Membranes

Research

18 pages, 2713 KiB

Open AccessArticle

Engineering Phosphatidylserine Containing Asymmetric Giant Unilamellar Vesicles

by Jake McDonough, Trevor A. Paratore, Hannah M. Ketelhohn, Bella C. DeCilio, Alonzo H. Ross and Arne Gericke

Membranes 2024, 14(9), 181; https://doi.org/10.3390/membranes14090181 - 23 Aug 2024

Cited by 1 | Viewed by 1724

Abstract

The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the [...] Read more.

The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the fabrication of large or giant unilamellar vesicles (GUVs) with an asymmetric lipid composition. Investigating the physicochemical properties of PS in such asymmetric lipid bilayers and studying its interactions with proteins necessitates the reliable fabrication of asymmetric GUVs (aGUVs) with a high degree of asymmetry that exhibit PS in the outer leaflet so that the interaction with peptides and proteins can be studied. Despite progress, achieving aGUVs with well-defined PS asymmetry remains challenging. Recently, a Ca²⁺-initiated hemifusion method has been introduced, utilizing the fusion of symmetric GUVs (sGUVs) with a supported lipid bilayer (SLB) for the fabrication of aGUVs. We extend this approach to create aGUVs with PS in the outer bilayer leaflet. Comparing the degree of asymmetry between aGUVs obtained via Ca²⁺ or Mg²⁺ initiated hemifusion of a phosphatidylcholine (PC) sGUVwith a PC/PS-supported lipid bilayer, we observe for both bivalent cations a significant number of aGUVs with near-complete asymmetry. The degree of asymmetry distribution is narrower for physiological salt conditions than at lower ionic strengths. While Ca²⁺ clusters PS in the SLB, macroscopic domain formation is absent in the presence of Mg²⁺. However, the clustering of PS upon the addition of Ca²⁺ is apparently too slow to have a negative effect on the quality of the obtained aGUVs. We introduce a data filtering method to select aGUVs that are best suited for further investigation. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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16 pages, 1914 KiB

Open AccessArticle

Breakdown of Phospholipid Asymmetry Triggers ADAM17-Mediated Rescue Events in Cells Undergoing Apoptosis

by Maria Sperrhacke, Sinje Leitzke, Björn Ahrens and Karina Reiss

Membranes 2023, 13(8), 720; https://doi.org/10.3390/membranes13080720 - 5 Aug 2023

Cited by 1 | Viewed by 1766

Abstract

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to [...] Read more.

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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17 pages, 6322 KiB

Open AccessArticle

Critical Role of Molecular Packing in Lo Phase Membrane Solubilization

by Nicolas Puff

Membranes 2023, 13(7), 652; https://doi.org/10.3390/membranes13070652 - 7 Jul 2023

Cited by 3 | Viewed by 1336

Abstract

Membrane solubilization induced by Triton X-100 (TX-100) was investigated. Different membrane compositions and phase states were studied along the detergent titration. Expected solubilization profiles were obtained but new information is provided. The fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids indicates that the liquid-ordered (Lo)/liquid-disordered (Ld) [...] Read more.

Membrane solubilization induced by Triton X-100 (TX-100) was investigated. Different membrane compositions and phase states were studied along the detergent titration. Expected solubilization profiles were obtained but new information is provided. The fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids indicates that the liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence is barely unaffected at sub-solubilizing detergent concentrations and highlights the vesicle-to-micelle transition. Moreover, the location of the NBD group in the bilayer emphasizes a detergent–membrane interaction in the case of the insoluble Lo phase membrane. It has also been shown that the molecular packing of the membrane loosens in the presence of TX-100, regardless of the solubilization profile. Motivated by studies on GPMVs, the solubilization of less ordered Lo phase membranes was considered in order to improve the effect of molecular packing on the extent of solubilization. Membranes composed of SM and Chol in an equimolar ratio doped with different amounts of PC were studied. The more ordered the Lo phase membrane is in the absence of detergent, the less likely it is to be solubilized. Furthermore, and in contrast to what is observed for membranes exhibiting an Lo/Ld phase coexistence, a very small decrease in the molecular packing of the Lo phase membrane radically modifies the extent of solubilization. These results have implications for the reliability of TX-100 insolubility as a method to detect ordered domains. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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14 pages, 2070 KiB

Open AccessArticle

Influence of Membrane Asymmetry on OmpF Insertion, Orientation and Function

by Annemarie Donoghue, Mathias Winterhalter and Thomas Gutsmann

Membranes 2023, 13(5), 517; https://doi.org/10.3390/membranes13050517 - 16 May 2023

Cited by 1 | Viewed by 1845

Abstract

The effect of asymmetric membranes containing lipopolysaccharides (LPS) on the outer membrane protein F (OmpF) reconstitution, channel orientation, and antibiotic permeation across the outer membrane was investigated. After forming an asymmetric planar lipid bilayer composed of LPS on one and phospholipids on the [...] Read more.

