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

IF 4.5 Q1 BIOCHEMICAL RESEARCH METHODS

Cell Reports Methods Pub Date : 2025-08-18 Epub Date: 2025-08-04 DOI:10.1016/j.crmeth.2025.101118

Chengchen Wu, Janeth Catalina Manjarrez-González, Muntaqa Choudhury, Noor Shamkhi, Siwen Ding, Vishnu M Nair, Viji M Draviam

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Abstract

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.

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超分辨率下的荧光恢复显示53BP1病灶的亚区室。

双链断裂（DSB）修复蛋白53BP1 （p53结合蛋白-1）介导远程DNA末端连接和异染色质维持。我们利用晶格结构照明显微镜（SIM）(双迭代SIM [diSIM，也称为SIM2]；~ 60 nm横向分辨率)，以及点阵光片显微镜和光漂白后荧光恢复（FRAP），揭示了53BP1焦点轮廓和组成的差异。致密型53BP1聚焦保持静止，而非晶态聚焦则发生动态形状变化。在超分辨率（SR）机制（FRAP-SR）中使用FRAP，我们发现无定形53BP1焦点快速恢复53BP1- egfp信号，显示出亚区室，表明单个焦点内蛋白质的不同迁移和功能。相比之下，紧凑病灶以单个区室的形式均匀地恢复53BP1-EGFP，但恢复率不均匀。在从DNA复制块释放的细胞中，53BP1-EGFP在无定形灶中的迁移率比致密灶高。我们讨论了不同53BP1迁移率和焦点轮廓的概念含义，以及FRAP-SR方法如何改变动态60-100 nm结构的研究。

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