Publications by authors named "Gerald W Feigenson"

57 Publications

On the small size of liquid-disordered + liquid-ordered nanodomains.

Biochim Biophys Acta Biomembr 2021 Jun 25;1863(10):183685. Epub 2021 Jun 25.

Cornell University Department of Molecular Biology and Genetics, Room 201 Biotechnology Building, 215 Tower Rd. Ithaca, New York 14853, United States. Electronic address:

Four-component phase diagrams reveal that Liquid-disordered + liquid-ordered (Ld + Lo) nanodomains are exclusively found adjacent to a three-phase region, and so cannot be a one-phase microemulsion. Of importance for understanding biological membranes, a small change in lipid bilayer composition can change the size of these coexisting phase domains hundreds of fold, between tens of nanometers and microns. Nanodomain diameter, measured from small angle neutron scattering, is in the range 15-35 nm, consistent with stabilization by repulsive dipole fields. Ld/Lo line tension controls the Ld + Lo domain size transition. Other than size, chemical and physical properties of the phase domains do not seem to change during the transition. Unfavorable lipid-lipid pairwise interactions, rather than phase thickness mismatch, seem to be the main reason for Ld + Lo immiscibility. Pairwise interactions of cholesterol-phospholipid seem to be favorable, whereas pairwise interactions of high-melting phospholipid with low-melting phospholipid are unfavorable. Measured Ld/Lo line tension, like the phase separation, is created mainly by unfavorable lipid-lipid pairwise interactions. Lipid dipole-dipole repulsion opposes these unfavorable lipid-lipid pairwise interactions and thus, in a sense, is the reason that nanodomains form. Bilayer physical and chemical properties measured from macroscopic domains of coexisting Ld + Lo phases should be good approximations for the properties of coexisting nanoscopic domains.
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http://dx.doi.org/10.1016/j.bbamem.2021.183685DOI Listing
June 2021

Dataset of asymmetric giant unilamellar vesicles prepared via hemifusion: Observation of anti-alignment of domains and modulated phases in asymmetric bilayers.

Data Brief 2021 Apr 3;35:106927. Epub 2021 Mar 3.

Cornell University, United States.

The data provided with this paper are confocal fluorescence images of symmetric giant unilamellar vesicles (GUVs) and asymmetric giant unilamellar vesicles (aGUVs). In this work, aGUVs were prepared using the hemifusion method and are labelled with two different fluorescent dyes, named TFPC and DiD. Both dyes show strong preference for the liquid-disordered (Ld) phase instead of the liquid-ordered (Lo) phase. The partition of these dyes favoring the Ld phase leads to bright Ld phase and dark Lo phase domains in symmetric GUVs observed by fluorescence microscopy. In symmetric vesicles, the bright and the dark domains of the inner and the outer leaflets are aligned. In aGUVs, the fluorescent probe TFPC exclusively labels the aGUV outer leaflet. Here, we show a dataset of fluorescence micrographs obtained using scanning fluorescence confocal microscopy. For the system chosen, the fluorescence signal of TFPC and DiD show anti-alignment of the brighter domains on aGUVs. Important for this dataset, TFPC and DiD have fluorescence emission centered in the green and far-red region of the visible spectra, respectively, and the dyes' fluorescence emission bands do not overlap. This dataset were collected in the same conditions of the dataset reported in the co-submitted work (Enoki, et al. 2021) where most of aGUVs show domains alignment. In addition, we show micrographs of GUVs displaying modulated phases and macrodomains. We also compare the modulated phases observed in GUVs and aGUVs. For these datasets, we collected a sequence of micrographs using confocal microscopy varying the -position, termed a -stack. Images were collected in a scanning microscope Nikon Eclipse C2+ (Nikon Instruments, Melville, NY). Additional samples used to measure the lipid concentrations and to prepare GUVs with accurate lipid fractions are also provided with this paper.
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http://dx.doi.org/10.1016/j.dib.2021.106927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973298PMC
April 2021

Investigation of the domain line tension in asymmetric vesicles prepared via hemifusion.

Biochim Biophys Acta Biomembr 2021 Jun 26;1863(6):183586. Epub 2021 Feb 26.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States of America.