The effect of asymmetric membranes containing lipopolysaccharides (LPS) on the outer membrane protein F (OmpF) reconstitution, channel orientation, and antibiotic permeation across the outer membrane was investigated. After forming an asymmetric planar lipid bilayer composed of LPS on one and phospholipids on the other side, the membrane channel OmpF was added. The ion current recordings demonstrate that LPS has a strong influence on the OmpF membrane insertion, orientation, and gating. Enrofloxacin was used as an example of an antibiotic interacting with the asymmetric membrane and with OmpF. The enrofloxacin caused the blockage of the ion current through the OmpF, depending on the side of addition, the transmembrane voltage applied, and the composition of the buffer. Furthermore, the enrofloxacin changed the phase behavior of the LPS-containing membranes, demonstrating that its membrane activity influences the function of OmpF and potentially the membrane permeability. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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12 pages, 22174 KiB

Open AccessArticle

Real Space and Time Imaging of Collective Headgroup Dipole Motions in Zwitterionic Lipid Bilayers

by Dima Bolmatov, C. Patrick Collier, Dmitry Zav’yalov, Takeshi Egami and John Katsaras

Membranes 2023, 13(4), 442; https://doi.org/10.3390/membranes13040442 - 18 Apr 2023

Cited by 9 | Viewed by 2672

Abstract

Lipid bilayers are supramolecular structures responsible for a range of processes, such as transmembrane transport of ions and solutes, and sorting and replication of genetic materials, to name just a few. Some of these processes are transient and currently, cannot be visualized in [...] Read more.

Lipid bilayers are supramolecular structures responsible for a range of processes, such as transmembrane transport of ions and solutes, and sorting and replication of genetic materials, to name just a few. Some of these processes are transient and currently, cannot be visualized in real space and time. Here, we developed an approach using 1D, 2D, and 3D Van Hove correlation functions to image collective headgroup dipole motions in zwitterionic phospholipid bilayers. We show that both 2D and 3D spatiotemporal images of headgroup dipoles are consistent with commonly understood dynamic features of fluids. However, analysis of the 1D Van Hove function reveals lateral transient and re-emergent collective dynamics of the headgroup dipoles—occurring at picosecond time scales—that transmit and dissipate heat at longer times, due to relaxation processes. At the same time, the headgroup dipoles also generate membrane surface undulations due a collective tilting of the headgroup dipoles. A continuous intensity band of headgroup dipole spatiotemporal correlations—at nanometer length and nanosecond time scales—indicates that dipoles undergo stretching and squeezing elastic deformations. Importantly, the above mentioned intrinsic headgroup dipole motions can be externally stimulated at GHz-frequency scale, enhancing their flexoelectric and piezoelectric capabilities (i.e., increased conversion efficiency of mechanical energy into electric energy). In conclusion, we discuss how lipid membranes can provide molecular-level insights about biological learning and memory, and as platforms for the development of the next generation of neuromorphic computers. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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17 pages, 2337 KiB

Open AccessArticle

A Small-Angle Neutron Scattering, Calorimetry and Densitometry Study to Detect Phase Boundaries and Nanoscale Domain Structure in a Binary Lipid Mixture

by Natalie Krzyzanowski, Lionel Porcar and Ursula Perez-Salas

Membranes 2023, 13(3), 323; https://doi.org/10.3390/membranes13030323 - 10 Mar 2023

Cited by 4 | Viewed by 1931

Abstract

Techniques that can probe nanometer length scales, such as small-angle neutron scattering (SANS), have become increasingly popular to detect phase separation in membranes. But to extract the phase composition and domain structure from the SANS traces, complementary information is needed. Here, we present [...] Read more.

