Developing methods to classify protein clusters as liquid-liquid phase separated biomolecular condensates in vivo.

Recently, it has been discovered that liquid-liquid phase separation (LLPS) occurs in the cell, driving the demixing of specific biomolecules to form concentrated droplets called condensates. LLPS is now believed to play a role in many key cellular processes including gene regulation. To test this hypothesis, we are studying the transcription factor Bicoid (Bcd) in fly embryos which has structural properties important for LLPS. Bcd also forms a spatial concentration gradient along the embryo which governs a spatial pattern of gene expression and provides a system to study the effect of concentration on condensate formation in vivo. By creating a dedicated confocal image acquisition and analysis workflow, Bcd clusters have been detected and shown to be quantitatively distinguishable from a non-clustering control protein. The diffusion coefficients of these clusters were also measured using particle tracking algorithms, indicating the existence of two populations, and suggesting that some of these clusters are bound to DNA. These results are supported by fluorescence correlation spectroscopy experiments that allow estimating the average number of Bcd molecules present in these clusters, while super-resolution Airyscan imaging enhances the ability to capture the formation and breaking down of these structures.