Quantification of nanocondensates formation at the single molecule level

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.