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

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 contactmediated 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.     

The feasibility of parameterizing four-state equilibria using relaxation dispersion measurements

Coupled equilibria play important roles in controlling information flow in biochemical systems, including allosteric molecules and multidomain proteins. In the simplest case, two equilibria are coupled to produce four interconverting states. In this study, we assessed the feasibility of determining the degree of coupling between two equilibria in a four-state system via relaxation dispersion measurements. A major bottleneck in this effort is the lack of efficient approaches to data analysis. To this end, we designed a strategy to efficiently evaluate the smoothness of the target function surface (TFS). Using this approach, we found that the TFS is very rough when fitting benchmark CPMG data to all adjustable variables of the four-state equilibria. After constraining a portion of the adjustable variables, which can often be achieved through independent biochemical manipulation of the system, the smoothness of TFS improves dramatically, although it is still insufficient to pinpoint the solution. The four-state equilibria can be finally solved with further incorporation of independent chemical shift information that is readily available. We also used Monte Carlo simulations to evaluate how well each adjustable parameter can be determined in a large kinetic and thermodynamic parameter space and how much improvement can be achieved in defining the parameters through additional measurements. The results show that in favorable conditions the combination of relaxation dispersion and biochemical manipulation allow the four-state equilibrium to be parameterized, and thus coupling strength between two processes to be determined.     

Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly

The SMN complex mediates the assembly of heptameric Sm protein rings on small nuclear RNAs (snRNAs), which are essential for snRNP function. Specific Sm core assembly depends on Sm proteins and snRNA recognition by SMN/Gemin2- and Gemin5-containing subunits, respectively. The mechanism by which the Sm proteins are gathered while preventing illicit Sm assembly on non-snRNAs is unknown. Here, we describe the 2.5?? crystal structure of Gemin2 bound to SmD1/D2/F/E/G pentamer and SMN's Gemin2-binding domain, a key assembly intermediate. Remarkably, through its extended conformation, Gemin2 wraps around the crescent-shaped pentamer, interacting with all five Sm proteins, and gripping its bottom and top sides and outer perimeter. Gemin2 reaches into the RNA-binding pocket, preventing RNA binding. Interestingly, SMN-Gemin2 interaction is abrogated by a spinal muscular atrophy (SMA)-causing mutation in an SMN helix that mediates Gemin2 binding. These findings provide insight into SMN complex assembly and specificity, linking snRNP biogenesis and SMA pathogenesis.     

Bcl10 phosphorylation-dependent droplet-like condensation positively regulates DNA virus-induced innate immune signaling

B-cell lymphoma 10 (Bcl10) is a scaffolding protein that functions as an upstream regulator of NF-κB signaling by forming a complex with Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and CARD-coiled coil protein family. This study showed that Bcl10 was involved in type I interferon (IFN) expression in response to DNA virus infection and that Bcl10-deficient mice were more susceptible to Herpes simplex virus 1 (HSV-1) infection than control mice. Mechanistically, DNA virus infection can trigger Bcl10 recruitment to the STING-TBK1 complex, leading to Bcl10 phosphorylation by TBK1. The phosphorylated Bcl10 undergoes droplet-like condensation and forms oligomers, which induce TBK1 phosphorylation and translocation to the perinuclear region. The activated TBK1 phosphorylates IRF3, which induces the expression of type I IFNs. This study elucidates that Bcl10 induces an innate immune response by undergoing droplet-like condensation and participating in signalosome formation downstream of the cGAS-STING pathway.