Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation
Our current understanding of biomolecular condensate formation is largely based on observing the final near-equilibrium condensate state. Despite expectations from classical nucleation theory, pre-critical protein clusters were recently shown to form under subsaturation conditions in vitro; if similar long-lived clusters comprising more than a few molecules are also present in cells, our understanding of the physical basis of biological phase separation may fundamentally change. Here, we combine fluorescence microscopy with photobleaching analysis to quantify the formation of clusters of NELF proteins in living, stressed cells. We categorise small and large clusters based on their dynamics and their response to p38 kinase inhibition. We find a broad distribution of pre-condensate cluster sizes and show that NELF protein cluster formation can be explained as non-classical nucleation with a surprisingly flat free-energy landscape for a wide range of sizes and an inhibition of condensation in unstressed cells.
Acknowledgements: We thank Ibrahim Cissé, Rosana Collepardo-Guevara, Stephan Grill and Rohit Pappu for helpful discussions, and Adam Klosin (Hyman lab) for supplying us with the plasmid of DDX4. This work was supported by the European Research Council (grant agreement No. 681891, T.H.) and the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy (CIBSS EXC-2189 Project ID 390939984, R.G., R.S. and T.H.) and the SFB1381 programme (Project ID 403222702, T.H.).
EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) (681891)