Correlated Fluorescence-Atomic Force Microscopy of Membrane Domains: Structure of Fluorescence Probes Determines Lipid Localization

Shaw, James E.; Epand, Richard M.; Li, Zaiguo; Bittman, Robert; Yip, Christopher M.

doi:10.1529/biophysj.105.073510

Cited by 181 publications

(177 citation statements)

References 49 publications

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“…We also find that glycan-patterned phase separation is thermally reversible -thus indicating that the effect is thermodynamic rather than kinetic -and that phase patterning results likely due to a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes [6][7][8] , potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes [9][10][11][12][13] , and may indicate a more intimate coupling between cellular lipidomes and glycomes than realized currently.…”

supporting

confidence: 69%

“…Lipid membranes were labelled 3 with trace amounts of fluorescent probes to visualize phase behaviour. Vesicle rupture was performed at 65 o C to ensure that the membranes, which consisted of up to five distinct lipid species, were fully mixed in a single macroscopically uniform liquid phase 11,13 . The glycan network-lipid membrane system was then cooled to room temperature for subsequent experiments.…”

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confidence: 99%

“…Liquid ordered (Lo) and solid ordered (So) domains coexisting with liquid disordered (Ld) domains on the glycan networks appear identical in shape, though other physical properties such as lipid diffusion coefficients vary as expected based on the known physical properties of these phases (SI, Supplementary Figure 4, 5). The lipid domains as a whole do not exhibit Brownian motion, have non-fluctuating boundaries, and do not coarsen over a two-day period, which differentiates their dynamics from multiphase lipid vesicles 11,25 and from solid-supported double bilayers 15,26,27 . Fourier analysis of the fluorescence intensity of the lipid phase-sensitive probes allows us to determine the characteristic length scale of the phase-separated domains, Llipid: we find that Llipid (green arrows) matches Lnetwork (red arrows) (Fig.…”

mentioning

confidence: 99%

“…We 7 conclude that the lipid domains are not trapped and that we are observing the inhomogeneous glycan network strongly modifying the characteristic length scale of phase separation, Llipid. While the phase behaviour of membranes with kinetically trapped domains is subject to several subtleties such as the incomplete dissolution of domains when temperature is cycled 26 , it is important to note, however, that unlike in previous reports 11,13,14,28,26 , we ensured that our vesicles were in a single uniform phase upon initial fusion onto the glycan networks. How do multiphase membranes behave on homogenous glycan networks, where there is no discernible Lnetwork?…”

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Glycans pattern the phase behaviour of lipid membranes

et al. 2012

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2Hydrated networks of glycans (polysaccharides) -in the form of cell walls, periplasms or gel-like matrices -are ubiquitously present adjacent to cellular plasma membranes 1-4 . Yet despite their abundance, the function of glycans in the extracellular milieu is largely unknown 5 . Here we show that the spatial configuration of glycans controls the phase behaviour of multiphase model lipid membranes: inhomogeneous glycan networks stabilize large lipid domains at the characteristic length scale of the network, whereas homogeneous networks suppress macroscopic lipid phase separation. We also find that glycan-patterned phase separation is thermally reversible -thus indicating that the effect is thermodynamic rather than kinetic -and that phase patterning results likely due to a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes 6-8 , potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes 9-13 , and may indicate a more intimate coupling between cellular lipidomes and glycomes than realized currently.Glycan-rich cell walls or extracellular matrices that are rigid in comparison to the plasma membrane surround most cells [1][2][3][4] . However, it is often reported that rigid supports cause non-'equilibrium' 14-16 behaviour of lipids and proteins in model lipid membranes. Indeed, while there are many reports of lipid membranes supported on non-biological polymers such as polyethylene glycol, polyacrylamide or polyethyleneimine [17][18][19][20] which seek to mitigate non-'equilibrium' behaviour, such systems have been used mainly to study the effects of polymer hydration 21 on the mobility of lipids 18,19 , or to preserve the function of transmembrane protein inclusions 17,22 . Furthermore, while it is widely acknowledged that a greater understanding of parameters that influence the size and length-scale of membrane domains is required [6][7][8][9][10][11][12][13]23,24 , as far as we know, systematic studies exploring the effects of biopolymers (the subject of this letter), or other more commonly used polymers in the field 17-20 on the phase behaviour of lipid membranes have not been conducted. To investigate the possible effects of biologically relevant polymers on the behaviour of membranes, we designed an in vitro solid-supported experimental platform that allows, through fluorescent labelling and confocal microscopy, the study of lipid membranes interacting with hydrated networks of glycans with arbitrary glycan composition and variable network configurations.We prepare glycan networks on flat hydrophilic surfaces using instability driven pattern formation (Supplementary Information (SI) for full details). Spontaneous rupture of giant lipid vesicles provided 2D lipid membrane patches that interact with the glycan networks. Lipid membranes were labelled 3 with trace amounts of fluorescent probes to visualize phase behaviour. Vesicle rupture was performed at 65 o C to ensu...

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Glycans pattern the phase behaviour of lipid membranes

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“…1D), 25-NBD-cholesterol (not shown), etc. (16,(30)(31)(32)(33)(34)(35)(36)(37)(38). Although there is considerable discussion regarding the solubility and orientation of these synthetic fluorescent sterol probes in membrane bilayers, especially 22-NBD-cholesterol and 25-NBD-cholesterol, at low concentrations some are thought to mimic the distribution and orientation of cholesterol (16,(35)(36)(37) (64,(69)(70)(71).…”

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Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

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Lipid Domains, Chemistry of

Epand

2008

Wiley Encyclopedia of Chemical Biology

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Biological membranes are composed largely of proteins and lipids. A wide range of molecular structures exists among these molecules. It is therefore not surprising that biological membranes are not uniform but rather cluster certain molecules in specific regions or domains. This behavior can be mimicked in model systems comprising a limited number of components to study in more detail the molecular nature of this domain formation. Two types of domains exist that have been more extensively studied. One is a membrane domain enriched in polyanionic lipids, such as phosphatidylinositol diphosphate. Such domains are formed by the presence of proteins with segments containing several cationic amino acid residues. Such proteins have been termed “pipmodulins.” Another kind of domain is formed as a consequence of the nonuniform distribution of cholesterol in the membrane. Caveolae represent one type of cholesterol‐rich domain that is well characterized. Other cholesterol‐rich domains are termed “rafts.” Characterization of rafts in model membranes is well documented, but the nature of cholesterol‐rich domains in biological membranes remains a subject of controversy. Several imaging and fluorescence methods are being employed to further characterize the size and lifetime of small raft domains in biological membranes.

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Correlated Fluorescence-Atomic Force Microscopy of Membrane Domains: Structure of Fluorescence Probes Determines Lipid Localization

Cited by 181 publications

References 49 publications

Glycans pattern the phase behaviour of lipid membranes

Glycans pattern the phase behaviour of lipid membranes

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Lipid Domains, Chemistry of

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