Techniques that can probe nanometer length scales, such as small-angle neutron scattering (SANS), have become increasingly popular to detect phase separation in membranes. But to extract the phase composition and domain structure from the SANS traces, complementary information is needed. Here, we present a SANS, calorimetry and densitometry study of a mixture of two saturated lipids that exhibits solidus–liquidus phase coexistence: 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC, tail-deuterated DPPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). With calorimetry, we investigated the phase diagram for this system and found that the boundary traces for both multilamellar vesicles (MLVs) as well as 50 nm unilamellar vesicles overlap. Because the solidus boundary was mostly inaccessible by calorimetry, we investigated it by both SANS and molecular volume measurements for a 1:1 dDPPC:DLPC lipid mixture. From the temperature behavior of the molecular volume for the 1:1 dDPPC:DLPC mixture, as well as the individual molecular volume of each lipid species, we inferred that the liquidus phase consists of only fluid-state lipids while the solidus phase consists of lipids that are in gel-like states. Using this solidus–liquidus phase model, the SANS data were analyzed with an unrestricted shape model analysis software: MONSA. The resulting fits show irregular domains with dendrite-like features as those previously observed on giant unilamellar vesicles (GUVs). The surface pair correlation function describes a characteristic domain size for the minority phase that decreases with temperature, a behavior found to be consistent with a concomitant decrease in membrane mismatch between the liquidus and solidus phases. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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Review

Jump to: Research, Other

29 pages, 6219 KiB

Open AccessReview

The Importance of Bilayer Asymmetry in Biological Membranes: Insights from Model Membranes

by Igor S. Oliveira, Guilherme X. Pinheiro, Maria Luana B. Sa, Pedro Henrique L. O. Gurgel, Samuel U. Pizzol, Rosangela Itri, Vera B. Henriques and Thais A. Enoki

Membranes 2025, 15(3), 79; https://doi.org/10.3390/membranes15030079 - 3 Mar 2025

Viewed by 971

Abstract

This mini-review intends to highlight the importance of bilayer asymmetry. Biological membranes are complex structures that are a physical barrier separating the external environment from the cellular content. This complex bilayer comprises an extensive lipid repertory, suggesting that the different lipid structures might [...] Read more.

This mini-review intends to highlight the importance of bilayer asymmetry. Biological membranes are complex structures that are a physical barrier separating the external environment from the cellular content. This complex bilayer comprises an extensive lipid repertory, suggesting that the different lipid structures might play a role in the membrane. Interestingly, this vast repertory of lipids is asymmetrically distributed between leaflets that form the lipid bilayer. Here, we discuss the properties of the plasma membrane from the perspective of experimental model membranes, consisting of simplified and controlled in vitro systems. We summarize some crucial features of the exoplasmic (outer) and cytoplasmic (inner) leaflets observed through investigations using symmetric and asymmetric membranes. Symmetric model membranes for the exoplasmic leaflet have a unique lipid composition that might form a coexistence of phases, namely the liquid disordered and liquid order phases. These phase domains may appear in different sizes and shapes depending on lipid composition and lipid–lipid interactions. In contrast, symmetric model membranes for the cytoplasmic leaflet form a fluid phase. We discuss the outcomes reported in the literature for asymmetric bilayers, which vary according to lipid compositions and, consequently, reflect different intra- and inter-leaflet interactions. Interestingly, the asymmetric bilayer could show induced domains in the inner leaflet, or it could decrease the tendency of the outer leaflet to phase separation. If cells regulate the lipid composition of the plasma membrane, they can adjust the existence and sizes of the domains by tuning the lipid composition. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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12 pages, 931 KiB

Open AccessReview

Asymmetric Distribution of Plasmalogens and Their Roles—A Mini Review

by Masanori Honsho and Yukio Fujiki

Membranes 2023, 13(9), 764; https://doi.org/10.3390/membranes13090764 - 29 Aug 2023

Cited by 6 | Viewed by 2577

Abstract

Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. The synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. Plasmalogens are transported to the post-Golgi compartment, including endosomes and plasma membranes, in a manner dependent [...] Read more.

Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. The synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. Plasmalogens are transported to the post-Golgi compartment, including endosomes and plasma membranes, in a manner dependent on ATP, but not vesicular transport. Plasmalogens are preferentially localized in the inner leaflet of the plasma membrane in a manner dependent on P4-type ATPase ATP8B2, that associates with the CDC50 subunit. Plasmalogen biosynthesis is spatiotemporally regulated by a feedback mechanism that senses the amount of plasmalogens in the inner leaflet of the plasma membrane and controls the stability of fatty acyl-CoA reductase 1 (FAR1), the rate-limiting enzyme for plasmalogen biosynthesis. The physiological consequences of such asymmetric localization and homeostasis of plasmalogens are discussed in this review. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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14 pages, 1627 KiB

Open AccessReview

Building Asymmetric Lipid Bilayers for Molecular Dynamics Simulations: What Methods Exist and How to Choose One?

by Emily H. Chaisson, Frederick A. Heberle and Milka Doktorova

Membranes 2023, 13(7), 629; https://doi.org/10.3390/membranes13070629 - 29 Jun 2023

Cited by 13 | Viewed by 5206

Abstract

The compositional asymmetry of biological membranes has attracted significant attention over the last decade. Harboring more differences from symmetric membranes than previously appreciated, asymmetric bilayers have proven quite challenging to study with familiar concepts and techniques, leaving many unanswered questions about the reach [...] Read more.

