Hybrid detectors improved time-lapse confocal microscopy of PML and 53BP1 nuclear body co-localization in DNA lesions

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Authors

FOLTÁNKOVÁ Veronika MATULA Pavel SOROKIN Dmitry KOZUBEK Stanislav BÁRTOVÁ Eva

Year of publication 2013
Type Article in Periodical
Magazine / Source Microscopy and Microanalysis
MU Faculty or unit

Faculty of Informatics

Citation
Web http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=8864223
Doi http://dx.doi.org/10.1017/S1431927612014353
Field Genetics and molecular biology
Keywords DNA damage;chromatin; 53BP1; PML bodies; hybrid detectors; confocal microscopy
Description We used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in gamma-irradiationinduced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes (PMTs), which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.
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