A modular platform for engineering function of natural and synthetic biomolecular condensates

Lasker, Keren; Boeynaems, Steven; V, Lam; Stainton, Emma; Jacquemyn, Maarten; Daelemans, Dirk; Villa, Elizabeth; Holehouse, Alex S.; Gitler, Aaron D.; Shapiro, Lucy

doi:10.1101/2021.02.03.429226

Cited by 27 publications

(37 citation statements)

References 78 publications

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“…Our findings provide a bridge that connects recent findings regarding dynamic clusters comprising of multivalent molecules (73,74). They also show that chemistry-or site-specific sticker-mediated interactions and solubility determining spacer-mediated interactions can be separable (27,29,43,44,48,49,(75)(76)(77)(78)(79)(80)(81), and suggested to be of importance for biological functions (82).…”

Section: Discussionsupporting

confidence: 85%

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Kar

Dar

Welsh

et al. 2022

Preprint

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Macromolecular phase separation is thought to be one of the processes that drive the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation, especially at low endogenous concentrations found in cells, are thought to follow the tenets of classical nucleation theory describing a sharp transition between a dense phase and a dilute phase by dispersed monomers. Here, we used in vitro biophysical studies to study subsaturated solutions of phase separating RNA binding proteins with intrinsically disordered prion like domains (PLDs) and RNA binding domains (RBDs). Surprisingly, we find that subsaturated solutions are characterized by heterogeneous distributions of clusters comprising tens to hundreds of molecules. These clusters also include low abundance mesoscale species that are several hundreds of nanometers in diameter. Our results show that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. Interestingly, however, cluster formation and phase separation can be decoupled from one another using solutes that impact the solubilities of phase separating proteins. They can also be decoupled by specific types of mutations. Overall, our findings implicate the presence of distinct, sequence-specific energy scales that contribute to the overall phase behaviors of RNA binding proteins. We discuss our findings in the context of theories of associative polymers.

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Section: Discussionsupporting

confidence: 85%

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Kar

Dar

Welsh

et al. 2022

Preprint

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“…Starting with unstructured and RNA-binding domains, shown to drive of many proteins (17), we show that both the disordered and the ordered domains in TmaR, the latter containing the RNA-binding domain, are required for its LLPS. Recently, condensation of PopZ in C. crescentus has been found to be driven by a structured domain, whereas the state of matter of these properties is determined by PopZ disordered region (50). Other results presented here show compliance with the molecular grammar developed for LLPS, such as the importance of arginines, often present in RNA-binding domains, for condensation (20).…”

Section: Discussionsupporting

confidence: 82%

Regulation of major bacterial survival strategies by transcript sequestration in a membraneless organelle

Szoke

Albocher

Goldberger

et al. 2022

Preprint

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Liquid-liquid phase separation (LLPS) of proteins was shown in recent years to regulate spatial organization of cell content without the need for membrane encapsulation in eukaryotes and prokaryotes. Yet evidence for the relevance of LLPS for bacterial cell functionality is largely missing. Here we show that the sugar metabolism-regulating clusters, recently shown by us to assemble in the E. coli cell poles by means of the novel protein TmaR, are formed via LLPS. A mutant screen uncovered residues and motifs in TmaR that are important for its condensation. Upon overexpression, TmaR undergoes irreversible liquid-to-solid transition, similar to the transition of disease-causing proteins in human, which impairs bacterial cell morphology and proliferation. Not only does RNA contribute to TmaR phase separation, but by ensuring polar localization and stability of flagella-related transcripts, TmaR enables cell motility and biofilm formation, thus providing a linkage between LLPS and major survival strategies in bacteria.

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“…The second approach that starts with a lower correlation threshold and includes subsequent iterative 3-D classification, is more accurate to find the final true-positive ribosomes and ribosome distributions (Lasker et al, 2021). However, it requires considerably more resources and expertise.…”

Section: Tomogram Collection and Processingmentioning

confidence: 99%

Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps

Gilbert

Thornburg

Lam

et al. 2021

Front. Mol. Biosci.

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JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A’s genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli’s, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome’s configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.

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A modular platform for engineering function of natural and synthetic biomolecular condensates

Cited by 27 publications

References 78 publications

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Regulation of major bacterial survival strategies by transcript sequestration in a membraneless organelle

Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps

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