990;Martens et al., 2000;Blasco, 2007). Accordingly, tumor cells need to compensate the loss of their telomere repeats in order to sustain an unlimited proliferative potential. In most cases this is accomplished by reactivating telomerase, a reverse transcriptase that synthesizes telomeric repeats at the chromosome ends (Greider and Blackburn, 1985;Chan and Blackburn, 2004;Collado et al., 2007;Johnson and Broccoli, 2007). However, a fraction of ϳ10 -15% of tumors is able to maintain their telomeres in the absence of telomerase activity. This process has been designated as alternative lengthening of telomeres (ALT; Bryan et al., 1995;Neumann and Reddel, 2006;Johnson and Broccoli, 2007). In yeast and mammals, it has been shown that the ALT mechanism involves homologous recombination events between telomere repeats (Dunham et al., 2000;Kass-Eisler and Greider, 2000;Lundblad, 2002;Muntoni and Reddel, 2005). It is characte...

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Section: Characterization Of U2os Cell Lines With Integrated Laco Arraysmentioning

confidence: 89%

Dynamics of Telomeres and Promyelocytic Leukemia Nuclear Bodies in a Telomerase-negative Human Cell Line

Jegou

Chung²,

Heuvelman³

et al. 2009

MBoC

Self Cite

121

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“…Because only a fraction of molecules will actually fluoresce at a given time, their precise location can be determined and a reconstructed superresolved image can be produced. In this work, we use a variation of SMLM called spectral precision determination microscopy (20,21) that allows for the use of conventional fluorophores, which enabled us to investigate chromatin organization via localizing DNA and particular histone modifications along the SC.…”

Section: Significancementioning

confidence: 99%

Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

Prakash

Fournier

Redl

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.

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“…In a previous study, we have shown that localisation microscopy (SPDM) can be accomplished with conventional fluorochromes such as Alexa dyes or fluorescein derivatives (Reymann et al 2008;Lemmer et al 2009;Cremer et al 2010). Besides their application in immunocytochemical staining methods, these dyes are widely used to specifically label chromatin structures by fluorescence in situ hybridisation (for review, see .…”

Section: Discussionmentioning

confidence: 99%

“…In the recent past, however, a variety of laser-optical farfield microscopy techniques based on fluorescence excitation has been developed to overcome the 'Abbe-limit' of 200 nm. Some well-known methods are confocal 4Pi-Laser Scanning Microscopy (Cremer and Cremer 1978;Hell et al , 2003Hänninen et al 1995;Egner et al 2002;Bewersdorf et al 2006;Baddeley et al 2006;Lang et al 2010), structured/patterned illumination microscopy (Heintzmann and Cremer 1999;Gustafsson 2000;Baddeley et al 2007;Schermelleh et al 2008), STED microscopy (Hell and Wichmann 1994;Schrader et al 1995;Hell 2007;Schmidt et al 2008) or localisation microscopy techniques using far-field fluorescence microscopy (Cremer et al 1996Bornfleth et al 1998;van Oijen et al 1998;Edelmann et al 1999, Edelmann andCremer 2000;Esa et al 2000;Lacoste et al 2000;Schmidt et al 2000;Heilemann et al 2002;Betzig et al 2006;Hess et al 2006;Egner et al 2007;Reymann et al 2008;Lemmer et al 2008). Using these techniques, an effective optical resolution in the 10-20 nm regime has been obtained.…”

Section: Introductionmentioning

confidence: 99%

“…We demonstrated recently that reversible photobleaching can be used for super-resolution imaging (SPDM) of cellular nanostructures labelled with conventional fluorochromes such as Alexa dyes (Reymann et al 2008;Baddeley et al 2009) or fluorescent proteins (Lemmer et al 2008Gunkel et al 2009). This effect may be obtained by appropriate physical conditions, such as an exciting illumination intensity in the 10 kW/cm 2 to several 100 kW/cm 2 range; only one exciting wavelength is necessary both for activation and read out of a given molecule type.…”

Section: Introductionmentioning

confidence: 99%

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Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures

2010

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The optical resolution of conventional far field fluorescence light microscopy is restricted to about 200 nm laterally and 600 nm axially and has been thought for many decades to be an insurmountable barrier for the quantitative spatial analysis of cellular and hence also nuclear constituents. Novel approaches in light microscopy have now overcome this barrier. Here, we report on a special method of localisation microscopy, spectral precision distance/ position determination microscopy and its combination with fluorescence in situ hybridization to analyse the spatial distribution of specific DNA sequences in human cell nuclei at the macromolecular optical resolution level. As an example, repetitive DNA sequence DYZ2 located within the heterochromatin region on human chromosome Yq12 was labelled with clone pHY2.1. Between 300 and 700 singleprobe molecules were resolved in individual chromatin domains, corresponding to a detected molecule density around 500/μm 2 , i.e., many times higher than resolvable by conventional fluorescence microscopy. A mean localisation accuracy of about 20 nm indicated a mean optical resolution in the 50 nm range. Beyond new perspectives for light microscopic studies of specific chromatin nanostructures, this may open a new avenue towards the general analysis of copy number of specific DNA sequences in small regions of individual interphase nuclei.

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High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

Cited by 68 publications

References 40 publications

Dynamics of Telomeres and Promyelocytic Leukemia Nuclear Bodies in a Telomerase-negative Human Cell Line

Dynamics of Telomeres and Promyelocytic Leukemia Nuclear Bodies in a Telomerase-negative Human Cell Line

Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures

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