Live cell imaging of nuclear actin filaments and heterochromatic repair foci in Drosophila and mouse cells

Pericentromeric heterochromatin largely comprises repeated DNA sequences prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins generating directed motions. Understanding this pathway requires the ability to detect nuclear actin filaments that are significantly less abundant than cytoplasmic filaments, and to image and track repair sites for long time periods. Here we describe an optimized protocol for live cell imaging of nuclear F-actin in response to IR in Drosophila cells, and for repair focus tracking in mouse cells, including imaging setup, image processing approaches, and analytical methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.