Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells

Hausmann, Michael; Wagner, Emma; Lee, Jinho; Schrock, Gerrit; Schaufler, Wladimir; Krufczik, Matthias; Papenfuß, Franziska; Port, Matthias; Bestvater, Felix; Scherthan, Harry

doi:10.1039/c7nr08145f

Cited by 50 publications

(108 citation statements)

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“…It has been shown that such foci can be separated into functionally relevant sub-foci or clusters of about the same size independent from the dose applied [23,28,29]. This has also been found for the cell nuclei analyzed here (see Figure S2).…”

Section: Resultssupporting

confidence: 78%

“…The blinking events registered above the fluorescent background are used to compute the exact positions of the individual fluorophores. Tests during former experiments [28,29,34] have revealed that the intensity of the maximum of a point signal must be at least four times higher than the background intensity to get registered as an event. The double of the average background intensity is subtracted from each pixel.…”

Section: Resultsmentioning

confidence: 99%

“…Nano-scaled analysis has reasoned several transmission electron and super-resolution light microscopic studies in order to elucidate the spatio-temporal internal organization of repair foci and their chromatin surroundings with molecular resolution [3,21,22,23,24,25,26,27,28,29]. Recently, it has been shown by super-resolution light microscopy, that γH2AX foci are built up by clusters that form nano-foci with different repair activities [23,28] and that inside these nano-foci repair proteins are well organized [23,24,29] whereas the chromatin environment is interacting in a characteristic arrangement [28,30,31]. In addition, it has been shown that after radiation exposure and DNA damaging, Alu heteroduplexes may undergo Alu/Alu recombination into a single chimeric Alu element by NHEJ [32].…”

Section: Introductionmentioning

confidence: 99%

“…During recent years, it has been demonstrated that single molecule localization microscopy (SMLM) [35] is an appropriate technique to elucidate conformations of molecular arrangements and their functional relevance in cell nuclei, cytosol, and on cell membranes [1,28,29,30,31,33,34,36,37,38]. An embodiment of SMLM [39] as being used in this article, applies standard fluorescent dyes for specific labeling that can be switched between spectral “on” and “off” states [40,41] to spatial separation of molecules (“reversible photo-bleaching”).…”

Section: Introductionmentioning

confidence: 99%

“…Approaches for a quantitative point density, distance, or cluster analysis exist for SMLM [23,28,29,33,34]. The analysis is restricted in scale to a certain order of magnitude and does not consider shape deformation.…”

Section: Introductionmentioning

confidence: 99%

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Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Hofmann

Krufczik

Heermann

et al. 2018

IJMS

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DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity.

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Section: Resultssupporting

confidence: 78%