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase. To better understand the interleaflet interactions that influence domains, we compared asymmetric model membranes that capture salient properties of the PM with simpler symmetric membranes. Using asymmetric giant unilamellar vesicles (aGUVs) prepared by hemifusion with a supported lipid bilayer, we investigate the domain line tension that characterizes the behavior of coexisting ordered + disordered domains. The line tension can be related to the contact perimeter of the different phases. Compared to macroscopic phase separation, the appearance of modulated phases was found to be a robust indicator of a decrease in domain line tension. Symmetric GUVs of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/cholesterol (chol) were formed into aGUVs by replacing the GUV outer leaflet with DOPC/chol = 0.8/0.2 in order to create a cytoplasmic leaflet model. These aGUVs revealed lower line tension for the ordered + disordered domains of the exoplasmic model leaflet.
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http://dx.doi.org/10.1016/j.bbamem.2021.183586DOI Listing
June 2021

PI(4,5)P Clustering and Its Impact on Biological Functions.

Annu Rev Biochem 2021 Jun 13;90:681-707. Epub 2021 Jan 13.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850, USA; email:

Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P] composes only 1-2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P in membranes. Physiological multivalent cations such as Ca and Mg can bridge between PI(4,5)P headgroups, forming nanoscopic PI(4,5)P-cation clusters. The distinct lipid environment surrounding PI(4,5)P affects the degree of PI(4,5)P clustering. In addition, diverse cellular proteins interacting with PI(4,5)P can further regulate PI(4,5)P lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P behavior in both cells and model membranes, with emphasis on both multivalent cation- and protein-induced PI(4,5)P clustering. Understanding the nature of spatially separated pools of PI(4,5)P is fundamental to cell biology.
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http://dx.doi.org/10.1146/annurev-biochem-070920-094827DOI Listing
June 2021

Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition.

Chem Phys Lipids 2020 10 15;232:104972. Epub 2020 Sep 15.

Cornell University Department of Molecular Biology and Genetics, Room 201 215 Tower Rd. Ithaca, New York, 14853, United States. Electronic address:

The eukaryotic plasma membrane (PM) exhibits lipid mixing heterogeneities known as lipid rafts. These lipid rafts, the result of liquid-liquid phase separation, can be modeled by coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. Four-lipid component systems with a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol) can give rise to domains of different sizes. These four-component systems have been characterized in experiments, yet there are few studies that model the asymmetric distribution of lipids actually found in the PM. We used molecular dynamics (MD) simulations to analyze the transition from nanoscopic to macroscopic domains in symmetric and in asymmetric model membranes. Using coarse-grained MD simulations, we found that asymmetry promotes macroscopic domain growth in a case where symmetric systems exhibit nanoscopic domains. Also, macroscopic domain formation in symmetric systems is highly dependent on registration of like phases in the cytoplasmic and exoplasmic leaflets. Using united-atom MD simulations, we found that symmetric Lo domains are only slightly more ordered than asymmetric Lo domains. We also found that large Lo domains in our asymmetric systems induce a slight chain ordering in the apposed cytoplasmic regions. The chol fractions of phase-separated Lo and Ld domains of the exoplasmic leaflet were unchanged whether the system was symmetric or asymmetric.
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http://dx.doi.org/10.1016/j.chemphyslip.2020.104972DOI Listing
October 2020

Mechanisms of PI(4,5)P2 Enrichment in HIV-1 Viral Membranes.

J Mol Biol 2020 09 31;432(19):5343-5364. Epub 2020 Jul 31.

Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for HIV-1 virus assembly. The viral membrane is enriched in PIP2, suggesting that the virus assembles at PIP2-rich microdomains. We showed previously that in model membranes PIP2 can form nanoscopic clusters bridged by multivalent cations. Here, using purified proteins we quantitated the binding of HIV-1 Gag-related proteins to giant unilamellar vesicles containing either clustered or free PIP2. Myristoylated MA strongly preferred binding to clustered PIP2. By contrast, unmyristoylated HIV-1 MA, RSV MA, and a PH domain all preferred to interact with free PIP2. We also found that HIV-1 Gag multimerization promotes PIP2 clustering. Truncated Gag proteins comprising the MA, CA, and SP domains (MACASP) or the MA and CA domains (MACA) induced self-quenching of acyl chain-labeled fluorescent PIP2 in liposomes, implying clustering. However, HIV-1 MA itself did not induce PIP2 clustering. A CA inter-hexamer dimer interface mutation led to a loss of induced PIP2 clustering in MACA, indicating the importance of protein multimerization. Cryo-electron tomography of liposomes with bound MACA showed an amorphous protein layer on the membrane surface. Thus, it appears that while protein-protein interactions are required for PIP2 clustering, formation of a regular lattice is not. Protein-induced PIP2 clustering and multivalent cation-induced PIP2 clustering are additive. Taken together, these results provide the first evidence that HIV-1 Gag can selectively target pre-existing PIP2-enriched domains of the plasma membrane for viral assembly, and that Gag multimerization can further enrich PIP2 at assembly sites. These effects could explain the observed PIP2 enrichment in HIV-1.
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http://dx.doi.org/10.1016/j.jmb.2020.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262684PMC
September 2020

