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SCIENCE CHINA Life Sciences, Volume 63 , Issue 7 : 953-985(2020) https://doi.org/10.1007/s11427-020-1702-x

Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases

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  • ReceivedFeb 28, 2020
  • AcceptedApr 20, 2020
  • PublishedApr 30, 2020

Abstract

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 biochemical 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.


Funded by

the National Natural Science Foundation of China(31871394,31670730)

the Shanghai Municipal Science and Technology Major Project(2018SHZDZX01)

ZJLab. Work in Xueliang Zhu’s laboratory was supported by grants from the National Natural Science Foundation of China(31420103916,31991192)

CAS(XDB19020102)

National Key R&D Program of China(2016YFA0501903,2019YFA0508402)

grants from the Beijing Municipal Science and Technology Committee(Z181100001318003)

the National Natural Science Foundation of China(31421002,31561143001,31630048,31790403)

the Ministry of Science and Technology of China(2017YFA0503401)

the Strategic Priority Research Program of the Chinese Academy of Sciences(CAS)

the Key Research Program of Frontier Sciences

CAS(QYZDY-SSW-SMC006)

the National Natural Science Foundation of China(91853113,31872716)

the Science and Technology Commission of Shanghai Municipality(18JC1420500)

the Shanghai Municipal Science and Technology Major Project(2019SHZDZX02)

the National Natural Science Foundation of China(11672317)


Acknowledgment

We are grateful to Dr. Isabel Hanson for editing work. Work in Hong Zhang’s laboratory was supported by grants from the Beijing Municipal Science and Technology Committee (Z181100001318003), the National Natural Science Foundation of China (31421002, 31561143001, 31630048, and 31790403), the Ministry of Science and Technology of China (2017YFA0503401), the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (XDB19000000) and the Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SMC006). Work in Xiong Ji’s laboratory was supported by funds from the Ministry of Science and Technology of China and the National Natural Science Foundation of China (2017YFA0506600 and 31871309). Work in Pilong Li’s laboratory was supported by funds from the Ministry of Science and Technology of China and the National Natural Science Foundation of China (2019YFA0508403 and 31871443). Work in Cong Liu’s laboratory was supported by grants from the Ministry of Science and Technology of China (2016YFA0501902), the National Natural Science Foundation of China (91853113 and 31872716), the Science and Technology Commission of Shanghai Municipality (18JC1420500), the Shanghai Municipal Science and Technology Major Project (2019SHZDZX02). Work in Jizhong Lou’s laboratory was supported by grants from the Ministry of Science and Technology of China (2019YFA0707000), the National Natural Science Foundation of China (11672317). Work in Wenyu Wen’s laboratory was supported by grants from the Ministry of Science and Technology of China (2019YFA0508401), the National Natural Science Foundation of China (31871394 and 31670730), the Shanghai Municipal Science and Technology Major Project (2018SHZDZX01) and ZJLab. Work in Xueliang Zhu’s laboratory was supported by grants from the National Natural Science Foundation of China (31420103916 and 31991192) and CAS (XDB19020102). Research in Mingjie Zhang’s laboratory was supported by grants from RGC of Hong Kong (AoE-M09-12 and C6004-17G) and National Key R&D Program of China (2016YFA0501903 and 2019YFA0508402).


Interest statement

The author(s) declare that they have no conflict of interest.


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  • Figure 1

    The forces driving phase separation and the material states of condensates. A, Two mechanisms for the formation of phase-separated liquid droplets. Top: interaction of intrinsically disordered regions (IDRs) within one protein species via different kinds of weak contacts (right). Bottom: binding of tandem interacting domains in two different proteins. In both cases, multivalent interactions lead to the formation of phase-separated liquid droplets at higher protein concentrations. B, The different material states of phase-separated condensates. Within the liquid droplets, the protein condensates are highly dynamic and reversibly assembled. The condensates can break up in response to certain changes in the solution conditions, such as protein concentration, temperature and ionic strength. The constituents inside liquid droplets have high mobility and exchange with the surrounding environment. With time, the liquid-like protein condensates may gradually transition into solid-like states, such as hydrogels. Compared to liquid droplets, protein condensates with gel-like structures are less dynamic, and the constituents inside can only undergo very limited exchange with their surroundings. However, the assembly of these hydrogel-like structures can also be partially reversed under certain conditions. In certain scenarios, liquid crystal-like structures can form in cells. The constituents inside liquid crystals are in an ordered arrangement and can realign in response to stimuli. Protein condensates can also transition into amyloid-like fibril structures or other types of aggregates, which are non-dynamic and extremely resistant to changes in solution conditions. The constituents inside amyloid-like fibrils and other aggregates are inert and immobile.

