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

Schmalz, Michael; Wieser, Ines; Schindler, Felix; Czada, Christina; Leitenstorfer, Alfred; Ferrando‐May, Elisa

doi:10.1364/ol.43.002877

Cited by 7 publications

(4 citation statements)

References 16 publications

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“…Twenty-four to forty hours after transfection, medium was replaced by phenolred-free medium for microscopic analysis. Microirradiation and subsequent time-lapse imaging was performed using an inverted laser-scanning microscope (Zeiss LSM700) equipped with a multi-color femtosecond fiber laser setup ( 66 ). In-situ bandwidth-limited optical pulses with a center wavelength of 1035 nm were focused onto the sample using a 63× oil immersion objective lens (N.A.…”

Section: Methodsmentioning

confidence: 99%

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response

Aberle

Krüger

Reber

et al. 2020

Nucleic Acids Research

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Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a ‘PAR code’.

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Section: Methodsmentioning

confidence: 99%

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response

Aberle

Krüger

Reber

et al. 2020

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…For microscopic analysis, cell culture medium was replaced by phenolred-free medium 24 to 40 h after transfection. A Zeiss LSM700 inverted laser-scanning microscope, equipped with a multi-color femtosecond fiber laser setup was employed for microirradiation and following time-lapse imaging (Schmalz et al 2018 ). In situ bandwidth-limited optical pulses (center wavelength 1035 nm) were focused onto the sample with a 63 × oil immersion objective lens (N.A.…”

Section: Aterial and Methodsmentioning

confidence: 99%

PARP1 and XRCC1 exhibit a reciprocal relationship in genotoxic stress response

et al. 2022

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PARP1 (aka ARTD1) acts as a prime sensor of cellular genotoxic stress response. PARP1 detects DNA strand breaks and subsequently catalyzes the formation of poly(ADP-ribose) (PAR), which leads to the recruitment of the scaffold protein XRCC1 during base excision and single strand break repair and the assembly of multi-protein complexes to promote DNA repair. Here, we reveal that the recruitment of either protein to sites of DNA damage is impeded in the absence of the other, indicating a strong reciprocal relationship between the two DNA repair factors during genotoxic stress response. We further analyzed several cellular and molecular endpoints in HeLa PARP1 KO, XRCC1 KO, and PARP1/XRCC1 double KO (DKO) cells after genotoxic treatments, i.e., PARylation response, NAD+ levels, clonogenic survival, cell cycle progression, cell death, and DNA repair. The analysis of NAD+ levels and cytotoxicity after treatment with the topoisomerase I inhibitor camptothecin revealed a hypersensitivity phenotype of XRCC1 KO cells compared to PARP1 KO cells—an effect that could be rescued by the additional genetic deletion of PARP1 as well as by pharmacological PARP inhibition. Moreover, impaired repair of hydrogen peroxide and CPT-induced DNA damage in XRCC1 KO cells could be partially rescued by additional deletion of PARP1. Our results therefore highlight important reciprocal regulatory functions of XRCC1 and PARP1 during genotoxic stress response.

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“…Another interesting example of the potential of LS for DDR study has been recently reported. In this case the authors employed lasers with three different wavelengths (515, 775, or 1035 nm) but exactly the same pulse length (80-81 fs) (Schmalz et al, 2018). Under conditions of similar average power, different alterations of the chromatin were observed in the irradiated cells.…”

Section: Dna Damage Response and Nuclear Perturbationmentioning

confidence: 99%

“…Therefore, by judiciously choosing the laser parameters (wavelength, pulse duration, power, etc.) it is possible to carry out relevant research on redox cell modulation with OT-like setups ( Westberg et al, 2016 ; Linz et al, 2016 ; Schmalz et al, 2018 ; Gomez-Godinez et al, 2020 ).…”

Section: Perspectivesmentioning

confidence: 99%

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

Blázquez‐Castro

Fernández‐Piqueras

Santos

2020

Front. Bioeng. Biotechnol.

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Light can be employed as a tool to alter and manipulate matter in many ways. An example has been the implementation of optical trapping, the so called optical tweezers, in which light can hold and move small objects with 3D control. Of interest for the Life Sciences and Biotechnology is the fact that biological objects in the size range from tens of nanometers to hundreds of microns can be precisely manipulated through this technology. In particular, it has been shown possible to optically trap and move genetic material (DNA and chromatin) using optical tweezers. Also, these biological entities can be severed, rearranged and reconstructed by the combined use of laser scissors and optical tweezers. In this review, the background, current state and future possibilities of optical tweezers and laser scissors to manipulate, rearrange and alter genetic material (DNA, chromatin and chromosomes) will be presented. Sources of undesirable effects by the optical procedure and measures to avoid them will be discussed. In addition, first tentative approaches at cellular-level genetic and organelle surgery, in which genetic material or DNA-carrying organelles are extracted out or introduced into cells, will be presented.

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

Cited by 7 publications

References 16 publications

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response

PARP1 and XRCC1 exhibit a reciprocal relationship in genotoxic stress response

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

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