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors.

Biophys J 2019 09 21;117(6):1037-1050. Epub 2019 Aug 21.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York.

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.
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http://dx.doi.org/10.1016/j.bpj.2019.07.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818168PMC
September 2019

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cell 2019 06 2;177(7):1757-1770.e21. Epub 2019 May 2.

Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA; Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA; Field of Biophysics, Cornell University, Ithaca, NY 14853, USA; Kavli Institute at Cornell for Nanoscale Science, Ithaca, NY 14853, USA. Electronic address:

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.
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http://dx.doi.org/10.1016/j.cell.2019.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768631PMC
June 2019

Molecular Dynamics Simulations Reveal Leaflet Coupling in Compositionally Asymmetric Phase-Separated Lipid Membranes.

J Phys Chem B 2019 05 1;123(18):3968-3975. Epub 2019 May 1.

The eukaryotic plasma membrane has an asymmetric distribution of its component lipids. Rafts that result from liquid-liquid phase separation are a feature of its exoplasmic leaflet, but how these exoplasmic leaflet domains are coupled to the cytoplasmic leaflet is not understood. These rafts can be studied in model membranes of three-component mixtures that produce coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. We conducted all-atom molecular dynamics simulations of compositionally asymmetric lipid bilayers that reflect a more realistic model of the plasma membrane. One leaflet contained phase-separated domains with phosphatidylcholine and cholesterol, representing the exoplasmic leaflet, whereas the other contained phosphatidylethanolamine, phosphatidylserine, and cholesterol, which are the predominant components of the cytoplasmic leaflet. Inspired by findings of domain alignment across the two leaflets in compositionally symmetric model membranes, we examined the coupling between the two leaflets to see how the single-phase cytoplasmic leaflet would respond to phase separation in the other leaflet and if information could be communicated across the membrane. We found the region of the single-phase leaflet apposing the Lo domain to be slightly more ordered and thicker than the region apposing the Ld domain. The region across from the Lo domain is somewhat enriched in cholesterol and significantly depleted of polyunsaturated lipids.
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http://dx.doi.org/10.1021/acs.jpcb.9b03488DOI Listing
May 2019

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles.

Biophys J 2019 03 25;116(5):860-873. Epub 2019 Jan 25.

Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York.

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.
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http://dx.doi.org/10.1016/j.bpj.2019.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400823PMC
March 2019

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes.

Biochim Biophys Acta Biomembr 2019 02 5;1861(2):478-485. Epub 2018 Dec 5.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States of America. Electronic address:

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld) + liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 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 (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld + Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.
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http://dx.doi.org/10.1016/j.bbamem.2018.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310626PMC
February 2019

Presence and Role of Midplane Cholesterol in Lipid Bilayers Containing Registered or Antiregistered Phase Domains.

J Phys Chem B 2018 08 21;122(34):8193-8200. Epub 2018 Aug 21.

Three-component lipid mixtures can produce coexisting liquid ordered and liquid disordered phases, a model for eukaryotic plasma membrane rafts. In compositionally symmetric bilayers with two phase-separated leaflets, phase domains of the two leaflets may align through registration, where domains are found across from domains of the same phase, or else antiregistration, where domains are found across from domains of the opposite phase. This alignment could serve as a method of information communication across the plasma membrane. We used coarse-grained molecular dynamics simulations to study ternary mixtures of a high-melting-temperature phospholipid, a low-melting-temperature phospholipid, and cholesterol. We found a significant presence of cholesterol molecules at the bilayer midplane rather than in a leaflet in some systems, corresponding to a lack of registration. Increasing the length of the acyl chains from 16 to 24 carbons in high-melting-temperature phospholipids or increasing the concentration of cholesterol from 20 to 35 mol % in the bilayer produced a transition from registration to antiregistration and gave rise to significant populations of midplane cholesterol.
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http://dx.doi.org/10.1021/acs.jpcb.8b03949DOI Listing
August 2018

Multivalent Cation-Bridged PI(4,5)P Clusters Form at Very Low Concentrations.

