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Methods Enzymol. 2018;601:359-389. doi: 10.1016/bs.mie.2017.11.033. Epub 2018 Feb 26.

Quantitative Methods to Investigate the 4D Dynamics of Heterochromatic Repair Sites in Drosophila Cells.

Author information

1
University of Southern California, Los Angeles, CA, United States.
2
University of Southern California, Los Angeles, CA, United States. Electronic address: chiolo@usc.edu.

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 postimaging using SoftWorX and Imaris software. We present a method to derive mean square displacement curves revealing some of the biophysical properties of the motion. Finally, we describe a method in R to identify tracts of directed motions (DMs) 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.

KEYWORDS:

Directed motions; Double-strand break repair; Drosophila; Heterochromatin; Homologous recombination; Live-cell imaging; Mean square displacement; Repair foci

PMID:
29523239
PMCID:
PMC6021022
DOI:
10.1016/bs.mie.2017.11.033
[Indexed for MEDLINE]
Free PMC Article

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