PT  - JOURNAL ARTICLE
AU  - Houx, Justin
AU  - Copie, Thomas
AU  - Gambin, Yann
AU  - Sierecki, Emma
TI  - Quantification of nanocondensates formation at the single molecule level
AID  - 10.1101/2024.05.05.592604
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.05.05.592604
4099  - http://biorxiv.org/content/early/2024/05/06/2024.05.05.592604.short
4100  - http://biorxiv.org/content/early/2024/05/06/2024.05.05.592604.full
AB  - Understanding the molecular mechanisms of biomolecular condensate formation through liquid-liquid phase separation is crucial for deciphering cellular cues in normal and pathological contexts. Recent studies have highlighted the existence of sub-micron assemblies, known as nanocondensates or mesoscopic clusters, in the organization of a significant portion of the proteome. However, as smaller condensates are invisible to classical microscopy, new tools must be developed to quantify their numbers and properties. Here, we establish a simple analysis framework using single molecule fluorescence spectroscopy to quantify the formation of nanocondensates diffusing in solution. We used the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system to show that we can recapitulate the phase separation diagram of the protein in various conditions. Single molecule spectroscopy reveals rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than described previously by microscopy. We demonstrate how straightforward fingerprinting of individual nanocondensates provides an exquisite quantification of their formation, size, density, and their temporal evolution. Overall, this study highlights the potential of single molecule spectroscopy to investigate the formation of biomolecular condensates and liquid-liquid phase separation mechanisms in protein systems.Competing Interest StatementThe authors have declared no competing interest.TDP-43TAR DNA-binding protein 43TARTrans-activation response elementLLPSLiquid-liquid phase separationLCDlow complexity domainTMAOTrimethylamine N-oxideLTELeishmania tarentolae extractTLTop leftTRTop rightBLBottom leftBRBottom rightFUSFused in sarcomacGAScyclic GMP–AMP synthase