Biophys J 2018 06;114(11):2630-2639

Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York. Electronic address:

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca, Mg, Zn, or trivalent ions Fe and Al. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.
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http://dx.doi.org/10.1016/j.bpj.2018.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129474PMC
June 2018

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

Biophys J 2018 05;114(9):2152-2164

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.
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http://dx.doi.org/10.1016/j.bpj.2018.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961461PMC
May 2018

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases.

Biophys J 2018 04;114(8):1921-1935

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (∼200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5-10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ∼5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.
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http://dx.doi.org/10.1016/j.bpj.2018.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937166PMC
April 2018

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

Biophys J 2017 Nov;113(9):2004-2015

Department of Biochemistry and Molecular Cell Biology, Cornell University, Ithaca, New York. Electronic address:

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.
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http://dx.doi.org/10.1016/j.bpj.2017.08.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685651PMC
November 2017

Line Tension Controls Liquid-Disordered + Liquid-Ordered Domain Size Transition in Lipid Bilayers.

Biophys J 2017 Apr;112(7):1431-1443

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.
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http://dx.doi.org/10.1016/j.bpj.2017.02.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390056PMC
April 2017

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag.

J Virol 2016 10 29;90(20):9518-32. Epub 2016 Sep 29.

Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York, USA

Unlabelled: The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes.

Importance: Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.
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http://dx.doi.org/10.1128/JVI.01102-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044813PMC
October 2016

Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.

Langmuir 2016 05 16;32(20):5195-200. Epub 2016 May 16.

Institute of Molecular Biosciences, Biophysics Division, NAWI Graz, University of Graz , Graz 8010, Austria.

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.
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http://dx.doi.org/10.1021/acs.langmuir.5b04562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910133PMC
May 2016

Effects of Transmembrane α-Helix Length and Concentration on Phase Behavior in Four-Component Lipid Mixtures: A Molecular Dynamics Study.

J Phys Chem B 2016 05 27;120(17):4064-77. Epub 2016 Apr 27.

Department of Molecular Biology and Genetics, Cornell University , Ithaca, New York 14853, United States.

We used coarse-grained molecular dynamics simulations to examine the effects of transmembrane α-helical WALP peptides on the behavior of four-component lipid mixtures. These mixtures contain a high-melting temperature (high-Tm) lipid, a nanodomain-inducing low-Tm lipid, a macrodomain-inducing low-Tm lipid and cholesterol to model the outer leaflet of cell plasma membranes. In a series of simulations, we incrementally replace the nanodomain-inducing low-Tm lipid by the macrodomain-inducing low-Tm lipid and measure how lipid and phase properties are altered by the addition of WALPs of different length. Regardless of the ratio of the two low-Tm lipids, shorter WALPs increase domain size and all WALPs increase domain alignment between the two leaflets. These effects are smallest for the longest WALP tested, and increase with increasing WALP concentration. Thus, our simulations explain the experimental observation that WALPs induce macroscopic domains in otherwise nanodomain-forming lipid-only mixtures (unpublished). Since the cell plasma membrane contains a large fraction of transmembrane proteins, these findings link the behavior of lipid-only model membranes in vitro to phase behavior in vivo.
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http://dx.doi.org/10.1021/acs.jpcb.6b00611DOI Listing
May 2016

Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin.

Food Chem 2016 Apr 22;197(Pt A):84-91. Epub 2015 Oct 22.

Institute of Food Science, Cornell University, Ithaca, NY 14853-7201, USA.

