Lipid Peroxides Promote Large Rafts: Effects of Excitation of Probes in Fluorescence Microscopy and Electrochemical Reactions during Vesicle Formation

Ayuyan, Artem G.; Cohen, Fredric S.

doi:10.1529/biophysj.106.087387

Cited by 231 publications

(248 citation statements)

References 59 publications

Supporting

Mentioning

232

Contrasting

Order By: Relevance

“…Noting that phase separation in vesicles labeled with fluorescent CTB and annexin V derivatives is similar to lipid-bound fluorophores (R-DOPE, DiIC16:0, and Nap) excludes the possibility that the fluid phase segregation we detect is due to membrane insertion of fluorescent lipids or to photodecomposition products of membrane-embedded fluorophores. Photoinduced lipid oxidation, which can interfere with membrane phase behavior (32), was minimized by the antioxidant DTT.…”

Section: Resultsmentioning

confidence: 99%

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

Baumgart¹,

Hammond

Sengupta

et al. 2007

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

760

854

View full text Add to dashboard Cite

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

show abstract

Section: Resultsmentioning

confidence: 99%

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

Baumgart¹,

Hammond

Sengupta

et al. 2007

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

760

854

View full text Add to dashboard Cite

show abstract

“…Here, a case that plagues the use of fluorescence microscopy is the introduction of artifactual visible domains by intense illumination (Zhao et al 2007b). This might be caused by free radical initiated polymerization that starts from the reactive excited singlet state (Ayuyan and Cohen 2006). More interesting examples come from aggregating ganglioside by binding cholera toxin B subunit, resulting in large domains (Hammond et al 2005).…”

Section: Discussionmentioning

confidence: 99%

Phase Separation in Lipid Membranes

Heberle¹,

Feigenson²

2011

Cold Spring Harbor Perspectives in Biology

220

180

View full text Add to dashboard Cite

Cell membranes show complex behavior, in part because of the large number of different components that interact with each other in different ways. One aspect of this complex behavior is lateral organization of components on a range of spatial scales. We found that lipidonly mixtures can model the range of size scales, from approximately 2 nm up to microns. Furthermore, the size of compositional heterogeneities can be controlled entirely by lipid composition for mixtures such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol or sphingomyelin (SM)/DOPC/POPC/cholesterol. In one region of special interest, because of its connection to cell membrane rafts, nanometer-scale domains of liquid-disordered phase and liquid-ordered phase coexist over a wide range of compositions. SCALES OF SPATIAL ORGANIZATION OF CELL MEMBRANESC ell membranes have daunting complexity, including but not limited to the scales of spatial organization that emerge from a complex system. Complex behavior arises in part from the large number of different kinds of lipids and proteins, the different ways in which these membrane components interact with each other and with the rest of the cell, and the dynamic processes that locally and dramatically change significant fractions of the membrane. To appreciate the nature of this behavior, various key aspects of the complexity must be examined. Although a complete picture has not yet emerged, the available data are compelling that compositional heterogeneity, rather than random mixing of membrane lipids and proteins, describes cell membranes (Lingwood and Simons 2010). Here, we focus on one specific subset of complex membrane behavior: lateral heterogeneity based on lipid -lipid interactions in multicomponent bilayer mixtures. The impetus for examining such mixtures is the apparent connection between the coexistence of liquiddisordered (Ld) and liquid-ordered (Lo) phases observed in some simple, lipid-only bilayers, and the properties of lipid rafts observed in some cellular membranes. One possible starting point for experimental study is to find the simplest system that shows a wide range of organization comparable to what is found in cells, that is, from nanometers to microns. This turns out

show abstract

“…NMR allows us to directly measure differences in ordering and partitioning of DPPC-d 62 between coexisting phases. In addition, NMR produces results free from artifacts associated with fluorescent probes (24) or oxidation of unsaturated lipid chains (15,25). We use our NMR data to identify quantitative miscibility phase boundaries and to solve for a large array of tie-lines.…”

mentioning

confidence: 99%

Critical fluctuations in domain-forming lipid mixtures

Veatch

Soubias

Keller

et al. 2007

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

430

525

View full text Add to dashboard Cite

Critical fluctuations are investigated in lipid membranes near miscibility critical points in bilayers composed of dioleoylphosphatidylcholine, chain perdeuterated dipalmitoylphosphatidylcholine, and cholesterol. Phase boundaries are mapped over the temperature range from 10°C to 60°C by deuterium NMR. Tie-lines and three-phase triangles are evaluated across two-phase and three-phase regions, respectively. In addition, a line of miscibility critical points is identified. NMR resonances are broadened in the vicinity of critical points, and broadening is attributed to increased transverse relaxation rates arising from modulation of chain order with correlation times on a microsecond time scale. We conclude that spectral broadening arises from composition fluctuations in the membrane plane with dimensions of <50 nm and speculate that similar fluctuations are commonly found in cholesterolcontaining membranes.lipid rafts ͉ liquid-immiscibility ͉ NMR relaxation ͉ phase diagram ͉ critical behavior T he first direct observation of immiscible liquid phases in model bilayer membranes was presented only recently, for membranes containing three or more components (1, 2). However, by the early 1970s, it was already clear that lipids in membranes with elevated cholesterol have high chain order yet diffuse as in a liquid (3-6). This liquid-ordered (L o ) phase of lipids (7) is distinguished from the familiar liquid-disordered phase (L ␣ or L d ) found in bilayers of pure lipids above the chain melting temperature (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Pure lipids below the chain melting temperature are in the solid or ''gel'' phase (S o ). Coexisting liquid-ordered and liquid-disordered phases are easily visualized in giant unilamellar vesicles by fluorescence microscopy when membranes contain cholesterol and two types of lipids: one with a high gel-fluid transition temperature and one with a low gel-f luid transition temperature (15-21). Coexisting liquid phases are also easily visualized in membranes of plasma membrane vesicles containing a wide spectrum of lipid and protein components (22).The research presented here represents a culmination of several years' work in our laboratories on ternary membranes of dioleoylphosphatidylcholine (DOPC), chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d 62 ), and cholesterol (Chol) (15-18, 23, 24). It has resulted in a straightforward thermodynamic description of micrometer-sized coexisting phases and of submicrometer composition fluctuations in membranes containing cholesterol. Here we use deuterium ( 2 H) NMR to assemble the DOPC/DPPC-d 62 /Chol phase diagram. NMR allows us to directly measure differences in ordering and partitioning of DPPC-d 62 between coexisting phases. In addition, NMR produces results free from artifacts associated with fluorescent probes (24) or oxidation of unsaturated lipid chains (15,25). We use our NMR data to identify quantitative miscibility phase boundaries and to solve for a large array of tie-lines. Tie-line endpoints yield quantitative co...

show abstract

Lipid Peroxides Promote Large Rafts: Effects of Excitation of Probes in Fluorescence Microscopy and Electrochemical Reactions during Vesicle Formation

Cited by 231 publications

References 59 publications

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

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

Phase Separation in Lipid Membranes

Critical fluctuations in domain-forming lipid mixtures

Contact Info

Product

Resources

About