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  1. Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are 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 that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here,more »we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis 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.« less
  2. 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 describemore »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.« less