We illustrate a method that uses bovine serum albumin (BSA) to control the receptor-accessible part of rebaudioside A (Reb A). The critical micelle concentration (CMC) of Reb A was found to be 4.5 mM and 5 mM at pH 3 and 6.7 respectively. NMR studies show that below its CMC, Reb A binds weakly to BSA to generate a Reb A-protein complex ("RPC"), which is only modestly stable under varying conditions of pH (3.0-6.7) and temperature (4-40°C) with its binding affinities determined to be in the range of 5-280 mM. Furthermore, saturation transfer difference (STD) NMR experiments confirm that the RPC has fast exchange of the bitterness-instigating diterpene of Reb A into the binding sites of BSA. Our method can be used to alter the strength of Reb A-receptor interaction, as a result of binding of Reb A to BSA, which may ultimately lead to moderation of its taste.
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http://dx.doi.org/10.1016/j.foodchem.2015.10.064DOI Listing
April 2016

Phase diagram of a polyunsaturated lipid mixture: Brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol.

Biochim Biophys Acta 2016 Jan 23;1858(1):153-61. Epub 2015 Oct 23.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Phospholipids having a polyunsaturated acyl chain are abundant in biological membranes, but their behavior in lipid mixtures is difficult to study. Here we elucidate the nature of such mixtures with this report of the first ternary phase diagram containing the polyunsaturated lipid SDPC in mixtures of BSM/SDPC/Chol (brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol). These mixtures show coexisting macroscopic liquid-disordered (Ld) and liquid-ordered (Lo) phase separation, with phase boundaries determined by FRET and by fluorescence microscopy imaging of giant unilamellar vesicles (GUVs). Surprisingly, SDPC mixes with BSM/Chol similarly to how DOPC and POPC mix with BSM/Chol. Notably, intermediate states are produced within the Ld+Lo liquid-liquid immiscibility region upon addition of fourth component POPC. These mixtures of BSM/SDPC/POPC/Chol exhibit nanoscopic Ld+Lo domains over a very large volume of composition space, possibly because Ld/Lo line tension is not high.
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http://dx.doi.org/10.1016/j.bbamem.2015.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664456PMC
January 2016

Pictures of the Substructure of Liquid-Ordered Domains.

Biophys J 2015 Sep;109(5):854-5

Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2015.07.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564828PMC
September 2015

Lipid bilayers: clusters, domains and phases.

Essays Biochem 2015 ;57:33-42

Department of Molecular Biology and Genetics, Field of Biophysics, Cornell University, Ithaca, NY 14853, U.S.A.

In the present chapter we discuss the complex mixing behaviour of plasma membrane lipids. To do so, we first introduce the plasma membrane and membrane mixtures often used to model its complexity. We then discuss the nature of lipid phase behaviour in bilayers and the distinction between these phases and other manifestations of non-random mixing found in one-phase mixtures, such as clusters, micelles and microemulsions. Finally, we demonstrate the applicability of Gibbs phase diagrams to the study of increasingly complex model membrane systems, with a focus on phase coexistence, morphology and their implications for the cell plasma membrane.
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http://dx.doi.org/10.1042/bse0570033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4377075PMC
October 2015

Multiscale modeling of four-component lipid mixtures: domain composition, size, alignment, and properties of the phase interface.

J Phys Chem B 2015 Mar 22;119(11):4240-50. Epub 2015 Jan 22.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, United States.

Simplified lipid mixtures are often used to model the complex behavior of the cell plasma membrane. Indeed, as few as four components-a high-melting lipid, a nandomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol)-can give rise to a wide range of domain sizes and patterns that are highly sensitive to lipid compositions. Although these systems are studied extensively with experiments, the molecular-level details governing their phase behavior are not yet known. We address this issue by using molecular dynamics simulations to analyze how phase separation evolves in a four-component system as it transitions from small domains to large domains. To do so, we fix concentrations of the high-melting lipid 16:0,16:0-phosphatidylcholine (DPPC) and chol, and incrementally replace the nanodomain-inducing low-melting lipid 16:0,18:2-PC (PUPC) by the macrodomain-inducing low-melting lipid 18:2,18:2-PC (DUPC). Coarse-grained simulations of this four-component system reveal that lipid demixing increases as the amount of DUPC increases. Additionally, we find that domain size and interleaflet alignment change sharply over a narrow range of replacement of PUPC by DUPC, indicating that intraleaflet and interleaflet behaviors are coupled. Corresponding united atom simulations show that only lipids within ∼2 nm of the phase interface are significantly perturbed regardless of domain composition or size. Thus, whereas the fraction of interface-perturbed lipids is negligible for large domains, it is significant for smaller ones. Together, these results reveal characteristic traits of bilayer thermodynamic behavior in four-component mixtures, and provide a baseline for investigation of the effects of proteins and other lipids on membrane phase properties.
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http://dx.doi.org/10.1021/jp511083zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4377067PMC
March 2015

Lattice simulations of phase morphology on lipid bilayers: renormalization, membrane shape, and electrostatic dipole interactions.

