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 that are 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 through 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, these structural constraints motivate a model of ALT in which t-loop resolution generates a C-circle template that drives rolling-circle amplification of telomeric repeats. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/724127v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@d6823org.highwire.dtl.DTLVardef@2fe34corg.highwire.dtl.DTLVardef@1f3a20forg.highwire.dtl.DTLVardef@14b4ec_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI3D MINFLUX combined with exchange DNA-PAINT resolves APB components at {bsim}3 nm isotropic precision, enabling direct visualization of molecular organization beyond the reach of conventional super-resolution microscopy. C_LIO_LIPML forms a partially open, [~]70 nm thick spherical shell in APBs with reduced molecular density compared to canonical PML nuclear bodies, providing the structural scaffold and coordinate system for mapping internal APB organization. C_LIO_LIDouble-stranded telomeric chromatin (TRF1-marked) fills the APB interior, with additional TRF1 domains outside the shell indicative of multi-telomere clustering at individual APBs. C_LIO_LIssC-rich repeats concentrate in the inner part of the PML shell while ssG-rich DNA repeats distribute broadly through the interior, revealing strand-specific spatial segregation. C_LIO_LIThe ssG-rich single-stranded repeats in APBs predominantly comprise DNA and partially colocalize with POT1, connecting their spatial distribution to single-stranded telomeric DNA substrates. C_LIO_LIThe spatial organization of APB components motivates a mechanistic model in which t-loop processing generates C-circle templates for rolling-circle amplification, while multi-telomere clustering facilitates inter-telomeric recombination. C_LI