Biology subjects

Caridi, C. P.

Publications and source records attributed to Caridi, C. P..

2 recordsLinked to original sources

bioRxiv2023

'Off-pore' nucleoporins relocalize heterochromatic breaks through phase separation

Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and "safe" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing a novel "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote. HighlightsO_LINup88 and Sec13 recruit Nup98 to heterochromatic DSBs downstream from Smc5/6 C_LIO_LINup88, Sec13 and Nup98 promote repair focus mobilization in heterochromatin off-pore C_LIO_LINup98 excludes Rad51 from repair sites inside the heterochromatin domain C_LIO_LIPhase separation by Nup98 is required and sufficient for relocalization and Rad51 exclusion C_LI

cell biology↗

bioRxiv2017

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

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.

cell biology↗