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Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles.

ABSTRACT: Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

SUBMITTER: Levental I

PROVIDER: S-EPMC3118457 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles.

Levental Ilya I Byfield Fitzroy J FJ Chowdhury Pramit P Gai Feng F Baumgart Tobias T Janmey Paul A PA

The Biochemical journal 20091111 2

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on c ...[more]

PMID: 19811449

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Project description:Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12 h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.

Dataset Information

Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles.

Publications

Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles.

Similar Datasets

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