Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

LIVE-CELL FLIM-FRET REVEALS DYNAMIC ATPASE-DRIVEN PRE-RIBOSOME REMODELING STATES Rajen K Goutam 1 ; Alexandria N Kerr 1 ; Kanda Borgognoni 2 ; Robin E Stanley 2 ; Sharonda J LeBlanc 1 ; 1 North Carolina State University, Physics and Astronomy, Raleigh, NC, USA 2 Molecular and Cellular Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, USA Ribosome biogenesis depends on ATP-driven remodeling events that reorganize large pre ribosomal complexes during maturation. AAA+ ATPases such as Rea1 and Rix7 coordinate these remodeling steps by repositioning or releasing assembly factors during 60S ribosomal subunit formation. Because these events are transient and heterogeneous across cells, their organization in living systems remains poorly understood. Here, we aimed to quantify ATPase associated proximity states directly in live cells. We investigated these remodeling processes in live Saccharomyces cerevisiae using fluorescence lifetime imaging microscopy with Förster resonance energy transfer (FLIM-FRET) combined with phasor-based analysis. This approach directly measures donor lifetime changes and resolves heterogeneous molecular proximity states without kinetic model fitting. We quantified nanoscale proximity between Rea1 and its partners Erb1 and Nop7, as well as between Rix7 and Nsa1, across logarithmic growth, stationary phase, and heat shock conditions. Our results resolved distinct donor-only and FRET-associated populations with clear condition-dependent lifetime shifts. FRET efficiency distributions contained dominant moderate-FRET populations, together with broader low- and high-FRET subpopulations. These results indicate that ribosome remodeling occurs through a continuum of dynamic molecular organizations rather than a single fixed state. Heat shock broadened these distributions and increased high-proximity populations across multiple ATPase assembly-factor pairs. This pattern is consistent with increased remodeling heterogeneity during stress and may reflect altered remodeling dynamics, delayed assembly-factor release, or stress-induced compaction of pre-ribosomal intermediates. Together, these findings show that ATPase-driven ribosome remodeling is dynamically reorganized across cellular conditions. More broadly, this work establishes a quantitative framework for studying dynamic macromolecular remodeling in living cells using FLIM-FRET and phasor analysis.

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