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bioRxiv · 10.64898/2026.05.07.723557

Triplet tumbling microscopy enables in situ quantification of protein complex assembly and dynamics

Lazzari-Dean, J. R.·Millett-Sikking, A.·Rao, P.·Jensvold, Z. D.·Baddock, H.·Ingaramo, M.·Nile, A. H.·York, A. G.·Preciado Lopez, M.
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Abstract

Protein-protein interactions (PPIs) mediate diverse cellular processes, but PPIs are typically characterized using reconstituted in vitro biochemical and biophysical approaches. Current approaches for PPI detection in living cells are limited in the scope of interactions they can capture and often require prior knowledge of the interacting partners. To close this gap, we developed triplet tumbling microscopy (TTM), which reveals the interactions of a tagged protein of interest in cells in real time. TTM reports protein complex size from rotational diffusion ("tumbling") by leveraging infrared-triggerable emission from triplet states to track tumbling over nanoseconds to hundreds of microseconds. These long-lived triplets overcome the size limitations of existing rotational diffusion-based approaches, enabling TTM to measure species from small protein complexes to organelle-scale beads. In living cells, we apply TTM to detect PPIs, quantify fraction bound, and distinguish protein complexes by size. We measure diverse types of interactions, including rapamycin-induced dimerization, p53 homo-oligomerization, and binding of the E3-ligase E6AP to the human papilloma virus 16 E6 protein. The required hardware is compatible with most fluorescent microscopes, making TTM a versatile way to extract molecular insights from the complex context of living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/723557v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c77bc1org.highwire.dtl.DTLVardef@8de44forg.highwire.dtl.DTLVardef@18803f2org.highwire.dtl.DTLVardef@bb3afe_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Lazzari-Dean, J. R., Millett-Sikking, A., Rao, P., Jensvold, Z. D., Baddock, H., Ingaramo, M., Nile, A. H., York, A. G., Preciado Lopez, M.. 2026-05-11. Triplet tumbling microscopy enables in situ quantification of protein complex assembly and dynamics. https://doi.org/10.64898/2026.05.07.723557

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