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Microfluidic characterization of macromolecular liquid-liquid phase separation.

ABSTRACT: Liquid-liquid phase separation plays important roles in the compartmentalization of cells. Developing an understanding of how phase separation is encoded in biomacromolecules requires quantitative mapping of their phase behavior. Given that such experiments require large quantities of the biomolecule of interest, these efforts have been lagging behind the recent breadth of biological insights. Herein, we present a microfluidic phase chip that enables the measurement of saturation concentrations over at least three orders of magnitude for a broad spectrum of biomolecules and solution conditions. The phase chip consists of five units, each made of twenty individual sample chambers to allow the measurement of five sample conditions simultaneously. The analytes are slowly concentrated via evaporation of water, which is replaced by oil, until the sample undergoes phase separation into a dilute and dense phase. We show that the phase chip lowers the required sample quantity by 98% while offering six-fold better statistics in comparison to standard manual experiments that involve centrifugal separation of dilute and dense phases. We further show that the saturation concentrations measured in chips are in agreement with previously reported data for a variety of biomolecules. Concomitantly, time-dependent changes of the dense phase morphology and potential off-pathway processes, including aggregation, can be monitored microscopically. In summary, the phase chip is suited to exploring sequence-to-binodal relationships by enabling the determination of a large number of saturation concentrations at low protein cost.

SUBMITTER: Bremer A

PROVIDER: S-EPMC7658026 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation.

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2021-12-23 | GSE174575 | GEO

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| S-EPMC8674230 | biostudies-literature

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Bacterial Heterochromatin Formation Through Liquid-Liquid Phase Separation

Project description:Bacterial Heterochromatin Formation Through Liquid-Liquid Phase Separation

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2022-10-20 | PXD033048 | Pride

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Project description:Proteinaceous liquid-liquid phase separation (LLPS) occurs when a polypeptide coalesces into a dense phase to form a liquid droplet (i.e., condensate) in aqueous solution. In vivo, functional protein-based condensates are often referred to as membraneless organelles (MLOs), which have roles in cellular processes ranging from stress responses to regulation of gene expression. Late embryogenesis abundant (LEA) proteins containing seed maturation protein domains (SMP; PF04927) have been linked to storage tolerance of orthodox seeds. The mechanism by which anhydrobiotic longevity is improved is unknown. Interestingly, the brine shrimp Artemia franciscana is the only animal known to express such a protein (AfrLEA6) in its anhydrobiotic embryos. Ectopic expression of AfrLEA6 (AWM11684) in insect cells improves their desiccation tolerance and a fraction of the protein is sequestered into MLOs, while aqueous AfrLEA6 raises the viscosity of the cytoplasm. LLPS of AfrLEA6 is driven by the SMP domain, while the size of formed MLOs is regulated by a domain predicted to engage in protein binding. AfrLEA6 condensates formed in vitro selectively incorporate target proteins based on their surface charge, while cytoplasmic MLOs formed in AfrLEA6-transfected insect cells behave like stress granules. We suggest that AfrLEA6 promotes desiccation tolerance by engaging in two distinct molecular mechanisms: by raising cytoplasmic viscosity at even modest levels of water loss to promote cell integrity during drying and by forming condensates that may act as protective compartments for desiccation-sensitive proteins. Identifying and understanding the molecular mechanisms that govern anhydrobiosis will lead to significant advancements in preserving biological samples.

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