The compositional asymmetry of biological membranes has attracted significant attention over the last decade. Harboring more differences from symmetric membranes than previously appreciated, asymmetric bilayers have proven quite challenging to study with familiar concepts and techniques, leaving many unanswered questions about the reach of the asymmetry effects. One particular area of active research is the computational investigation of composition- and number-asymmetric lipid bilayers with molecular dynamics (MD) simulations. Offering a high level of detail into the organization and properties of the simulated systems, MD has emerged as an indispensable tool in the study of membrane asymmetry. However, the realization that results depend heavily on the protocol used for constructing the asymmetric bilayer models has sparked an ongoing debate about how to choose the most appropriate approach. Here we discuss the underlying source of the discrepant results and review the existing methods for creating asymmetric bilayers for MD simulations. Considering the available data, we argue that each method is well suited for specific applications and hence there is no single best approach. Instead, the choice of a construction protocol—and consequently, its perceived accuracy—must be based primarily on the scientific question that the simulations are designed to address. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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21 pages, 2326 KiB

Open AccessReview

A Guide to Your Desired Lipid-Asymmetric Vesicles

by Mona Krompers and Heiko Heerklotz

Membranes 2023, 13(3), 267; https://doi.org/10.3390/membranes13030267 - 23 Feb 2023

Cited by 14 | Viewed by 3551

Abstract

Liposomes are prevalent model systems for studies on biological membranes. Recently, increasing attention has been paid to models also representing the lipid asymmetry of biological membranes. Here, we review in-vitro methods that have been established to prepare free-floating vesicles containing different compositions of [...] Read more.

Liposomes are prevalent model systems for studies on biological membranes. Recently, increasing attention has been paid to models also representing the lipid asymmetry of biological membranes. Here, we review in-vitro methods that have been established to prepare free-floating vesicles containing different compositions of the classic two-chain glycero- or sphingolipids in their outer and inner leaflet. In total, 72 reports are listed and assigned to four general strategies that are (A) enzymatic conversion of outer leaflet lipids, (B) re-sorting of lipids between leaflets, (C) assembly from different monolayers and (D) exchange of outer leaflet lipids. To guide the reader through this broad field of available techniques, we attempt to draw a road map that leads to the lipid-asymmetric vesicles that suit a given purpose. Of each method, we discuss advantages and limitations. In addition, various verification strategies of asymmetry as well as the role of cholesterol are briefly discussed. The ability to specifically induce lipid asymmetry in model membranes offers insights into the biological functions of asymmetry and may also benefit the technical applications of liposomes. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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Other

Jump to: Research, Review

13 pages, 682 KiB

Open AccessPerspective

Ordered Domain (Raft) Formation in Asymmetric Vesicles and Its Induction upon Loss of Lipid Asymmetry in Artificial and Natural Membranes

by Erwin London

Membranes 2022, 12(9), 870; https://doi.org/10.3390/membranes12090870 - 9 Sep 2022

Cited by 8 | Viewed by 2373

Abstract

Lipid asymmetry, the difference in the lipid composition in the inner and outer lipid monolayers (leaflets) of a membrane, is an important feature of eukaryotic plasma membranes. Investigation of the biophysical consequences of lipid asymmetry has been aided by advances in the ability [...] Read more.

Lipid asymmetry, the difference in the lipid composition in the inner and outer lipid monolayers (leaflets) of a membrane, is an important feature of eukaryotic plasma membranes. Investigation of the biophysical consequences of lipid asymmetry has been aided by advances in the ability to prepare artificial asymmetric membranes, especially by use of cyclodextrin-catalyzed lipid exchange. This review summarizes recent studies with artificial asymmetric membranes which have identified conditions in which asymmetry can induce or suppress the ability of membranes to form ordered domains (rafts). A consequence of the latter effect is that, under some conditions, a loss of asymmetry can induce ordered domain formation. An analogous study in plasma membrane vesicles has demonstrated that asymmetry can also suppress domain formation in natural membranes. Thus, it is possible that a loss of asymmetry can induce domain formation in vivo. Full article

(This article belongs to the Special Issue Advances in Symmetric and Asymmetric Lipid Membranes)

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