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Hybrid Detectors Improved Time-Lapse Confocal Microscopy of PML and 53BP1 Nuclear Body Colocalization in DNA Lesions

Published online by Cambridge University Press:  15 February 2013

Veronika Foltánková
Affiliation:
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ-612 65, Brno, Czech Republic
Pavel Matula
Affiliation:
Faculty of Informatics, Masaryk University, Brno, Botanická 68a, 602 00, Brno, Czech Republic
Dmitry Sorokin
Affiliation:
Faculty of Informatics, Masaryk University, Brno, Botanická 68a, 602 00, Brno, Czech Republic
Stanislav Kozubek
Affiliation:
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ-612 65, Brno, Czech Republic
Eva Bártová*
Affiliation:
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ-612 65, Brno, Czech Republic
*
*Corresponding author. E-mail: bartova@ibp.cz
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Abstract

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 γ-irradiation-induced 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, 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.

Type
Biological Applications
Copyright
Copyright © Microscopy Society of America 2013

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