SCIENCE CHINA Life Sciences •REVIEW• July 2020 Vol.63 No.7: 953–985 https://doi.org/10.1007/s11427-020-1702-x Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases Hong Zhang1,2*, Xiong Ji3*, Pilong Li4*, Cong Liu5*, Jizhong Lou2,6*, Zheng Wang1, Wenyu Wen7*, Yue Xiao8, Mingjie Zhang9* & Xueliang Zhu8* 1National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; 3Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; 4Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China; 5Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China; 6Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 7State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; 8State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China; 9Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China Received February 28, 2020; accepted April 20, 2020; published online April 30, 2020 Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contact- mediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation (LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization, gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin- and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre- and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as bio- chemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases. *Corresponding authors (Hong Zhang, email: [email protected] (lead contact); Xiong Ji, email: [email protected]; Pilong Li, email: [email protected]; Cong Liu, email: [email protected]; Jizhong Lou, email: [email protected]; Wenyu Wen, email: [email protected]; Mingjie Zhang, email: [email protected] (lead contact); Xueliang Zhu, email: [email protected]) © Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020 life.scichina.com link.springer.com 954 Zhang, H., et al. Sci China Life Sci July (2020) Vol.63 No.7 phase separation, phase transition, transcription, asymmetric division, postsynaptic density, autophagy Citation: Zhang, H., Ji, X., Li, P., Liu, C., Lou, J., Wang, Z., Wen, W., Xiao, Y., Zhang, M., and Zhu, X. (2020). Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases. Sci China Life Sci 63, 953–985. https://doi.org/10.1007/s11427-020-1702-x Introduction The physical principles underlying LLPS Numerous protein interactions and biochemical reactions Basic principles underlying LLPS occur simultaneously within the limited spaces inside eu- The LLPS phenomena of polymers have been extensively karyotic cells. Multiple mechanisms have been identified studied in the fields of polymer chemistry and soft matter that ensure the spatiotemporal specificity and efficiency of physics. Biomacromolecules are polymers and hence the these cellular processes. Organelles delineated by phospho- physical basis of biomacromolecular LLPS appears to be the lipid membranes provide relatively confined spaces which same. The in-depth explanation of the theoretical basis of allow various signaling pathways and biological interactions LLPS, known as the Flory-Higgins theory, has been nicely to proceed efficiently and specifically. The membrane-bound summarized (Flory, 1953; Michaeli et al., 1957). We can organelles are connected via vesicle-mediated trafficking rationalize the condensation process of a biomacromolecular and distinct membrane contacts, thereby forming elaborate phase separation system using a simple thermodynamics cellular rection and signaling compartments essential for the argument. Phase separation systems can contain one or more well-being of cells. type(s) of biomacromolecular component(s), but for sim- Biomacromolecules such as proteins and nucleic acids can plicity, we will use a system comprising one type of bio- coacervate into liquid-like membrane-less condensates via macromolecule in a solution for further discussion. liquid-liquid phase separation (LLPS), which provides an- Biomacromolecules in a solution interact with each other and other means for concentrating and segregating cellular solvent molecules in a manner that reduces the free energy of components in a spatiotemporally defined manner for di- the system. Generally, this means that the biomacromole- verse functional processes. The phase-separated condensates cules will tend to be distributed uniformly throughout the are also called aggregates, bodies, granules and membrane- solution volume in monomeric and small-sized complexes to less compartments. There is mounting evidence that protein maximize the entropy. If biomacromolecules can engage in condensates fulfill a range of distinct physiological functions more energetically favorable interactions among one another in living cells. To mention a few examples, phosphorylation- in a condensed solution than in a dilute solution, the extra induced phase separation of T-cell receptor (TCR) and its energy output can compensate for the entropic penalty due to downstream signaling proteins enriches signaling compo- the clustering of biomacromolecules. The higher the bio- nents and expels inhibitory regulators, thus ensuring sub- macromolecule concentration is, the lower the entropic sequent signal transduction (Su et al., 2016); gel-like penalty is. The critical concentration of the biomacromole- assembly of postsynaptic density (PSD) scaffold proteins cule solution is defined as the concentration at which the free may facilitate synaptic signal transduction, synaptic devel- energy generated from the extra interactions of a molecule in opment and plasticity (Zeng et al., 2016; Zeng et al., 2018); a condensate versus the interactions in dilute solution is phase separation of PGL granules modulated by mTORC1 equal to the entropy penalty caused by constraining it within signaling ensures their efficient degradation by autophagy or a condensate. At concentrations below the critical con- their retention as an adaptation to heat stress during devel- centration, the solution is homogeneous. At concentrations opment (Zhang et al., 2018a; Wang and Zhang, 2019); the above the critical concentration, the solution undergoes cell fate determinants Numb and Pon form condensates on phase separation to yield a dilute solution phase and a con- the basal cortex of the inner surface of the plasma membrane densed, biomacromolecule-rich phase. A number of recent during asymmetric cell division of Drosophila neuroblasts reviews have formally dealt with the thermodynamics of (NBs) (Shan et al., 2018). Phase-separated protein con- phase separation (Banani et al., 2017; Shin and Brangwynne, densates also intimately interact with membrane-bound or- 2017). Here we introduce recent progress in understanding ganelles via lipid-binding proteins and membrane-anchored the mechanisms driving the formation of biomacromolecular proteins (Banjade and Rosen, 2014; Case et al., 2019a; Liao condensates. et al., 2019; Ma and Mayr, 2018; Milovanovic et al., 2018; Yamasaki et al., 2020). The field of biomacromolecular Multivalent interactions underlying