Insolubility of lipids in Triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

London, Erwin; Brown, Deborah A.

doi:10.1016/s0304-4157(00)00007-1

Cited by 632 publications

(518 citation statements)

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“…Different detergents, extraction procedures and cell/tissue sources have been used (Table 3). As already indicated by others (Banerjee et al, 1995;Chamberlain, 2004;Edidin, 2001a;London and Brown, 2000;Schuck et al, 2003), the detergent insolubility of proteins depends highly on the detergent and the extraction conditions used. DeBruin et al (2005) In addition to the detergent dependence of the results, it is not clear whether proteins…”

Section: Perspectivesmentioning

confidence: 83%

“…Within these phases, specific lipids (and proteins) may dynamically associate with each other to form functionally relevant platforms that are important in processes as diverse as membrane protein sorting, signaling and (caveolae-mediated) endocytosis. An operational basis for this lateral functional compartmentalization was given by the discovery that a specific set of membrane components was insoluble in cold (4°C) non-ionic detergent, toctylphenoxypolyethoxyethanol (Triton X-100, TX-100), resulting in a detergentresistant membrane (DRM) fraction that could be recovered by density gradient flotation (Brown and Rose, 1992;Brown and London, 2000;London and Brown, 2000;Simons and Ikonen, 1997). Resistance to detergent extraction has since become an operational definition of membrane rafts, and 'raft association' is defined as the partitioning of proteins and lipids into DRMs.…”

Section: The Raft Conceptmentioning

confidence: 99%

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Rafts in oligodendrocytes: Evidence and structure–function relationship

Gielen

Baron

vandeVen

et al. 2006

Glia

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Section: Perspectivesmentioning

confidence: 83%

Section: The Raft Conceptmentioning

confidence: 99%

Rafts in oligodendrocytes: Evidence and structure–function relationship

Gielen

Baron

vandeVen

et al. 2006

Glia

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“…Furthermore, a comment is needed here on another biochemical technique that is widely used to study lipid rafts. Detergent-resistant membrane (DRM) fractions that are prepared from model membranes and cell membranes are enriched in cholesterol and sphingomyelin, and contain a subset of lipid-anchored and integral plasmamembrane proteins [8][9][10] . These observations, among other things, have led to the assumption that L o domains, lipid rafts and DRMs can be considered as synonymous terms…”

Section: Box 1 How Does Cholesterol Drive Domain Formation In Membranes?mentioning

confidence: 99%

“…A DRM fraction is enriched in cholesterol and sphingomyelin and contains >240 proteins, including glycosylphosphatidylinositol (GPI)-anchored proteins, caveolin and a subset of transmembrane proteins [8][9][10]55 . The interpretation of DRM experiments is predicated on the assumption that liquid ordered (L o ) domains that exist in intact membranes at 37°C and their associated proteins are faithfully purified by cold-detergent extraction [8][9][10] . It is clear, however, that the association of a protein with a DRM fraction should not be considered sole evidence that a protein is associated with L o domains in intact cells.…”

Section: Box 2 Limitations Of Biochemical Approaches To Study Lipid Rmentioning

confidence: 99%

Lipid rafts: contentious only from simplistic standpoints

Hancock

2006

Nat Rev Mol Cell Biol

740

700

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The hypothesis that lipid rafts exist in plasma membranes and have crucial biological functions remains controversial. The lateral heterogeneity of proteins in the plasma membrane is undisputed, but the contribution of cholesterol-dependent lipid assemblies to this complex, non-random organization promotes vigorous debate. In the light of recent studies with model membranes, computational modelling and innovative cell biology, I propose an updated model of lipid rafts that readily accommodates diverse views on plasma-membrane micro-organization.A widely accepted hypothesis in contemporary cell biology is that freely diffusing, stable, lateral assemblies of sphingolipids and cholesterol, which are termed lipid rafts 1-3 , constitute an important organizing principle for the plasma membrane. The basic concept is that lipid rafts can facilitate selective protein-protein interactions by selectively excluding or including proteins. This lipid-based sorting mechanism has been widely implicated in the assembly of transient signalling platforms and more permanent structures such as the immunological synapse, as well as in the sorting of proteins for entry into specific exocytic and endocytic trafficking pathways [1][2][3] . Despite the undoubted theoretical utility of lipid rafts to many cell biological processes, the basic hypothesis that stable lipid rafts exist at all in biological membranes is under intense scrutiny 4,5 . This is partly because lipid rafts, if they exist in resting cell membranes, are too small to be resolved by fluorescent microscopy and have no defined ultrastructure; therefore, proving their existence is problematic. This article will consider the biophysical properties of model membranes that underpin the lipid raft hypothesis, and the limitations of the biochemical approaches that have been used to study rafts in biological membranes that largely account for the current debate on the hypothesis mentioned above. After examining the challenges that are involved in correlating observations, which have been made in model and cellular membranes, I focus on synthesizing recent data on the size of lipid domains in model membranes with observations that have been obtained by imaging intact plasma membranes. These imaging approaches include single particle tracking (SPT), single fluorophore video tracking (SFVT), fluorescence resonance energy transfer (FRET), homo-FRET and electron microscopy (EM). On the one hand, these studies challenge the simplistic null hypothesis that lipid-based assemblies, such as lipid rafts, do not exist in biological membranes. On the other hand, it is timely to reconsider the raft hypothesis in the light of these new data because the consensus model that emerges is more complex than a simplistic notion of stable, freely diffusing lipid rafts. Some intriguing new questions aboutCompeting interests statement The author declares no competing financial interests. DATABASES

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“…The definition of such lipid rafts is quite variable and sometimes controversial (Mayor & Rao, 2004;Munro, 2003;Simons & Gerl, 2010)-biochemists and cell biologists have somewhat different approaches in this regard. Biochemists regard lipid rafts as the membrane fractions which are insoluble or less soluble in nonionic detergents, e.g., Triton X-100, CHAPS, Brij 96, or Lubrol WX (London & Brown, 2000), although even these different detergents produce variable "raft" fractions. In this regard, Brij 96 and Lubrol WX have milder solubilizing potential than Triton X-100 (Schuck, Honsho, Ekroos, Shevchenko, & Simons, 2003); thus, "Lubrol rafts" may contain different membrane lipids and proteins than "Triton rafts."…”

Section: Article In Pressmentioning

confidence: 99%