Baumgart, T. et al. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc. Natl Acad. Sci. USA 104, 3165-3170

Department of Chemistry and Chemical Biology, Cornell University, Итак, New York, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2007; 104(9):3165-70. DOI: 10.1073/pnas.0611357104
Source: PubMed


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 approximately 25 degrees C. At 37 degrees 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 FcepsilonRI 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.

Download full-text


Available from: David Holowka
  • Source
    • "Cholesterol depletions were carried out by treating the cells with 10mM MβCD at 37 o C for 30 mins, followed by exogenous labeling of fluorescent probes. Stable membrane blebs for FCS measurements were generated by incubating the cells with 10% ethanol (v/v) in HEPES based buffer for 15-20 mins at 37 o C (Baumgart et al., 2007). Actin perturbations were effected by incubating the cells with actin-polymerization inhibitor Latrunculin A (LatA) at 2μM for 15 minutes before the FCS measurements. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Molecular diffusion at the surface of living cells is thought to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy (FCS) we measure the diffusion coefficient of a variety of cell-surface molecules over a temperature range 24-37°C. Predictably, exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature independent diffusion over this range, and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature independent behavior, thereby directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature-dependent when the filamentous dynamic actin nucleator, formin, is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus, the cell surface diffusion of GPI-anchored proteins and transmembrane protein that associate with actin, are driven by the active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface.
    Full-text · Article · Sep 2015 · Molecular biology of the cell
  • Source
    • "Because of their large size (up to 10 mm in diameter), they are easily observable by light microscopy (Sezgin et al., 2012a,b; Levental and Levental, 2015a,b). Most importantly, at certain temperatures, GPMVs separate into coexisting liquid ordered and liquid disordered phases (Baumgartet al., 2007; Sezgin et al., 2012a,b; Levental and Levental, 2015a,b). This capacity provides a powerful tool to study protein partitioning to ordered domains in biological membranes—a close proxy for lipid rafts in vivo. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Increasing evidence supports the existence of lateral nanoscopic lipid domains in plasma membranes, known as lipid rafts. These domains preferentially recruit membrane proteins and lipids to facilitate their interactions and thereby regulate transmembrane signaling and cellular homeostasis. The functionality of raft domains is intrinsically dependent on their selectivity for specific membrane components; however, while the physicochemical determinants of raft association for lipids are known, very few systematic studies have focused on the structural aspects that guide raft partitioning of proteins. In this review, we describe biophysical and thermodynamic aspects of raft-mimetic liquid ordered phases, focusing on those most relevant for protein partitioning. Further, we detail the variety of experimental models used to study protein-raft interactions. Finally, we review the existing literature on mechanisms for raft targeting, including lipid post-translational modifications, lipid binding, and transmembrane domain features. We conclude that while protein palmitoylation is a clear raft-targeting signal, few other general structural determinants for raft partitioning have been revealed, suggesting that many discoveries lay ahead in this burgeoning field. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Aug 2015 · Chemistry and Physics of Lipids
  • Source
    • "Melittin also exhibits anticancer properties [28] [29] that may be mediated by the activation of the ubiquitous enzyme, phospholipase A2 (PLA2) [30] [31], which catalyzes hydrolysis of sn-2 acyl bond of phospholipids resulting in the release of fatty acids [32]. This lipid–peptide interaction leads to changes in structure of cell plasma membranes causing vesiculation or blebbing [33]. These spherical outgrowths from the plasma membrane are due to the detachment of plasma membranes that are devoid of cytoskeleton material [34] [35]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labelling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labelled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, haemolysis and cytotoxicity, but not essential for cell membrane disruption. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jun 2015 · Biochimica et Biophysica Acta
Show more