Publications by authors named "Thais A Enoki"

10 Publications

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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

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

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

Antimicrobial Peptide K-W-Hya1 Induces Stable Structurally Modified Lipid Domains in Anionic Membranes.

Langmuir 2018 02 22;34(5):2014-2025. Epub 2018 Jan 22.

Instituto de Física da Universidade de São Paulo, São Paulo, SP, CEP 05508-090, Brasil.

Considering the known different mode of action of antimicrobial peptides in zwitterionic and anionic cell membranes, the present work compares the action of the antimicrobial peptide K-W-Hya1 (KIFGAIWPLALGALKNLIK-NH) with zwitterionic and negatively charged model membranes, namely, liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, and a mixture of the two. Differential scanning calorimetry (DSC), steady state fluorescence of the Trp residue, dynamic light scattering (DLS), and measurement of the leakage of an entrapped fluorescent dye (carboxyfluorescein, CF) were performed with large unilamellar vesicles (LUVs). All techniques evidenced the different action of the peptide in zwitterionic and anionic vesicles. Trp fluorescence spectroscopy shows that the differences are related not only to the partition of the cationic peptide in zwitterionic and anionic membranes, but also to the different penetration depth of the peptide into the lipid bilayers: Trp goes deeper into negatively charged membranes, both in the gel and fluid phases, than into zwitterionic ones. DSC shows that the peptide is strongly attached to anionic bilayers, giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disturbed, possibly a stable or transient polar pore, considering the leakage of CF. This contrasts with the homogeneous effect produced by the peptide in zwitterionic membranes, probably related to peptide-membrane diffusion. Moreover, in mixed bilayers (PC:PG), the peptide sequesters negatively charged lipids, creating peptide-rich anionic lipid regions, strongly disturbing the membrane. The distinct structural interaction displayed by the peptide in PC and PG membranes could be related to the different mechanisms of action of the peptide in anionic prokaryotic and zwitterionic eukaryotic cell membranes.
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http://dx.doi.org/10.1021/acs.langmuir.7b03408DOI Listing
February 2018

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

Light scattering on the structural characterization of DMPG vesicles along the bilayer anomalous phase transition.

Chem Phys Lipids 2012 Dec 16;165(8):826-37. Epub 2012 Nov 16.

Instituto de Física, Universidade de São Paulo, CP 66318, CEP 05314-970, São Paulo, SP, Brazil.

Highly charged vesicles of the saturated anionic lipid dimyristoyl phosphatidylglycerol (DMPG) in low ionic strength medium exhibit a very peculiar thermo-structural behavior. Along a wide gel-fluid transition region, DMPG dispersions display several anomalous characteristics, like low turbidity, high electrical conductivity and viscosity. Here, static and dynamic light scattering (SLS and DLS) were used to characterize DMPG vesicles at different temperatures. Similar experiments were performed with the largely studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). SLS and DLS data yielded similar dimensions for DMPC vesicles at all studied temperatures. However, for DMPG, along the gel-fluid transition region, SLS indicated a threefold increase in the vesicle radius of gyration, whereas the hydrodynamic radius, as obtained from DLS, increased 30% only. Despite the anomalous increase in the radius of gyration, DMPG lipid vesicles maintain isotropy, since no light depolarization was detected. Hence, SLS data are interpreted regarding the presence of isotropic vesicles within the DMPG anomalous transition, but highly perforated vesicles, with large holes. DLS/SLS discrepancy along the DMPG transition region is discussed in terms of the interpretation of the Einstein-Stokes relation for porous vesicles. Therefore, SLS data are shown to be much more appropriate for measuring porous vesicle dimensions than the vesicle diffusion coefficient. The underlying nanoscopic process which leads to the opening of pores in charged DMPG bilayer is very intriguing and deserves further investigation. One could envisage biotechnological applications, with vesicles being produced to enlarge and perforate in a chosen temperature and/or pH value.
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http://dx.doi.org/10.1016/j.chemphyslip.2012.11.002DOI Listing
December 2012
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