Composition-dependent thermodynamics of intracellular phase separation

Riback, Joshua A.; Zhu, Lian; Ferrolino, Mylene C.; Tolbert, Michele; Mitrea, Diana M.; Sanders, David W.; Wei, Ming; Kriwacki, Richard W.; Brangwynne, Clifford P.

doi:10.1038/s41586-020-2256-2

Cited by 503 publications

(648 citation statements)

References 28 publications

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“…Our results may provide further insight to the origins and functionality of natural condensates. For instance, though often overlooked, fluorescent protein labelling of eukaryotic condensates often results in foci void of fluorescence (12,16,33), which is reminiscent of our observations of multilayering. The compositional control by ligand sorting may be relevant to natural organelles, such as ribonucleoprotein granules (20) that rapidly shift their preferences in response to growth conditions.…”

Section: Discussionmentioning

confidence: 87%

“…The compositional control by ligand sorting may be relevant to natural organelles, such as ribonucleoprotein granules (20) that rapidly shift their preferences in response to growth conditions. Turnover-governed preferences over the same pair of interacting molecules may explain how systems like nucleoli (16,33) select reactants over structurally-alike products. Moreover, we demonstrate that condensates may have diverse effects on cellular phenotypic variability beyond the established noise reduction role.…”

Section: Discussionmentioning

confidence: 99%

“…The solute partitioned by condensates needs to interact with downstream molecules to output functions. In the presence of heterotypic interactions, solutes may no longer have a fixed concentration in the aqueous phase (33), raising the question of whether noise is still buffered in such multicomponent systems. This process can be mimicked in our ligand-sorting framework.…”

Section: Phase Separation Provide Diverse Mechanisms To Control Reactmentioning

confidence: 99%

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Spatial engineering ofE. coliwith addressable phase-separated RNAs

Guo

Ryan

Mallet

et al. 2020

Preprint

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AbstractBiochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here we demonstrate Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS) as synthetic microdomains within the Escherichia coli. TEARS are assembled from RNA-binding protein recruitment domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures and a non-equilibrium mechanism controlling their composition and reaction robustness. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates and scaffolding protein-protein interactions. TEARS are a simple and versatile tool for spatially controlling E. coli biochemistry.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Phase Separation Provide Diverse Mechanisms To Control Reactmentioning

confidence: 99%

See 1 more Smart Citation

Spatial engineering ofE. coliwith addressable phase-separated RNAs

Guo

Ryan

Mallet

et al. 2020

Preprint

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“…Through both biochemical reconstitution and computational approaches, we revealed a non-monotonic mechanism of regulating LAT clustering by PLC1. Compositional control emerges as an important mechanism for regulating the physical feature and chemical activities of liquid-like condensates (Banani et al, 2016;Ditlev et al, 2019;Riback et al, 2020). The concentration of PLC1 may have been tuned to control cluster size and stability, with the purpose of transducing signaling to the physiological needs.…”

Section: Discussionmentioning

confidence: 99%

PLCγ1 promotes phase separation of the T cell signaling clusters

Zeng

Palaia

et al. 2020

Preprint

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SummaryThe T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here we report that phospholipase PLCγ1 promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly crosslinks LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK and SLP76 activation. Intriguingly, a non-monotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.

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“…Changes in the phase boundary for an LLPS system following variation of solution conditions affords insights to the thermodynamic processes driving protein condensation and the factors that modulate them. 15 As such, the generation of phase diagrams is a vital step to an understanding of protein phase-separation behaviour. However, given the large variety of proteins undergoing LLPS and the environmental conditions which regulate their behaviour, there is a need for experimental methods that enable rapid and high-resolution characterisation of LLPS phase diagrams.…”

Section: Introductionmentioning

confidence: 99%

High Resolution Biomolecular Condensate Phase Diagrams with a Combinatorial Microdroplet Platform

Arter

Krainer

et al. 2020

Preprint

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The assembly of intracellular proteins into biomolecular condensates via liquid-liquid phase separation (LLPS) has emerged as a fundamental process underlying the organisation and regulation of cellular space and function. Physicochemical characterisation of the parameters that control and modulate phase separation is therefore essential for an improved understanding of protein phase behaviour, including for the therapeutic modulation of LLPS phenomena. A fundamental measure with which to describe protein phase behaviour in chemical space is the phase diagram. Characterisation of phase diagrams requires measuring the presence or absence of the condensed phase under a multitude of conditions and, as such, is associated with significant consumption of time and sample volume even when performed in microwell format.However, due to the rapidly increasing number of biologically and disease-relevant condensate systems, experimental techniques that enable high-throughput analysis of protein phase behaviour are required. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for the rapid and high-resolution acquisition of protein phase diagrams. Using this platform, we demonstrate characterisation of the phase behaviour of a pathologically relevant mutant of the protein fused in sarcoma (FUS) in a highly parallelised manner, with significantly improved assay throughput and reduced sample consumption. We demonstrate the capability of the platform by finding the phase boundary at which FUS transitions from a one-phase to a two-phase state as modulated by 1,6-hexanediol, and estimate the free-energy landscape of this system using Flory-Huggins theory. These results thus provide a basis for the rapid acquisition of phase diagrams through the application of microdroplet techniques and pave the way for a wide range of applications, enabling rapid characterisation of the effect of environmental conditions and coacervate species on the thermodynamics of phase separation.

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Composition-dependent thermodynamics of intracellular phase separation

Cited by 503 publications

References 28 publications

Spatial engineering ofE. coliwith addressable phase-separated RNAs

Spatial engineering ofE. coliwith addressable phase-separated RNAs

PLCγ1 promotes phase separation of the T cell signaling clusters

High Resolution Biomolecular Condensate Phase Diagrams with a Combinatorial Microdroplet Platform

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