Fluorescence Anisotropy Measurements of Lipid Order in Plasma Membranes and Lipid Rafts from RBL-2H3 Mast Cells

412

307

A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid͞cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ؎ 0.4 Å, 39.5 ؎ 0.4 Å, 35.6 ؎ 0.6 Å, and 42.5 ؎ 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.

Section: Effect Of Cholesterol On the Bilayer Thickness Of Exocytic Psupporting

confidence: 90%

Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

Mitra

Ubarretxena-Belandia

Taguchi

et al. 2004

412

307

“…Ours were formed through cell swelling and blebbing, as in oncosis (53). These blebs have been shown to be free of cellular organelles, with lipid compositions representative of the plasma membrane, with phospholipid/cholesterol ratios of Ϸ2:1 (6,16,24). Our GPMVs from RBL cells contain constitutively active tyrosine kinase Lyn (N. Smith, D.A.H., and B.A.B., unpublished data).…”

Section: Discussionmentioning

confidence: 80%

Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

Baumgart¹,

Hammond

Sengupta

et al. 2007

760

854

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 Ϸ25°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 liquidordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcRI 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.liquid-disordered ͉ liquid-ordered ͉ membrane domains ͉ membrane heterogeneity ͉ rafts

“…Glycosylphosphatidylinositol (GPI)-anchored proteins in the exoplasmic leaflet (10,11) and lipid-anchored proteins in the cytoplasmic leaflet (12)(13)(14) of cell membranes were shown to be distributed nonrandomly in the plasma membrane with cluster sizes of 4-200 nm, but whether these correlate with lipid domains in a phase-separating system is not known. Indications for distinct lipid environments in living cells come from the measurements of different viscous drags for different bead-coupled proteins in the plasma membrane of fibroblasts (15) and from fluorescence anisotropy measurements of diphenyl chain-labeled phosphatidylcholine revealing liquid-ordered environments in the plasma membrane of mast cells (16) and in vivo images of liquid-ordered domains in macrophages labeled with 6-lauroyl-2-dimethylaminonaphthalene (17). However, the interpretations of the results obtained in all three cases may be questioned because of the invasive nature of the techniques.…”

mentioning

confidence: 99%

Phase coexistence and connectivity in the apical membrane of polarized epithelial cells

Meder

Moreno

Verkade

et al. 2006

158

125

Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature (Ϸ25°C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37°C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12°C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phaseseparated system with a continuous (percolating) raft phase <25°C in which isolated domains of the nonraft phase are dispersed, whereas at 37°C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.fluorescence recovery after photobleaching ͉ rafts ͉ paurdan ͉ liquid-ordered ͉ Madin-Darby-canine kidney T he domain organization of biological membranes is presently under intense scrutiny. In particular, the existence and role of sphingomyelin-and cholesterol-rich lipid bilayer phases, commonly known as rafts, have drawn much attention (1-5). Phase diagrams of model membrane systems made from ternary mixtures of sphingomyelin, 1-palmitoyl-2-oleoylphosphatidylcholine, and cholesterol show regions of fluid-fluid phase coexistence (6). The two fluid phases of special relevance are a liquid-ordered phase, characterized by high conformational and low translational order, and a liquid-disordered phase, characterized by low conformational and translational order (7,8). The coexistence of these two phases has been visualized by several laboratories in giant unilamellar vesicles and supported lipid bilayers prepared from synthetic lipids. More importantly, giant unilamellar vesicles prepared from cell membrane lipid extracts (9) also show visible fluid-fluid phase coexistence. However, there was no direct evidence for phase separation in native cell membranes (2, 5). Glycosylphosphatidylinositol (GPI)-anchored proteins in the exoplasmic leaflet (10, 11) and lipid-anchored proteins in the cytoplasmic leaflet (12-14) of cell membranes were shown to b...