Scaffolds were defined as the drivers of LLPS essential for the structural integrity of MLOs, and the major components which, alone or with co-scaffolds, undergo LLPS (1-4). A minimum set of six experimental tests, including the assembly of spherical droplets, the observation of fusion events, and the identification of mutations that abolish or inhibit LLPS in vitro and in cells have been proposed for rigorous analysis of LLPS processes (1). For each known scaffold protein, descriptions on performed assays of the minimum set of experiments were presented on its gene page. For example, the human fused in sarcoma (FUS), a well-characterized RNA-binding protein undergoing LLPS involved in formation of multiple biomolecular condensates (5,6-8), forms liquid-like droplets both in cells and at near physiological conditions in vitro. All the six experimental tests were performed to analyze the LLPS properties of FUS (9).
References
1. Alberti, S., Gladfelter, A. and Mittag, T. (2019) Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell, 176, 419-434. PMID: 30682370
2. Banani, S.F., Lee, H.O., Hyman, A.A. and Rosen, M.K. (2017) Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285-298. PMID: 28225081
3. Banani, S.F., Rice, A.M., Peeples, W.B., Lin, Y., Jain, S., Parker, R. and Rosen, M.K. (2016) Compositional control of phase-separated cellular bodies. Cell, 166, 651-663. PMID: 27374333
4. Hyman, A.A. and Simons, K. (2012) Cell biology. Beyond oil and water--phase transitions in cells. Science, 337, 1047-1049. PMID: 22936764
5. Hofweber, M., Hutten, S., Bourgeois, B., Spreitzer, E., Niedner-Boblenz, A., Schifferer, M., Ruepp, M.D., Simons, M., Niessing, D., Madl, T. et al. (2018) Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. Cell, 173, 706-719 e713. PMID: 29677514
6. Murray, D.T., Kato, M., Lin, Y., Thurber, K.R., Hung, I., McKnight, S.L. and Tycko, R. (2017) Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains. Cell, 171, 615-627 e61. PMID: 28942918
7. Guo, L., Kim, H.J., Wang, H., Monaghan, J., Freyermuth, F., Sung, J.C., O'Donovan, K., Fare, C.M., Diaz, Z., Singh, N. et al. (2018) Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains. Cell, 173, 677-692 e20. PMID: 29677512
8. Yoshizawa, T., Ali, R., Jiou, J., Fung, H.Y.J., Burke, K.A., Kim, S.J., Lin, Y., Peeples, W.B., Saltzberg, D., Soniat, M. et al. (2018) Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. Cell, 173, 693-705 e22. PMID: 29677513
9. Patel, A., Lee, H.O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M.Y., Stoynov, S., Mahamid, J., Saha, S., Franzmann, T.M. et al. (2015) A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell, 57, 162, 1066-1077. PMID: 26317470
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