Liquid-liquid phase separation within fibrillar networks

Abstract

Complex fibrillar networks mediate liquid-liquid phase separation of biomolecular condensates within the cell. Mechanical interactions between these condensates and the surrounding networks are increasingly implicated in the physiology of the condensates and yet, the physical principles underlying phase separation within intracellular media remain poorly understood. Here, we elucidate the dynamics and mechanics of liquid-liquid phase separation within fibrillar networks by condensing oil droplets within biopolymer gels. We find that condensates constrained within the network pore space grow in abrupt temporal bursts. The subsequent restructuring of condensates and concomitant network deformation is contingent on the fracture of network fibrils, which is determined by a competition between condensate capillarity and network strength. As a synthetic analog to intracellular phase separation, these results further our understanding of the mechanical interactions between biomolecular condensates and fibrillar networks in the cell.

Fig. 1. Kinetics of liquid–liquid phase separation within fibrillar networks.

a Schematic depicting the phase separation of oil condensates within a fibrillar network. Blue represents water, tan represents ethanol, green represents agarose fibrils, and red represents decane. b Bright-field and fluorescence confocal microscopy time series showing the condensation of decane (bright-field and red) within a 0.8% w/w agarose network (green) (Supplementary Movies 1 and 2). Growth of the condensate within the fibrillar network occurs between (b_1–4), followed by network deformation (b₅), and finally fracture of the restraining element indicated by the arrow in (b₅), which leads to fluid rearrangement and expansion of a cavity within the network (b₆). c Evolution of the condensate in-plane area vs. time, where the in-plane area is determined from the bright-field images. Numbered red circles correspond to panels (b_1–3). Abrupt jumps are observed at t = 26 and 66 s, at numerals I and II. The corresponding regions are marked in (b₂) and (b₃). d Total in-plane area of all condensates in the full field-of-view movie as a function of time (Supplementary Movies 3 and 4). Area growth ceases by approximately t = 500 s, indicating that the oil solute has been fully depleted.

Fig. 2. Condensate growth via abrupt interface jumps.

a Fluorescence confocal microscopy time series showing abrupt growth jumps of a decane condensate (red) within a 0.3% w/w agarose network (green). The yellow shapes identify regions I and II which appear in abrupt interface jumps. b Evolution of condensate in-plane area vs. time. Numbered red circles correspond to (a_1–4). Abrupt growth jumps are observed at gray numerals I and II, corresponding to the regions marked in (a₂) and (a₄). c Width of the condensate lobe between the blue markers of (a₁), plotted vs. time. Sudden reductions in the lobe width are experienced at t = 54 and 74 s. These reductions occur concurrently with the growth jumps in (b), indicating that regions I and II form via fluid redistribution from pre-existing lobes. Vertical dashed lines between (b) and (c) are guides to the eye.

Fig. 3. Fibril fracture and network compaction by restructuring condensates.

a, b Confocal fluorescence microscopy time series of a decane condensate (red) restructuring into a roughly spherical droplet after fracturing restraining fibrils in a 0.8% w/w agarose network (green). Numbered white arrows in (b_1–3) indicate the individual network elements which restrain the condensate. c Time evolution of the condensate width as measured between the blue markers in (a₁). Numbered red circles correspond to panels (a_1–5). At t = 152 s, the left restraining element in (b₁) fractures, allowing for the condensate to expand slightly. At t = 628 s, restraining element 2 fractures, allowing the tortuous condensate to minimize its surface area by restructuring into a roughly spherical droplet. The inset shows a zoom-in to the fracture event in which fibril elongation is highlighted in green. d Profiles of the edge of the oil droplet at different times throughout the cavity formation process (example profile shown in blue in a₁). Profiles are colored according to the intensity of the network fluorescence along the profile, normalized to the mean background intensity. The white curves, perpendicular to the colored profiles, are expansion trajectories. e Spatial profile of the relative network material density, ρ, plotted along the black trajectory at t = 444, 632, and 692 s (corresponding to b_3–5). The network fluorescence intensity is used as a proxy for ρ and is normalized to the mean background intensity. The distance along the trajectory, λ, is normalized to the mesh radius, ξ/2 = 0.65 μm. The circle, star, and triangle mark the peak density and correspond to the markers in (b_3–5, d, and f). f Peak density extracted from (d) as a function of λ along the black and orange expansion trajectories, depicting the rise in peak ρ as the restructuring condensate drives the cavity to expand and compactifies the surrounding network.

Fig. 4. Structure of the densified network around restructured condensates.

a, b Cross-sectional SEM images of a 2.0% w/w agarose gel after oil phase separation, network fracture, and cavity expansion. The sample is frozen in liquid-nitrogen-cooled liquid ethane to circumvent freezing artifacts, and the oil and aqueous phases are subsequently removed by lyophilization. c Bright-field and confocal fluorescence microscopy images showing the densified network (green) precluding the coalescence of two adjacent oil droplets (bright-field) in a 1.3% w/w gel.

Fig. 5. Dissolution experiments demonstrate that network deformation is plastic.

a Bright-field and confocal fluorescence microscopy time series showing the dissolution of a decane condensate (bright-field) in a 0.3% w/w agarose gel (green). The network cavity in (a₃) has a 20% smaller area than the cavity in (a₁). No surfactant is present in this experiment. b Confocal fluorescence microscopy images depicting a decane condensate (red) in a 0.8% w/w agarose gel (green), before and after dissolution. The surfactant laureth-4 has been added at a concentration of 5% v/v to reduce the oil–water interfacial tension, ${\gamma }_{\mathrm{ow}}$, and prevent network fracture. The network is deformed slightly by the condensate, and this deformation persists after oil dissolution; white arrows are a guide to identify this mesh-scale network deformation.

Fig. 6. Competition between condensate capillarity and network strength.

a Morphology state diagram of oil condensates as a function of gel concentration and interfacial tension, γ_ow, which is varied via addition of the surfactant Triton X-100. The diagram is colored according to the average circularity of condensates present in a given sample (colorbar below diagram). Red dots correspond to samples which exhibit network fracture, as inferred by the presence of any condensates which possess a circularity above 0.95. The schematic shapes depict representative condensate morphologies which appear in the corresponding samples ①–④. b–e Confocal fluorescence micrographs of decane (red) and the agarose network (green), as well as probability distribution function histograms of the condensates’ circularity for samples ①–④.