Fluorescence recovery in the super-resolution regime reveals subcompartments of 53BP1 foci

Double-strand break (DSB) repair protein 53BP1 (p53 binding protein-1) mediates long-range DNA end-joining and heterochromatin maintenance. We exploit lattice structured illumination microscopy (SIM) (dual iterative SIM [diSIM, also called SIM²]; ∼60 nm lateral resolution) alongside lattice light-sheet microscopy and fluorescence recovery after photobleaching (FRAP) and reveal differences in 53BP1 foci contour and composition. Compact 53BP1 foci remain stationary, while amorphous foci undergo dynamic shape changes. Using FRAP in the super-resolution (SR) regime (FRAP-SR), we show that amorphous 53BP1 foci recover 53BP1-EGFP signals rapidly exhibiting subcompartments, indicating differential protein mobilities and functions within a single foci. In contrast, compact foci recover 53BP1-EGFP uniformly as a single compartment but show heterogeneous recovery rates. In cells released from a DNA replication block, 53BP1-EGFP shows increased mobility in amorphous foci compared to compact foci. We discuss the conceptual implications of different 53BP1 mobilities and foci contours and how the FRAP-SR method transforms studies of dynamic 60-100 nm structures.