Carnahan, C. F. | bioRxiv Science

bioRxiv2025

Apolipoprotein interaction induces shape remodeling and lipid phase separation in giant unilamellar vesicles

Apolipoprotein A-I (ApoA-I) - a 243-residue amphipathic protein containing an N-terminal globular domain and a primarily helical C-terminal lipid binding domain - is a principal protein component of high-density lipoprotein (HDL) or "good" cholesterol, which is an essential component of lipid homeostasis in humans. Synthesized in the liver and intestine and excreted in the blood, ApoA-I undergoes complex, cooperative, and dynamic self-assembly with membrane lipids, producing unlipi-dated (or weakly lipidated), nascent discoidal, and mature HDL states. In vitro studies demonstrate that the reconstitution of purified protein and lipids restores this cooperative self-assembly. However, the kinetic pathways by which these mesoscopic, proteolipidic assemblies form remain incompletely understood. Here, we monitor the dynamics of ApoA-I-membrane interactions through real-time monitoring of morphological changes, which ensue when ApoA-I is incubated with minimal giant unilamellar vesicles (GUVs) composed of single phospholipids or phase-separating phospholipid-cholesterol mixtures. Our fluorescence microscopy measurements reveal that the interaction initiates a gross, morphological remodeling of the parent vesicle proceeding through discrete stages involving membrane poration, solute leakage, vesiculation, and lipid-lipid phase separation. Our atomic force microscopy measurements confirm that the outcome includes discoidal nanoparticles. This qualitative phenomenology is robust and fully reproducible for different protein mutants and alleles (WT APOA-1, {Delta}49ApoA-I, ApoE-3, and ApoE-4) and other lipid mixtures (including mixtures containing phosphoserine lipids). Our molecular simulations recapitulate the essential shape changes and further reveal the composition dependence of the interactions. Together, these findings outline key steps in protein-lipid interactions that facilitate the assembly of mesoscopic reconstituted lipoproteins and nanodiscs.

biophysics↗

bioRxiv2024

Regulating biocondensates within synthetic cells via segregative phase separation

Living cells orchestrate a myriad of biological reactions within a highly complex and crowded environment. A major factor responsible for such seamless assembly are the preferential interactions between the constituent macromolecules, either associative or segregative, that can drive de-mixing to produce co-existing phases, and thus provide a dynamic intracellular compartmentalization. But how these two types of interactions, occurring simultaneously within the cytoplasmic space, influence each other is still largely unknown. This makes understanding and applying the molecular interactions that interfere with each other in such crowded environments crucial when engineering increasingly complex synthetic cells. Here, we show that the interplay between segregative and associative phase separation within cell-mimicking vesicles can lead to rich dynamics between them. Using on-chip microfluidic systems, we encapsulate the associative and segregative components in cell-sized containers and trigger their phase separations to create hierarchical structures that act as molecular recruiters, membrane targeting agents, and initiators of condensation. The obtained multiphase architecture provides an isolated microenvironment for condensates, restricting their molecular communication as well as diffusive motion, and leading to budding-like behaviour at the lipid membrane. In conclusion, we propose segregative phase separation as a universal condensate regulation strategy in managing molecular distribution, condensate location, as well as membrane interaction. We believe our approach will facilitate controlling the behaviour of membraneless organelles within synthetic cells.

synthetic biology↗