Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles.
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.
Levental
Ilya
I
Department of Bioengineering, University of Pennsylvania, 1010 Vagelos Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA.
Byfield
Fitzroy J
FJ
Chowdhury
Pramit
P
Gai
Feng
F
Baumgart
Tobias
T
Janmey
Paul A
PA
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry*; D004058:Diffusion; D021962:Membrane Microdomains / Q000737:chemistry; D051379:Mice; D041681:NIH 3T3 Cells; D013050:Spectrometry, Fluorescence
D002784:Cholesterol