Single-stranded telomeric repeats segregate into spatial compartments within ALT-associated PML bodies

Alternative lengthening of telomeres (ALT)-associated PML bodies (APBs) concentrate single-stranded (ss) telomeric DNA and RNA species critical for recombination-based telomere maintenance. However, how these species are organized inside APBs has remained invisible at microscopic resolution. Here, we map the nanoscale topology of APB components using 3D MINFLUX super-resolution microscopy combined with multiplexed exchange DNA-PAINT labeling at [~]3 nm localization precision. We discover that ssC-rich and ssG-rich telomeric repeats occupy distinct spatial compartments within a partially open, ~70 nm-thick PML protein shell: ssC-rich repeats concentrate at the inner shell surface, while ssG-rich repeats distribute broadly throughout the interior alongside TRF1-marked double-stranded telomeric chromatin. The ssG-rich signal is predominantly DNA and frequently colocalizes with POT1 assemblies. Additional TRF1 clusters outside the shell indicate multi-telomere association. Together, the spatial segregation of single-stranded telomeric species within APBs imposes structural constraints on different mechanisms of ALT-mediated telomere elongation and defines features that distinguish them.