More than just oil droplets in water: surface tension and viscosity of protein condensates quantified by micropipette aspiration

Wang, H; Fm, Kelley; Milovanović, Dragomir; Bs, Schuster; Z, Shi

doi:10.1101/2021.05.28.446248

Cited by 6 publications

(2 citation statements)

References 52 publications

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“…Considering that RGG−RGG condensates have a viscosity (1.6 Pa s) lower than those of most other IDP condensates, 52 we expect the NanoLuc reaction to be diffusion-limited in most condensates that have a comparable or higher viscosity. We believe our model system can be easily translated to other condensate platforms by replacing the RGG domain with the pathological IDR, or incorporating the NanoLuc into composite condensates.…”

Section: ■ Results and Discussionmentioning

confidence: 98%

“…The Stokes−Einstein equation gives an estimate of the diffusion coefficient: D = kT/(6πηa), where k is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the diffusion medium, and a is the radius of the diffusing molecule. Using the reported viscosity value of RGG−RGG condensates (1.6 Pa s) 52 and an estimated simple radius of furimazine calculated from the topological polar surface area (0.2 nm), we estimate a diffusion coefficient of furimazine in RGG-based condensates of ≈0.7 μm 2 /s. This indicates that for a condensate with a radius of 1 μm, the diffusion time scale of the substrate is on the order of 1−2 s; for a condensate with a radius of 5 μm, however, the diffusion time scale of the substrate is on the order of 35 s. We believe this diffusion-limited behavior is potentially useful, as condensate viscosity might affect the light output when screened by a microplate reader.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

et al. 2021

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Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid–liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their propensity to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of a novel enzymatic activity in protein coacervates with the light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of Caenorhabditis elegans LAF-1, we functionalized condensates with enzymatic activity in vitro and show that enzyme localization to coacervates enhances assembly and activity of split enzymes. To build condensates that function as light-emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and displayed significantly increased reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. Because our model condensates have low viscosities, we predict NanoLuc diffusion-limited behavior in most other condensates and thus propose the condensate-Nanoluc system as a potential strategy for high-throughput screening of condensate targeting drugs. By splitting the NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. In addition, we demonstrate control of the spatial localization of the enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity and that this activity can be spatially and temporally controlled via biochemical reconstitution and design of protein surfactants.

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Section: ■ Results and Discussionmentioning

confidence: 98%

Section: ■ Results and Discussionmentioning

confidence: 99%

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

et al. 2021

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Multiphase condensates from a kinetically arrested phase transition

Erkamp

Šneideris

Ausserwöger

et al. 2022

Preprint

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The formation of biomolecular condensates through liquid-liquid phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but contain an internal structure consisting of distinct sub-compartments with different compositions. In many instances, such compartments inside the condensate are depleted in the biopolymers that make up the condensate. Here, we describe that this multiphase structure arises from a kinetically arrested phase transition. The combination of a change in composition coupled with a slow response to this change can lead to the spontaneous formation of multiple emulsions consisting of several inner cores within a polymer-rich middle phase. In the case of liquid-like biomolecular condensates, the slow diffusion of biopolymers causes nucleation of biopolymer-poor liquid inside of the condensate to achieve the new equilibrium composition. This framework shows that multiphase condensates can be a result of kinetic trapping, rather than thermodynamic stability, and provides and avenue to understand and control the internal structure of condensates in vitro and in vivo.

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Membrane curvature governs the distribution of Piezo1 in live cells

Yang

Arnold

et al. 2022

Preprint

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Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells, leading to depletion of Piezo1 from filopodia. Quantification of the curvature-dependent sorting of Piezo1 directly reveals its nano-geometry in situ. Piezo1 density on filopodia increases upon activation, independent of Ca2+, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that is abolished with channel activation.

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More than just oil droplets in water: surface tension and viscosity of protein condensates quantified by micropipette aspiration

Cited by 6 publications

References 52 publications

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Multiphase condensates from a kinetically arrested phase transition

Membrane curvature governs the distribution of Piezo1 in live cells

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