Measuring the non-equilibrium fluctuations of biological liquid-liquid phase separation

In biological systems, liquid-liquid phase separation (LLPS) is used to facilitate biochemical reactions, provide cellular compartmentalization, and sequester condition-specific factors. Whether biological LLPS events are active processes remains a difficult question to address because experiments that alter cellular ATP levels also affect the liquid properties of LLPS states. Understanding when different biological LLPS phenomena are active will provide insight into how the cell controls and drives their formation. The first instance of biological LLPS defined and one of the best characterized is the P granule of C. elegans. P granules exist in the cytosol of C. elegans germ-line cells and specify polarity during early embryonic development. To detect active processes in P granules, we measure irreversibility in the positional trajectories of single-walled carbon nanotube probes targeted to the P granules of C. elegans embryos. These extremely bright, photostable, infrared fluorophores provide sufficiently long single-particle trajectories for irreversibility calculations. By measuring irreversibility in the fluctuations of P granules we hope to understand if and when P granules are active liquid states and to directly connect this to a cellular chemical energy input.