Phospholipid membrane formation templated by coacervate droplets

Abstract

We report formation of coacervate-supported phospholipid membranes by hydrating a dried lipid film in the presence of coacervate droplets. In contrast to traditional giant lipid vesicles formed by gentle hydration in the absence of coacervates, the coacervate-templated membrane vesicles are more uniform in size, shape, and apparent lamellarity. Due to their fully-coacervate model cytoplasm, these simple artificial cells are macromolecularly crowded and can be easily pre-loaded with high concentrations of proteins or nucleic acids. Coacervate-supported membranes were characterized by fluorescence imaging, polarization, fluorescence recovery after photobleaching of labeled lipids, lipid quenching experiments, and solute uptake experiments. Our findings are consistent with the presence of lipid membranes around the coacervates, with many droplets fully coated with what appear to be continuous lipid bilayers. Within the same population, other coacervate droplets are coated with membranes having defects or pores that permit solute entry, and still others are coated with multilayered membranes. These membranes surrounding protein-based coacervate droplets provided protection from a protease added to the external solution. The simplicity of producing artificial cells having a coacervate model cytoplasm surrounded by a model membrane is at the same time interesting as a potential mechanism for prebiotic protocell formation and appealing for biotechnology. We anticipate that such structures could serve as a new type of model system for understanding interactions between intracellular phases and cell- or organelle membranes, which are implicated in a growing number of processes ranging from neurotransmission to signaling.

A self-templated route to monodisperse complex droplets as artificial extremophile-mimic from coacervate-liposome interplay

“…The assembly of phospholipid membrane on coacervates represents a further step to construct synthetic cells with both hallmarks of cell envelope and cytoplasm and may bridge the gap between coacervate-based protocell models and membrane bounded biological cells. Bare coacervates have recently been utilized templates to incubate with lipids in ethanol or hydrated lipid film, resulting in polydisperse giant phospholipid vesicles with apparent lamellarity 37,38 . In this work, through the freeze-thaw treatment of monodisperse coacervates coated by DOPC/DOPS/Chol (37/3/10) liposomes, we demonstrate the first example for the bulk assembly of monodisperse GUVs (Fig.…”

“…4b). Recent work indicated that coacervate-supported phospholipid membrane contained defects and thus was leaky 37 . Here, we show that the membrane permeability of coacervate-supported GUVs can be tunable.…”

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering

“…However, relevant life-like properties driven by protein networks observed in single compartment LLPS system, such as division, have also been observed (Song et al 2018). Nevertheless, further study of enzymatic cascades or networks could be complemented by recent studies demonstrating a wide range of multiphase membraneless droplet systems (Mountain and Keating 2020Keating , 2021Lu and Spruijt 2020;Sato et al 2020). These systems should be subjects of further study, perhaps even as a mechanism to show prebiotically relevant catalytic ribozyme cascades within a primitive compartment.…”

“…Encapsulation or assembly of LLPS systems within liposomes or vesicles could result in the formation of an artificial cell with similarities to membraneless compartment-containing modern cells. For example, various aspects of the structure and function of a number of liposome-encapsulated LLPS systems have been studied (Helfrich et al 2002;Long et al 2005;Li et al 2008;Crowe and Keating 2018;Liu et al 2019;Love et al 2020;Cakmak et al 2021). While these studies share a common goal, they often differ in the construction of each system.…”

“…Other studies utilized chemical and environmental changes, such as pH, to induce phase separation of encapsulated components of LLPS droplets within pre-assembled liposomes (Love et al 2020;Last et al 2020). Finally, direct assembly of lipid membrane constituents around pre-assembled phase-separated droplets, akin to LLPS-scaffolded membranes, has also been performed (Tang et al 2014;Cakmak et al 2021). In particular, the latter two techniques would have been quite relevant to primitive LLPS-in-vesicle systems, as environmental oscillations could lead to pH or temperature changes.…”