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• W2765764597 abstract "Abstract Heterochromatin is mostly composed of long stretches of repeated DNA sequences prone to ectopic recombination during double-strand break (DSB) repair. In Drosophila , ‘safe’ homologous recombination (HR) repair of heterochromatic DSBs relies on a striking relocalization of repair sites to the nuclear periphery. Central to understanding heterochromatin repair is the ability to investigate the 4D dynamics (movement in space and time) of repair sites. A specific challenge of these studies is preventing phototoxicity and photobleaching effects while imaging the sample over long periods of time, and with sufficient time points and Z-stacks to track repair foci over time. Here we describe an optimized approach for high-resolution live imaging of heterochromatic DSBs in Drosophila cells, with a specific emphasis on the fluorescent markers and imaging setup used to capture the motion of repair foci over long time periods. We detail approaches that minimize photobleaching and phototoxicity with a DeltaVision widefield deconvolution microscope, and image-processing techniques for signal recovery post-imaging using SoftWorX and Imaris software. We present a method to derive mean square displacement (MSD) curves revealing some of the biophysical properties of the motion. Finally, describe a method in R to identify tracts of directed motions in mixed trajectories. These approaches enable a deeper understanding of the mechanisms of heterochromatin dynamics and genome stability in the three-dimensional context of the nucleus, and have broad applicability in the field of nuclear dynamics." @default.
• W2765764597 created "2017-11-10" @default.
• W2765764597 creator A5008912019 @default.
• W2765764597 creator A5030876345 @default.
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• W2765764597 date "2017-11-06" @default.
• W2765764597 modified "2023-09-27" @default.
• W2765764597 title "Quantitative methods to investigate the 4D dynamics of heterochromatic repair sites in Drosophila cells" @default.
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• W2765764597 doi "https://doi.org/10.1101/214601" @default.
• W2765764597 hasPublicationYear "2017" @default.
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