Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method

Ijavi, Mahdiye; Style, Robert W.; Emmanouilidis, Leonidas; Kumar, Anil; Meier, Sandro M.; Torzynski, Alexandre L.; Allain, Frédéric; Barral, Yves; Steinmetz, Michel O.; Dufresne, Eric R.

doi:10.1039/d0sm01319f

Cited by 42 publications

(52 citation statements)

References 46 publications

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“…2) is the slope measured from fitting the data linearly. The value of for Bik1 is measured as 2.25 ± 0.37 ( ⁄ ) which is consistent with the previously reported values of viscosity and surface tension for Bik1, 18.2 ( • ) and 7 ( ⁄ ), respectively (Ijavi et al, 2021).…”

Section: Droplet Fusion Analysissupporting

confidence: 91%

“…which is valid where the viscosity of the protein condensed phase ( ) is much bigger than the viscosity of the protein dilute phase ( ). For instance, viscosity of the Bik1 droplet phase has been reported previously as ≈ 18.2 ( • ) which is four orders of magnitude higher than the Bik1 dilute phase (Ijavi et al, 2021).…”

Section: Droplet Fusion Analysismentioning

confidence: 88%

“…Furthermore, previous studies have indicated that Kar9 carries at least one binding site for the Bik1 dimer in its C-terminal domain (Moore et al, 2006), which is consistent with our finding that Kar9 phase separates together with Bik1 (Figure 1C). Since Bik1 also undergoes phase separation on its own (Figure 1C; (Ijavi et al, 2021)), it must contain at least three homodimerization interfaces. Additionally, the CAP-Gly domain of Bik1 was shown to interact with the C-terminal tail of Bim1 (Stangier et al, 2018).…”

Section: Self-association Of Kar9mentioning

confidence: 99%

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High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

Meier

Farcas

Kumar

et al. 2021

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Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential notably during eukaryotic cell division. Here, we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIPs components Kar9 (functional homologue of APC, MACF, and SLAIN), Bim1 (orthologue of EB1), and Bik1 (orthologue of CLIP-170). Our data indicate that a redundant, multivalent web of interactions links the three +TIPs together to form a "Kar9 body" at the tip of a single cytoplasmic microtubule. They further suggest that this body is a liquid-like condensate, allowing it to persist on both growing and shrinking microtubule tips, and functions as a mechanical coupling device between microtubules and actin cables during mitosis. Our study underlines the power of dissecting the web of low-affinity interactions driving liquid-liquid phase separation of proteins in order to demonstrate the importance and establish the functional roles of condensation processes in vivo.

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Section: Droplet Fusion Analysissupporting

confidence: 91%

Section: Droplet Fusion Analysismentioning

confidence: 88%

Section: Self-association Of Kar9mentioning

confidence: 99%

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High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

Meier

Farcas

Kumar

et al. 2021

Preprint

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“…e Delta surface tension integrated along the droplets' edges overlapped on the fluid velocity field (black arrows) for the droplets in panel b . f Box plot of surface tension difference between the supernatant and droplet phase as a function of the pH of the supernatant, calculated with the sessile drop method 50 (inset). The central mark indicates the median, the top and bottom edges of the box the 25th and 75th percentile, the whiskers the maximum and minimum value of the data.…”

Section: Resultsmentioning

confidence: 99%

Sustained enzymatic activity and flow in crowded protein droplets

et al. 2021

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Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

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“…Finally, we note that in our model, the absence of an explicit solvent implies that, by construction, low-valency proteins will show lower interfacial free energy than highvalency ones. This is a reasonable approximation since high-valency proteins are usually expected to present higher pure component critical points and higher surface tensions with the solvent than low-valency proteins [95] (or engineered peptides [50]). However, high-valency proteins with specific domains enriched in hydrophilic residues, still could exhibit lower interfacial free energy with the solvent than low-valency hydrophobic species.…”

Section: Discussionmentioning

confidence: 99%

Valency and Binding Affinity Variations Can Regulate the Multilayered Organization of Protein Condensates with Many Components

et al. 2021

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Biomolecular condensates, which assemble via the process of liquid–liquid phase separation (LLPS), are multicomponent compartments found ubiquitously inside cells. Experiments and simulations have shown that biomolecular condensates with many components can exhibit multilayered organizations. Using a minimal coarse-grained model for interacting multivalent proteins, we investigate the thermodynamic parameters governing the formation of multilayered condensates through changes in protein valency and binding affinity. We focus on multicomponent condensates formed by scaffold proteins (high-valency proteins that can phase separate on their own via homotypic interactions) and clients (proteins recruited to condensates via heterotypic scaffold–client interactions). We demonstrate that higher valency species are sequestered to the center of the multicomponent condensates, while lower valency proteins cluster towards the condensate interface. Such multilayered condensate architecture maximizes the density of LLPS-stabilizing molecular interactions, while simultaneously reducing the surface tension of the condensates. In addition, multilayered condensates exhibit rapid exchanges of low valency proteins in and out, while keeping higher valency proteins—the key biomolecules involved in condensate nucleation—mostly within. We also demonstrate how modulating the binding affinities among the different proteins in a multicomponent condensate can significantly transform its multilayered structure, and even trigger fission of a condensate into multiple droplets with different compositions.

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Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method

Abstract: Phase separated macromolecules play essential roles in many biological and synthetic systems. We extend sessile droplet tensiometry to work with small quantities of such ultra-low surface tension droplets.

Cited by 42 publications

References 46 publications

High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast

Sustained enzymatic activity and flow in crowded protein droplets

Valency and Binding Affinity Variations Can Regulate the Multilayered Organization of Protein Condensates with Many Components

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