The analysis of protein recruitment to laser microirradiation-induced DNA damage in live cells: Best practices for data analysis

Bianchini, Roberto; Kurz, Ebba U.

doi:10.1016/j.dnarep.2023.103545

Cited by 1 publication

(5 citation statements)

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“…By imaging Z-stacks, we accounted for axial movement or focus shift during the acquisition. In accordance with recently published protocols, we also corrected the image for photo bleaching [ 21 , 22 , 23 ]. Additionally we corrected for lateral movement of cells.…”

Section: Resultsmentioning

confidence: 99%

“…In this study, we used a combination of UV-A laser (355 nm) micro-irradiation to induce DNA damage and live-cell imaging to visualize DDR in different cell models with stable, endogenous expression of eGFP-tagged wild-type FUS protein. We developed a workflow to characterize and model the recruitment kinetics of FUS-eGFP in response to laser-induced DNA damage in more detail, which is technically based on similar recent reports [ 21 , 22 , 23 ]. However, we additionally aimed to develop an openly available resource for semi-automated analysis.…”

Section: Discussionmentioning

confidence: 99%

“…The herein presented workflow for image acquisition, detection of nuclei and DDS is a robust tool for processing UV laser ablation experiments. In accordance with other recently published laser micro-irradiation protocols and guidelines, we conducted photo-bleaching correction [ 21 , 22 , 23 ] and acquired images with a high framerate to resolve the rapid recruitment dynamics of our protein of interest. Additionally, we imaged Z-stacks to account for omni-directional recruitment of FUS, focus drift as well as axial movement of the region of interest (ROI), a problem which can be encountered during laser micro-irradiations [ 21 ].…”

Section: Discussionmentioning

confidence: 99%

“…In accordance with other recently published laser micro-irradiation protocols and guidelines, we conducted photo-bleaching correction [ 21 , 22 , 23 ] and acquired images with a high framerate to resolve the rapid recruitment dynamics of our protein of interest. Additionally, we imaged Z-stacks to account for omni-directional recruitment of FUS, focus drift as well as axial movement of the region of interest (ROI), a problem which can be encountered during laser micro-irradiations [ 21 ]. Our algorithm conducts stack registration to compensate lateral movement, which allows for the acquisition of videos over a longer time period, which has been a major limitation in the study of long recruitment events [ 23 ].…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we generated iPSC lines in which endogenous wild-type FUS is c-terminally tagged eGFP to visualize FUS during the early response to UV laser micro irradiation-induced DNA damage in various cell types. We additionally developed a robust workflow, to measure the kinetics of FUS protein recruitment to and dissociation from the DDS, similar to what was reported very recently [ 21 , 22 , 23 ]. Our obtained multiparametric results describe the dynamics of FUS kinetics at laser-induced DDS in a quantitative and comprehensive manner.…”

Section: Introductionmentioning

confidence: 99%

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Cell-Type-Dependent Recruitment Dynamics of FUS Protein at Laser-Induced DNA Damage Sites

Niu,

Pal,

Szewczyk

et al. 2024

IJMS

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Increased signs of DNA damage have been associated to aging and neurodegenerative diseases. DNA damage repair mechanisms are tightly regulated and involve different pathways depending on cell types and proliferative vs. postmitotic states. Amongst them, fused in sarcoma (FUS) was reported to be involved in different pathways of single- and double-strand break repair, including an early recruitment to DNA damage. FUS is a ubiquitously expressed protein, but if mutated, leads to a more or less selective motor neurodegeneration, causing amyotrophic lateral sclerosis (ALS). Of note, ALS-causing mutation leads to impaired DNA damage repair. We thus asked whether FUS recruitment dynamics differ across different cell types putatively contributing to such cell-type-specific vulnerability. For this, we generated engineered human induced pluripotent stem cells carrying wild-type FUS-eGFP and analyzed different derivatives from these, combining a laser micro-irradiation technique and a workflow to analyze the real-time process of FUS at DNA damage sites. All cells showed FUS recruitment to DNA damage sites except for hiPSC, with only 70% of cells recruiting FUS. In-depth analysis of the kinetics of FUS recruitment at DNA damage sites revealed differences among cellular types in response to laser-irradiation-induced DNA damage. Our work suggests a cell-type-dependent recruitment behavior of FUS during the DNA damage response and repair procedure. The presented workflow might be a valuable tool for studying the proteins recruited at the DNA damage site in a real-time course.

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Section: Resultsmentioning

confidence: 99%