Salt-dependent rheology and surface tension of protein condensates using optical traps

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquid-like phases. These protein condensates serve as simple models for the investigation of the more complex membrane-less organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties.Comment: 5 pages, 3 figures, 1 supplemen

Liquid-Liquid Phase Separation in an Elastic Network

Living and engineered systems rely on the stable coexistence of two interspersed liquid phases. Yet surface tension drives their complete separation. Here we show that stable droplets of uniform and tuneable size can be produced through arrested phase separation in an elastic matrix. Starting with an elastic polymer network swollen by a solvent mixture, we change the temperature or composition to drive demixing. Droplets nucleate and grow to a stable size that is tuneable by the network cross-linking density, the cooling rate, and the composition of the solvent mixture. We discuss thermodynamic and mechanical constraints on the process. In particular, we show that the threshold for macroscopic phase separation is altered by the elasticity of the polymer network, and we highlight the role of internuclear correlations in determining the droplet size and polydispersity. This phenomenon has potential applications ranging from colloid synthesis and structural colour to phase separation in biological cells.Comment: 6 figure

Multivalency ensures persistence of a +TIP body at specialized microtubule ends

Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential for cell division and morphogenesis. Here we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIP proteins Kar9 (functional homologue of APC, MACF and SLAIN), Bim1 (orthologous to EB1) and Bik1 (orthologous to CLIP-170). A multivalent web of redundant interactions links the three +TIPs together to form a ‘+TIP body’ at the end of chosen microtubules. This body behaves as a liquid condensate that allows it to persist on both growing and shrinking microtubule ends, and to function as a mechanical coupling device between microtubules and actin cables. Our study identifes nanometre-scale condensates as efective cellular structures and underlines the power of dissecting the web of low-afnity interactions driving liquid–liquid phase separation in order to establish how condensation processes support cell function.ISSN:1465-7392ISSN:1476-467

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

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.ISSN:1744-683XISSN:1744-684

A solid beta-sheet structure is formed at the surface of FUS droplets during aging

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of beta-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.ISSN:1552-4450ISSN:1552-446