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

Levental, Ilya; Byfield, Fitzroy J.; Chowdhury, P. K.; Gai, Feng; Baumgart, Tobias; Janmey, Paul A.

doi:10.1042/bj20091283

Cited by 152 publications

(160 citation statements)

References 33 publications

(46 reference statements)

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“…When chilled below room temperature, these membranes phase separated into L o -like and L d -like phases. The temperature and cholesterol dependence of this phase separation resembled that of simple model systems (Levental et al 2009). Other studies of plasma membranes blown up into giant spheres using a swelling procedure that separated the membranes from the influence of cytoskeletal and membrane trafficking processes showed cholesterol-dependent coalescence into micrometer-scale phases on 2]), or they contain acyl chains in addition to their TMD (3).…”

Section: Phase Separation In Plasma Membranesmentioning

confidence: 97%

Membrane Organization and Lipid Rafts

Simons¹,

Sampaio²

2011

Cold Spring Harbor Perspectives in Biology

886

806

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Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or interact with the lipids on either side of the two leaflets. Although recent advances in lipid analytics show that membranes in eukaryotic cells contain hundreds of different lipid species, the function of this lipid diversity remains enigmatic. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a twodimensional liquid with fascinating properties designed to perform the functions cells require. To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. This principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity. Here we will review the emerging principles of membrane architecture with special emphasis on lipid organization and domain formation.

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Section: Phase Separation In Plasma Membranesmentioning

confidence: 97%

Membrane Organization and Lipid Rafts

Simons¹,

Sampaio²

2011

Cold Spring Harbor Perspectives in Biology

886

806

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“…[12][13][14][15][16][17][18] In this report, we measure the collective mobility of a number of membrane components as a function of temperature in living cell membranes by fluorescence correlation spectroscopy (FCS). [19][20] Since collective mobility exhibits a discontinuity as a system passes through a miscibility phase transition, [21][22] these observations are expected to reveal the phase transition, even in cases where the phase domains are too small to resolve by direct imaging. We support this by a variety of Monte-Carlo and atomistic simulations, which show that FCS measurements of tagged particle diffusion would capture such transitions.…”

Section: Introductionmentioning

confidence: 99%

Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

et al. 2015

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Lipid:cholesterol mixtures derived from cell membranes, as well as their synthetic reconstitutions, exhibit well defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid:cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid phase separation is not generally observed in cell membranes and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte-Carlo and atomistic simulations on model lipid membrane systems. However, temperature dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes show a consistently smooth increase of the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14 -37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical paramters, such as tension, in the membrane.

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“…The microscopic phases observed in these studies are likely the result of coalescence of nanoscopic assemblies (lipid rafts) present in cellular membranes under physiological conditions (4). This coalescence into a condensed "raft phase" (5) allows microscopic investigation of the composition (1,6,7) and physical properties (8,9) of the underlying raft assemblies.…”

mentioning

confidence: 99%

Raft domains of variable properties and compositions in plasma membrane vesicles

Levental

Grzybek

Simons

2011

Proc. Natl. Acad. Sci. U.S.A.

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225

279

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Biological membranes are compartmentalized for functional diversity by a variety of specific protein-protein, protein-lipid, and lipidlipid interactions. A subset of these are the preferential interactions between sterols, sphingolipids, and saturated aliphatic lipid tails responsible for liquid-liquid domain coexistence in eukaryotic membranes, which give rise to dynamic, nanoscopic assemblies whose coalescence is regulated by specific biochemical cues. Microscopic phase separation recently observed in isolated plasma membranes (giant plasma membrane vesicles and plasma membrane spheres) (i) confirms the capacity of compositionally complex membranes to phase separate, (ii) reflects the nanoscopic organization of live cell membranes, and (iii) provides a versatile platform for the investigation of the compositions and properties of the phases. Here, we show that the properties of coexisting phases in giant plasma membrane vesicles are dependent on isolation conditions-namely, the chemicals used to induce membrane blebbing. We observe strong correlations between the relative compositions and orders of the coexisting phases, and their resulting miscibility. Chemically unperturbed plasma membranes reflect these properties and validate the observations in chemically induced vesicles. Most importantly, we observe domains with a continuum of varying stabilities, orders, and compositions induced by relatively small differences in isolation conditions. These results show that, based on the principle of preferential association of raft lipids, domains of various properties can be produced in a membrane environment whose complexity is reflective of biological membranes.T he recent discovery of phase separation in plasma membrane (PM) vesicles isolated from mammalian cells (1-3) is the most convincing evidence of functionally relevant coexistence of liquid domains in biological membranes. The microscopic phases observed in these studies are likely the result of coalescence of nanoscopic assemblies (lipid rafts) present in cellular membranes under physiological conditions (4). This coalescence into a condensed "raft phase" (5) allows microscopic investigation of the composition (1, 6, 7) and physical properties (8, 9) of the underlying raft assemblies.The current conception of lipid rafts in cell biology is of mesoscopic, isolated domains with defined properties and compositions. How lipid rafts in cells correspond to simple lipid model systems-which show complete, microscopic separation of a condensed, ordered phase (L o ) rich in saturated lipids and sterols from a disordered (L d ) phase depleted of these components and enriched in unsaturated glycerophospholipids (10, 11)-is yet to be determined. Large-scale membrane domains are not observable in intact cells without significant perturbation (e.g., by antibody cross-linking; ref. 12), so the submicroscopic membrane architecture has been inferred from biochemical fractionation of whole cells or spectroscopic techniques that probe the level of individual molecules. In con...

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

Cited by 152 publications

References 33 publications

Membrane Organization and Lipid Rafts

Membrane Organization and Lipid Rafts

Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

Raft domains of variable properties and compositions in plasma membrane vesicles

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