TY - JOUR A1 - Eryilmaz, Marion A1 - Schmitt, Eberhard A1 - Krufczik, Matthias A1 - Theda, Franziska A1 - Lee, Jin-Ho A1 - Cremer, Christoph A1 - Bestvater, Felix A1 - Schaufler, Wladimir A1 - Hausmann, Michael A1 - Hildenbrand, Georg T1 - Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines JF - Cancers N2 - In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair. KW - Krebs, Medizin KW - Strahlentherapie KW - Brustkrebs Y1 - 2018 U6 - https://doi.org/https://doi.org/10.3390/cancers10010025 SN - 2072-6694 VL - 10 IS - 1 PB - MDPI AG ER - TY - JOUR A1 - Hausmann, Michael A1 - Ilić, Nataša A1 - Pilarczyk, Götz A1 - Lee, Jin-Ho A1 - Logeswaran, Abiramy A1 - Borroni, Aurora A1 - Krufczik, Matthias A1 - Theda, Franziska A1 - Waltrich, Nadine A1 - Bestvater, Felix A1 - Hildenbrand, Georg A1 - Cremer, Christoph A1 - Blank, Michael T1 - Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research JF - International Journal of Molecular Sciences N2 - Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2017 U6 - https://doi.org/https://doi.org/10.3390/ijms18102066 SN - 1422-0067 VL - 18 IS - 10 PB - MDPI AG ER - TY - JOUR A1 - Krufczik, Matthias A1 - Sievers, Aaron A1 - Hausmann, Annkathrin A1 - Lee, Jin-Ho A1 - Hildenbrand, Georg A1 - Schaufler, Wladimir A1 - Hausmann, Michael T1 - Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure JF - International Journal of Molecular Sciences N2 - Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements—dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA–DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets. KW - Genom Y1 - 2017 U6 - https://doi.org/https://doi.org/10.3390/ijms18051005 SN - 1422-0067 VL - 18 IS - 5 PB - MDPI AG ER - TY - JOUR A1 - Pagáčová, Eva A1 - Štefančíková, Lenka A1 - Schmidt-Kaler, Franz A1 - Hildenbrand, Georg A1 - Vičar, Tomáš A1 - Depeš, Daniel A1 - Lee, Jin-Ho A1 - Bestvater, Felix A1 - Lacombe, Sandrine A1 - Porcel, Erika A1 - Roux, Stéphane A1 - Wenz, Frederik A1 - Kopecna, Olga A1 - Falková, Iva A1 - Hausmann, Michael A1 - Falk, Martin T1 - Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy JF - International Journal of Molecular Sciences N2 - From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the “physics” behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2–3 nm). Next, we introduced a novel super-resolution approach—single molecule localization microscopy (SMLM)—to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns. KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2019 U6 - https://doi.org/https://doi.org/10.3390/ijms20030588 SN - 1422-0067 VL - 20 IS - 3 PB - MDPI AG ER - TY - CHAP A1 - Hausmann, Michael A1 - Lee, Jin-Ho A1 - Sievers, Aaron A1 - Krufczik, Matthias A1 - Hildenbrand, Georg T1 - COMBinatorial Oligonucleotide FISH (COMBO-FISH) with Uniquely Binding Repetitive DNA Probes T2 - The Nucleus KW - Genom Y1 - 2020 SN - 9781071607626 U6 - https://doi.org/https://doi.org/10.1007/978-1-0716-0763-3_6 SN - 1064-3745 SP - 65 EP - 77 PB - Springer US CY - New York, NY ER - TY - JOUR A1 - Bobkova, Elizaveta A1 - Depes, Daniel A1 - Lee, Jin-Ho A1 - Jezkova, Lucie A1 - Falkova, Iva A1 - Pagacova, Eva A1 - Kopecna, Olga A1 - Zadneprianetc, Mariia A1 - Bacikova, Alena A1 - Kulikova, Elena A1 - Smirnova, Elena A1 - Bulanova, Tatiana A1 - Boreyko, Alla A1 - Krasavin, Evgeny A1 - Wenz, Frederik A1 - Bestvater, Felix A1 - Hildenbrand, Georg A1 - Hausmann, Michael A1 - Falk, Martin T1 - Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy JF - International Journal of Molecular Sciences N2 - DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10–20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site. KW - Genom KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2018 U6 - https://doi.org/https://doi.org/10.3390/ijms19123713 SN - 1422-0067 VL - 19 IS - 12 PB - MDPI AG ER - TY - GEN A1 - Lee, Jin-Ho A1 - Bobkova, Elizaveta A1 - Gier, Theresa A1 - Gote, Martin A1 - Schmidt-kaler, Fanz A1 - Brieger, Emily A1 - Maus, Emanuel A1 - Krufczik, Matthias A1 - Chojowski, Robert A1 - Korn, Friederike A1 - Sarah, Schumann A1 - Scherthan, Harry A1 - Falkova, Iva A1 - Falk, Martin A1 - Hausmann, Michael A1 - Hildenbrand, Georg T1 - Mechanisms and Challenges for Understanding Radiation Induced Changes in Chromatin Nanoarchitecture T2 - DRO 2018 N2 - The three-dimensional architecture of genomes acts as an additional level of mode for fundamental biological processes such as DNA damage response. In this context, nanoprobing and super-resolution microscopy are powerful methods for structural analyses of genomic targets in native chromatin of single cells at resolutions of single antibodies, proteins, histones, short DNA stretches, etc. We used multi-color chromatin nanoprobing and single molecule localization microscopy of established DNA damage and chromatin markers in 3D-conserved nuclei of different cell types exposed to various types and doses of ionizing radiation. Similarly, effect of gold nanoparticles on extent and direction of cellular radiation response was assessed. Our studies revealed the nanoarchitecture of damage foci with respect to γH2AX, Mre11 or 53BP1 and their molecular rearrangements during repair processes. Nanoscopy of genomic Alu resulted in linear-quadratic dose-effects for low to higher dose ranges and in changes of H3K9me3 distribution around Alu clusters upon radiation exposure. Preliminary results show post-irradiation time dependent changes in Alu chromatin. Moreover, gold nanoparticles incorporated into cells seem to act by an interplay of radiation enhancement and chromatin remodeling leading to altered radiosensitivity. Our results contribute to the understanding of cellular radiation responses, thereby laying the basis for improved biological dosimetry and radiotherapies in future. N2 - Poster KW - Genom Y1 - 2019 U6 - https://doi.org/https://doi.org/10.13140/RG.2.2.31391.20647 ER - TY - CHAP A1 - Hausmann, Michael A1 - Lee, Jin-Ho A1 - Hildenbrand, Georg T1 - 3D DNA FISH for analyses of chromatin-nuclear architecture T2 - Epigenetics Methods KW - Genom Y1 - 2020 SN - 9780128194140 U6 - https://doi.org/https://doi.org/10.1016/B978-0-12-819414-0.00020-3 SP - 399 EP - 418 PB - Elsevier ER -