FRAP-in-SR: Fluorescence recovery in the Super-Resolution regime reveals subcompartments of 53BP1 foci

We combine Lattice Structured Illumination Microscopy ( di SIM or SIM ² with ∼60 nm resolution), Lattice Light-sheet microscopy and Fluorescence Recovery After Photobleaching (FRAP) to explore 53BP1 dynamics in Retinal Pigment Epithelial cells. 53BP1 forms liquid condensates during double-strand DNA repair, long-range DNA end-joining and heterochromatin maintenance. Our super-resolution movies reveal differences in 53BP1 foci contour: some foci are compact and stationary while others appear amorphous, dynamically changing shapes. To explore them, we developed FRAP in the Super-Resolution regime (FRAP-SR). 53BP1 foci with an amorphous loose contour display subcompartments that recover 53BP1-eGFP signals rapidly, indicating differential protein mobilities and 53BP1 functions within a single foci. In contrast, 53BP1-eGFP foci with a compact contour recover uniformly as single foci but show higher heterogeneity in 53BP1-eGFP recovery rates compared to foci that recover as multiple subcompartments. In cells released from aphidicolin, amorphous foci show faster 53BP1 recovery compared to compact foci. We discuss the conceptual implications of different 53BP1 mobilities, and how the FRAP-SR method transforms studies of dynamic 60-100 nm structures.