  • Figure 2

    Examples of phase separation of cytoskeleton-related proteins. A, In the presence of Filamin, short actin filaments form tactoids in vitro through LLPS. B, LLPS of signaling proteins triggers local F-actin assembly in T-cells at the immunological synapse or in podocytes at the slit diaphragms. C, LLPS of BuGZ and Tacc3 induces the spindle matrix and the liquid-like meiotic spindle domain respectively to facilitate MT polymerization and spindle assembly. D, Liquid droplets of Tau facilitate MT polymerization and bundling in vitro.

  • Figure 3

    Phase separation in transcription regulation. A, Interactions of the IDRs in two HP1 homologs (human HP1α and fly HP1a) (left) and CBX2-PRC1 (right) lead to the formation of phase-separated liquid droplets at higher protein concentrations and low salt concentrations in vitro. RING1B, PHC1, PCGHx and CBX2 are components of PRC1 complex. IDR, intrinsically disordered region; CD, chromodomain; CSD, chromo shadow domain; NTE, N-terminal extension; CTE, C-terminal extension; SAM, sterile alpha motif domain. B, Nucleosome arrays undergo phase separation under physiological conditions. C, HP1 can form complexes with a plethora of proteins, e.g., SUV39H1 and TRIM28, via their CSD-binding motifs (HP1-boxes). These complexes often contain multiple H3K9me3-recognizing CDs and can undergo phase separation with H3K9me3-marked nucleosome arrays. D, Step-by-step functions of phase separation in transcription complex assembly. (1) Transcription factors (TFs) bind to distal control elements (enhancers or super enhancers) based on their DNA-binding domains and DNA remodelers. (2) TFs interact with cofactors (mediators or chromatin regulators) to form condensates through their IDRs or multivalent domains. These condensates modify chromatin structures to facilitate the recruitment of additional factors. (3) Condensates of TFs and cofactors dynamically assemble at the promoter region to promote a high level of transcription initiation. This involves the recruitment of general transcription factors, and the formation of dynamic transcriptional condensates based on interactions of the C-terminal domain (CTD) of Pol II.

  • Figure 4

    LLPS-mediated basal condensation of the cell fate determinant Numb during ACD of Drosophila NBs. In mitotic cells, the Baz/Par6/aPKC complex (yellow) forms a condensed crescent apically with Insc, Pins and Gαi (red), whereas the cell fate determinants Numb, Pros and Brat (green) and their adaptors Pon and Mira (cyan) concentrate basally. The specific and multivalent interaction between Numb and Pon leads to LLPS of the Numb-Pon complex, thus driving their basal condensation. The right panel shows the interaction between Numb PTB and one Pon repeating motif (top) and phase-separated droplets formed by Numb PTB and a Pon fragment containing three repeating motifs in vitro.

  • Figure 5

    Assembly of pre- and post-synaptic density signaling complexes via liquid-liquid phase separation. A, A diagram showing that the postsynaptic protein complex is likely formed via phase separation-mediated assembly of multiple proteins (adapted from Zeng et al., 2018). B, Role of phase separation-mediated condensation of the TARP/PSD-95 complex in AMPA Receptor (AMPAR) synaptic transmission (adapted from Zeng et al., 2019). C, Formation of active pre-synaptic protein condensates via phase separation (adapted from Wu et al., 2019a).

  • Figure 6

    Phase separation and transition specify autophagic degradation of PGL granules. A, The autophagy pathway in multicellular organisms. Nascent autophagosomes fuse with vesicles derived from the endolysosomal compartments to form amphisomes, which further proceed into degradative autolysosomes. IM, isolation membrane; EE, early endosome; MVB, multivesicular body; LE, late endosome. B, LLPS-mediated assembly of PGL granules. PGL-1 and PGL-3 are post-translationally modified by EPG-11 and mTORC1. The receptor protein SEPA-1 mediates aggregation of PGL-1 and PGL-3, which is also modulated by PTMs. The scaffold protein EPG-2 or a gelation mutant of PGL-1 promotes the transition of PGL granules into a gel-like state, which is essential for their autophagic degradation. Under heat stress conditions, assembly of PGL granules is promoted, while levels of EPG-2, which undergoes normal autophagic degradation, are not sufficient to make PGL granules amenable to autophagic degradation.

  • Figure 7

    Schematic view of phase transition between different states, and the relationship between aberrant phase separation and neurodegenerative diseases. RNA-binding proteins (RBPs) undergo reversible LLPS to form liquid-like condensates, which can further mature into irreversible aggregates composed of pathological fibrils. This process underpins neurodegenerative diseases. The different states have distinct material properties, with the dynamics and reversibility decreasing as the condensates transition from a liquid-like to a solid-like state. In biological contexts, the LLPS process is precisely regulated by protein quality control systems, protein PTMs and cellular transportation systems. Different chaperones and PTMs may prevent protein phase separation, while disease-associated mutations and certain pathological PTMs may increase the probability that RBPs will form solid-like condensates, thus leading to diseases.

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