Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles
27 Feb 2007-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 104, Iss: 9, pp 3165-3170
TL;DR: It is demonstrated that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains, and GPMVs now provide an effective approach to characterize biological membrane heterogeneities.
Abstract: The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below ≈25°C. At 37°C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FceRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.
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TL;DR: The evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity is reviewed.
Abstract: Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.
3,811 citations
TL;DR: The membrane raft hypothesis formalized a physicochemical principle for a subtype of lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed membrane domains that selectively recruit certain lipids and proteins.
Abstract: Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.
1,349 citations
TL;DR: How the field has matured and an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in signalling, viral infection and membrane trafficking are presented.
Abstract: Ten years ago, we wrote a Review on lipid rafts and signalling in the launch issue of Nature Reviews Molecular Cell Biology. At the time, this field was suffering from ambiguous methodology and imprecise nomenclature. Now, new techniques are deepening our insight into the dynamics of membrane organization. Here, we discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in signalling, viral infection and membrane trafficking.
1,151 citations
Cites background from "Large-scale fluid/fluid phase separ..."
...So if one were to leave a plasma membrane in a resting state (no lipid synthesis or metabolism, no exocytosis or endocytosis, no interactions with peripheral proteins and no actin cytoskeleton), would membrane components phase-separate into large domains with time? This question has yet to be answered, but lipid-based phase separation into liquid-ordered-like and liquid-disordered-like phases has been seen in isolated giant plasma membrane vesicles (GPMVs) formed by membrane blebbin...
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TL;DR: The emerging principles of membrane architecture are reviewed with special emphasis on lipid organization and domain formation, which combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity.
Abstract: 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.
931 citations
Cites background from "Large-scale fluid/fluid phase separ..."
...Baumgart et al. (2007) showed that cells treated with paraformaldehyde and dithiothreitol produced membrane blebs that could be isolated as giant plasma membrane vesicles....
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TL;DR: In this paper, the authors provided a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity by combining 14 types of headgroups and 11 types of tails asymmetrically distributed across the two leaflets, closely mimicking an idealized mammalian plasma membrane.
Abstract: The detailed organization of cellular membranes remains rather elusive. Based on large-scale molecular dynamics simulations, we provide a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity. Our plasma membrane model consists of 63 different lipid species, combining 14 types of headgroups and 11 types of tails asymmetrically distributed across the two leaflets, closely mimicking an idealized mammalian plasma membrane. We observe an enrichment of cholesterol in the outer leaflet and a general non-ideal lateral mixing of the different lipid species. Transient domains with liquid-ordered character form and disappear on the microsecond time scale. These domains are coupled across the two membrane leaflets. In the outer leaflet, distinct nanodomains consisting of gangliosides are observed. Phosphoinositides show preferential clustering in the inner leaflet. Our data provide a key view on the lateral organization of lipids in one of life's fundamental structures, the cell membrane.
656 citations
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TL;DR: A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer that function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.
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