Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

Abstract

Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D‐confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two‐photon photoactivation of PA‐GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time‐dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

“…In the study by Kruhlak et al, this distinction was reached by adding a photosensitizer. Recently, we have presented an alternative strategy based on the use of femtosecond laser pulses of different wavelengths (Tomas et al, 2012). In a first step, DNA strand breaks are introduced via irradiation at λ = 1050 nm in a defined subnuclear volume, as described above.…”

“…The spatial precision of non-linear excitation also enables to vary the position of the photoactivation spot with respect to the damaged region. Using this method, we could show that DNA strand breaks lead to an increase in the mobility of histone H1.2 in the time range of 2 min after infliction of damage and that this change is spatially confined because distant chromatin is not affected (Tomas et al, 2012). The spatial and temporal flexibility of this assay will enable to visualize how the chromatin response to DNA damage emanates within the cell nucleus thus contributing to dissect the spatiotemporal complexity of this fundamental biological process.…”

“…The approximation of a uniform distribution can be made only if the area in which fluorescence redistribution is measured is much smaller than the DNA damaged one, and only for short observation times during which the protein does not move outside of this region. In our assay, this condition is fulfilled since the protein of interest is photoactivated in a small spot (diameter 1 μm) within a significantly larger zone containing the damage (6 μm 2 ) (Tomas et al, 2012). These caveats concerning the temporal and spatial equilibrium criteria apply in general to all methods combining microirradiation with fluorescence photoperturbation.…”

“…It is interesting to note that in the case of histone H1, these authors do not detect subpopulations with higher and lower mobility, contrarily to what was reported previously in FRAP studies (Raghuram et al, 2010; Stasevich et al, 2010). Since the bleaching laser may induce phototoxic effects including DNA damage (Dobrucki et al, 2007; Solarczyk et al, 2012) and the mobility of the protein is increased in the presence of such lesions (Tomas et al, 2012), the partial fluorescence recovery observed in FRAP experiments can be due to a change of the protein's mobility within the bleached area leading to a local decrease in concentration rather than to the presence of an immobile fraction. These effects have to be considered when choosing experimental conditions for photoperturbation studies.…”

Highly standardized multicolor femtosecond fiber system for selective microphotomanipulation of deoxyribonucleic acid and chromatin

“…The latter feature is absolutely crucial to rule out any influence of temporal duration and spectral phase on the observed effects which may be caused by, for instance, multiphoton intrapulse interference. The choice of wavelengths was guided by previous proof-of principle studies showing that (1) fs laser pulses at λ 1050 nm induced DNA strand breaks [6], and (2) fs laser pulses at λ 750-780 nm can efficiently photoactivate green fluorescent protein (PA-GFP) via two-photon absorption [10,11]. Finally, λ 515 nm matches the two-photon absorption maximum of DNA and should thus efficiently generate pyrimidine dimers.…”