Phys Rev E Stat Nonlin Soft Matter Phys 2014 Feb 3;89(2):022702. Epub 2014 Feb 3.

Field of Biophysics, Cornell University, Ithaca, New York 14850, USA.

When liquid phases coexist at equilibrium but are not driven to minimize domain interfacial contact energy, the resulting patterns of phase domains can have important implications for living cells. In this study we explore some of the interactions and conditions that produce the stable patterned phases that are observed in model lipid mixtures. By use of Monte Carlo simulations we find that background curvature is important for the formation of patterned (modulated) phases. The interactions that stabilize nanoscopic phase separation are still not well understood. We show that inclusion of an electrostatic dipole repulsion with decay lengths as short as two to four lipid diameters can break up domains at the nanometer scale and that the location of the miscibility critical point is sensitive to this interaction. The use of a coarse-grained simulation raises questions about comparing parameters in simulations performed at different length scales. Using renormalization group techniques we show how to reconcile this problem, treating line tension as a running coupling constant.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4391078PMC
http://dx.doi.org/10.1103/PhysRevE.89.022702DOI Listing
February 2014

Hybrid and nonhybrid lipids exert common effects on membrane raft size and morphology.

J Am Chem Soc 2013 Oct 26;135(40):14932-5. Epub 2013 Sep 26.

Biology and Soft Matter and §Biosciences Divisions, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37831, United States.

Nanometer-scale domains in cholesterol-rich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chain-asymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain. Hybrid lipids generate nanodomains in some model membranes and are also abundant in the PM. It was proposed that they align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. We used small-angle neutron scattering and fluorescence techniques to detect nanoscopic and modulated liquid phase domains in a mixture composed entirely of nonhybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains and strongly implying a common mechanism for the overall control of raft size and morphology. We discuss implications of these findings for theoretical descriptions of nanodomains.
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http://dx.doi.org/10.1021/ja407624cDOI Listing
October 2013

Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol.

Biochim Biophys Acta 2013 Sep 7;1828(9):2204-14. Epub 2013 Jun 7.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

We report the first 4-component phase diagram for the lipid bilayer mixture, DSPC/DOPC/POPC/chol (distearoylphosphatidylcholine/dioleoylphosphatidylcholine/1-palmitoyl, 2-oleoylphosphatidylcholine/cholesterol). This phase diagram, which has macroscopic Ld+Lo phase domains, clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has nanoscopic phase domains of Ld+Lo. Our studies start from two published ternary phase diagrams, and show how these can be combined into a quaternary phase diagram by study of a few hundred samples of intermediate compositions.
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http://dx.doi.org/10.1016/j.bbamem.2013.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738200PMC
September 2013

Limited perturbation of a DPPC bilayer by fluorescent lipid probes: a molecular dynamics study.

J Phys Chem B 2013 May 19;117(17):4844-52. Epub 2013 Apr 19.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, United States.

The properties of lipid bilayer nanometer-scale domains could be crucial for understanding cell membranes. Fluorescent probes are often used to study bilayers, yet their effects on host lipids are not well understood. We used molecular dynamics simulations to investigate perturbations in a fluid DPPC bilayer upon incorporation of three indocarbocyanine probes: DiI-C18:0, DiI-C18:2, or DiI-C12:0. We find a 10-12% decrease in chain order for DPPC in the solvation shell nearest the probe but smaller effects in subsequent shells, indicating that the probes significantly alter only their local environment. We also observe order perturbations of lipids directly across from the probe in the opposite leaflet. Additionally, the DPPC headgroup phosphorus-to-nitrogen vector of lipids nearest the probe exhibits preferential orientation pointing away from the DiI. We show that, while DiI probes perturb their local environment, they do not strongly influence the average properties of "nanoscopic" domains containing a few hundred lipids.
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http://dx.doi.org/10.1021/jp400289dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280801PMC
May 2013
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