Quantitative methods to investigate the 4D dynamics of heterochromatic repair sites in Drosophila cells

• DOI: 10.1101/214601
• Publication Date: 2017-11-14T11:25:11Z
• Authors: Christopher P. Ca...; Laetitia Delabaere; Harianto Tjong; Hannah Hopp; Devika Das; Frank Alber; Irene Chiolo
• Keywords: 0301 basic medicine; 0303 health sciences; Recombinational DNA Repair; DNA; 03 medical and health sciences; Imaging, Three-Dimensional; Microscopy, Fluorescence; Heterochromatin; Animals; DNA Breaks, Double-Stranded; Drosophila; Software

AbstractHeterochromatin 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.