Phase diagrams and tie lines in giant unilamellar vesicles

Blosser, Matthew C.; Cornell, Caitlin E.; Rayermann, Scott; Keller, Sarah L.

doi:10.1201/9781315152516-18

Cited by 4 publications

(6 citation statements)

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“…For example, mixing long, saturated lipids with short, unsaturated or methylated lipids typically results in thick Ld phases and thin Lo phases (16)(17)(18)(19)(20). Existing phase diagrams and tie-lines, which are reviewed in (34,35) and discussed further in the Supplementary Information, can be leveraged to quantitatively and accurately measure the relative amounts of Ld and Lo phases.…”

Section: Discussionmentioning

confidence: 99%

Direct imaging of liquid domains in membranes by cryo-electron tomography

Cornell

Mileant

Thakkar

et al. 2020

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

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Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.

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Section: Discussionmentioning

confidence: 99%

Direct imaging of liquid domains in membranes by cryo-electron tomography

Cornell

Mileant

Thakkar

et al. 2020

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

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“…Two-component systems are also favored because their tie-lines are parallel at different temperatures. In contrast, in a lipid membrane with three components, multiple challenges arise: I) the direction of each tie-line must be experimentally determined for every new set of molecules (39); II)the directions of tie-lines found by measuring area fractions may only approximate the directions found by measuring mole fractions (50); III) when the amount of only one component is changed, the system lies on an entirely new tie-line; and IV) the direction of the tie-line changes with temperature (20). As a result, when different labs seek to compare results in lipid bilayers, it is easy for them to verify that their systems lie along the same tie-line when they use 2-component membranes, and difficult when they use 3-component membranes.…”

Section: Resultsmentioning

confidence: 99%

“…Micron-scale, reversible, liquid-liquid phase separation has previously been observed in more complex membranes of living cells (1,36), where it has biological importance. For example, in yeast, phase separation of vacuole membranes promotes docking of lipid droplets during periods of nutrient restriction (37)(38)(39). Furthermore, yeast actively modify their lipid compositions to maintain their vacuole membranes near a miscibility transition (40).…”

Section: Discussionmentioning

confidence: 99%

“…The lipid di(14:0)-cholesteryl-6-O-phosphatidyl-α-d-glucopyranoside (CPG) was synthesized as previously described (45). The lipids 1-palmitoyl(16:0)-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1-oleoyl(18:0)-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), cholesteryl 6-O-palmitoyl-β-D-galactopyranoside (BbGL-1), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl(18:1)-phosphocholine (DPPC), diphytanoyl(16:0-4Me)-phosphocholine (diPhyPC), and dipalmitoyl(16:0)-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-DPPE) were purchased from Avanti Polar Lipids (Alabaster, AL).…”

Section: Methodsmentioning

confidence: 99%

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Sterol-lipids enable large-scale, liquid-liquid phase separation in bilayer membranes of only 2 components

Wilson,

Nguyen,

Gervay-Hague

et al. 2024

Preprint

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Despite longstanding excitement and progress toward understanding liquid-liquid phase separation in natural and artificial membranes, fundamental questions have persisted about which molecules are required for this phenomenon. Except in extraordinary circumstances, the smallest number of components that has produced large-scale, liquid-liquid phase separation in bilayers has stubbornly remained at three: a sterol, a phospholipid with ordered chains, and a phospholipid with disordered chains. This requirement of three components is puzzling for two reasons: (1) the Gibbs Phase Rule states that only two components are necessary, and (2) only two components are required for liquid-liquid phase separation in lipid monolayers, which resemble half of a bilayer. Inspired by reports that sterols interact closely with lipids with ordered chains, we tested whether phase separation would occur in bilayers in which a sterol and lipid were replaced by a single, joined sterol-lipid. By evaluating a panel of sterol-lipids, we discovered a minimal bilayer of only two components (PChemsPC and diPhyPC) that demixes into micron-scale, liquid phases. In this system, the sterol-lipid behaves as a 3:1 ratio of cholesterol to phospholipid. Our system gives the computation and theory community a two-component membrane that maps directly onto simplified theories and that can be used to validate simulation force fields. It suggests a new role for sterol-lipids in nature, and it gives experimental communities a membrane in which tie-lines (and, therefore, the lipid composition of each phase) are easily determined and will be consistent across multiple laboratories.Significance StatementA wide diversity of bilayer membranes, from those with hundreds of lipids (e.g., vacuoles of living yeast cells) to those with very few (e.g., artificial vesicles) phase separate into micron-scale liquid domains. The number of components required for liquid-liquid phase separation has been perplexing: only two should be necessary, but more are required except in extraordinary circumstances. What minimal set of molecular characteristics leads to liquid-liquid phase separation in bilayer membranes? This question inspired us to search for single, joined “sterol-lipid” molecules to replace both a sterol and a phospholipid in membranes undergoing liquid-liquid phase separation. By producing phase-separating membranes with only two components, we mitigate experimental challenges in determining tie-lines and in maintaining constant chemical potentials of lipids.

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“…For example, mixing long, saturated lipids with short, unsaturated or methylated lipids typically results in thick Ld phases and thin Lo phases (16)(17)(18)(19)(20). Existing phase diagrams and tie-lines, which are reviewed in (34,35) (38)). A remaining challenge is that the mCherry probe appears to strongly partition to the air-water interface of cryo-ET grids, and any protein at this location has the potential to denature.…”

Section: Discussionmentioning

confidence: 99%

Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography

Cornell¹,

Mileant

Thakkar

et al. 2020

Preprint

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Images of micron-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been lacking, leading to an inability to answer key questions. For example, researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two new methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under native solution conditions. The first method is optimized for probe-free applications:domains are directly distinguished from the surrounding membrane by their thickness. This technique measures area fractions of domains with quantitative accuracy, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future highperformance, dual-use probes. These developments have led to the first direct and quantitative imaging of submicron membrane domains under native conditions. 3 SIGNIFICANCE STATEMENT:Fluorescence micrographs that capture the sizes, shapes, and distributions of liquid domains in model membranes have provided high standards of evidence to prove (and disprove) theories of how micron-scale domains form and grow. Corresponding theories about smaller domains have remained untested, partly because experimental methods of identifying submicron domains in vesicles under native solvent conditions have not been available. Here we introduce two such methods. Both leverage cryo-electron tomography to observe membrane features far smaller than the diffraction limit of light. The first method is probe-free and identifies differences in thicknesses between liquid domains and their surrounding membranes. The second method identifies membrane regions labeled by an electron-dense, fluorescent protein, which enables direct comparison of fluorescence micrographs with cryo-electron tomograms. 4 INTRODUCTION:

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Phase diagrams and tie lines in giant unilamellar vesicles

Cited by 4 publications

References 1 publication

Direct imaging of liquid domains in membranes by cryo-electron tomography

Direct imaging of liquid domains in membranes by cryo-electron tomography

Sterol-lipids enable large-scale, liquid-liquid phase separation in bilayer membranes of only 2 components

Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography

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