Liquid-Liquid Phase Separation inside Giant Vesicles Drives Shape Deformations and Induces Lipid Membrane Phase Separation

Abstract

An astounding variety of cellular contexts converge to the process of liquid-liquid phase separation for the creation of new functional levels of organization. But the kinetic pathways by which intracellular phase separation proceeds – typically in physically confined and macromolecularly crowded volumes of topologically closed cellular and intracellular compartments –remain incompletely understood. Here, we monitor the dynamics of liquid-liquid phase separation of mixtures of phase-separating polymers (i.e., polyethyleneglycol and dextran) inside all-synthetic, cell-sized giant unilamellar vesicles in real-time. We dynamically trigger phase separation by subjecting an initially homogeneous polymer solution inside vesicles to an abrupt osmotic quench. The latter removes water and elevates polymer concentrations in the phase-coexistence regime thereby initiating a segregative phase separation of the polymers. We find that the ensuing relaxation – en route to the new equilibrium – is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The early trajectory of droplet coarsening exhibit significant acceleration, but a competing process of membrane-droplet interactions – one in which the membrane boundary is preferentially wetted by one of the incipient phases – dynamically arrests the progression and deforms the membrane. As a result, a novel multi-bud morphology, reminiscent of cellular blebs, decorate the vesicle surface. Furthermore, when the vesicles are composed of phase-separating mixtures of common lipids, the three-dimensional liquid-liquid phase separation within the vesicular interior becomes coupled to the membrane’s compositional degrees of freedom producing microphase-separated membrane textures. This coupling of bulk and surface phase separation processes suggests a new physical principle by which liquid-liquid phase separation inside living cells might be dynamically regulated and materially communicated inside-out to the cellular boundaries.

Endocytosis of Coacervates into Liposomes

“…9 Several groups have reported small coacervates encapsulated in liposomes without apparent wetting, 10 but coacervate droplets can also partially wet 11 and remodel membranes in such structures. 12 In a different study, small phospholipid vesicles were found to assemble at the surface of large complex coacervate droplets without apparent deformation. 13 However, when similar coacervate droplets were added to dried lipid films 14 or mixed with ethanolic lipid solutions, 15 membrane remodeling was observed, resulting in the assembly of a continuous, coacervate-supported phospholipid bilayer.…”

“…Membranes and coacervates have also been combined in the field of artificial cells to create hierarchically organized compartments or hybrid protocells . Several groups have reported small coacervates encapsulated in liposomes without apparent wetting, but coacervate droplets can also partially wet and remodel membranes in such structures . In a different study, small phospholipid vesicles were found to assemble at the surface of large complex coacervate droplets without apparent deformation .…”

Interfacing Coacervates with Membranes: From Artificial Organelles and Hybrid Protocells to Intracellular Delivery

“…Keating and co-workers studied phase ordering inside GUVs to understand how molecules phase separate and stabilize over time inside cells and whether it is possible to mimic this in vitro. [73] They used polyethylene glycol (PEG) and dextran that undergo segregative phase separation, forming a homogenous phase at low concentrations that separates into two co-existing phases, each with one polymer, beyond a threshold concentration. These components can be induced to phase separate inside GUVs by abrupt hypertonic stress, which elevates the polymer concentrations inside the GUVs as the water is expelled out.…”

Biomolecular condensates modulate membrane lipid packing and hydration

“…Recently, membranes have been reported to control the size of intracellular condensates and modify their material properties 14 . Furthermore, the crosstalk between membranes and condensates can promote phase separation coupling in the lipid and the protein phases 15,16,17 also shown in cytoplasm mimetic systems 18 . While the field of membrane-condensate interactions is rapidly gaining momentum, important cues are still missing in our understanding of the underlying mechanisms associated with the resulting structural changes and remodeling.…”