Differential Detection of Phospholipid Fluidity, Order, and Spacing by Fluorescence Spectroscopy of Bis-pyrene, Prodan, Nystatin, and Merocyanine 540

Wilson-Ashworth, Heather A.; Bahm, Quinn; Erickson, Joshua; Shinkle, Aaron; Vu, Mai; Woodbury, Dixon J.; Bell, John D.

doi:10.1529/biophysj.106.090860

Cited by 57 publications

(79 citation statements)

References 47 publications

(77 reference statements)

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“…To independently measure fluidity changes in membranes due to ethanol, we took advantage of the fact that nystatin, used for the fusion assay above, also fluoresces in membranes (64). The fluorescence of nystatin has been shown to reflect membrane fluidity (65). When a membrane is more rigid, nystatin partitions deeper into the bilayer and its fluorescence increases.…”

Section: Ethanol Changes Membrane Fluiditymentioning

confidence: 99%

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Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Paxman

Hunt

Hallan

et al. 2017

Biophysical Journal

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Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol's effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC 50 ¼ 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.

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Section: Ethanol Changes Membrane Fluiditymentioning

confidence: 99%

“…It is known that ethanol increases membrane fluidity and that cholesterol decreases fluidity by increasing the order and packing of the lipids (65). Furthermore, ethanol's disordering effect is more potent in cholesterol-containing membranes (66,67).…”

Section: Possible Mechanisms For Inhibition Of Fusion By Trans Additimentioning

confidence: 99%

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Paxman

Hunt

Hallan

et al. 2017

Biophysical Journal

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“…Finally, for microscopic arrangements membrane dynamics can be assessed by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). In general, the ability of fluorescent membrane probes to resolve different physical properties makes it interesting to combine them and their readouts for complementary information 13,14 .This also motivates additional fluorescence readouts for membrane dynamic studies to be considered, representing additional complementary information. Merocyanine 540 (MC540) is an anionic lipophilic polymethine dye, which binds superficially to the outer leaflets of membranes 15,16 , and which is sensitive to subtle differences in lipid packing 17 .…”

Section: Introductionmentioning

confidence: 99%

Trans–Cis Isomerization of Lipophilic Dyes Probing Membrane Microviscosity in Biological Membranes and in Live Cells

et al. 2015

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PostprintThis is the accepted version of a paper published in Analytical Chemistry. This paper has been peerreviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Chmyrov, V., Spielmann, T., Hevekerl, H., Widengren, J. (2015) Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells. Corresponding AuthorJerker Widengren: jerker@biomolphysics.kth.se.ABSTRACT Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins, and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540) was first analyzed by Fluorescence Correlation Spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics could then also be monitored of MC540 in lipid vesicles, and the influence of lipid polarity, membrane curvature and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photo-induced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. Based on these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics, and that the TRAST imaging technique can provide spatial maps of photo-induced isomerization as well as both photo-induced and thermal back-isomerization, resolving differences in local membrane micro-viscosity in live cells.

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“…Fluorescence spectroscopy and microscopy provide indirect information at lower resolution, but can be used in both model systems and living membranes with manageable perturbation to the bilayer. The ability of fluorescent membrane probes to resolve physical properties can be improved by making multiple measurements on the same system with various probes that sense different properties (12,13).…”

mentioning

confidence: 99%

“…An initial step toward that end was to develop the use of multiple probes to assess properties of different thermodynamic phases in a well-characterized system. Accordingly, we have used the fluorescent probes nystatin, prodan, bispyrene, merocyanine 540 (MC540), diphenylhexatriene (DPH), and laurdan to map lipid behavior across the dipalmitoylphosphotidylcholine (DPPC)-cholesterol phase diagram (13). This report takes the next steps toward achieving our goal.…”

mentioning

confidence: 99%

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

Stott

McLemore

et al. 2008

Journal of Lipid Research

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The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-b-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquiddisordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquidordered behavior observed in the various types of liposomes as well as some interesting

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Differential Detection of Phospholipid Fluidity, Order, and Spacing by Fluorescence Spectroscopy of Bis-pyrene, Prodan, Nystatin, and Merocyanine 540

Cited by 57 publications

References 47 publications

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Trans–Cis Isomerization of Lipophilic Dyes Probing Membrane Microviscosity in Biological Membranes and in Live Cells

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

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