scholarly journals Reconstitution of Functional Integrin αIIbβ3 and Its Activation in Plasma Membrane-Mimetic Lipid Environments

Membranes ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 499
Author(s):  
Una Janke ◽  
Alexandra Mitlehner ◽  
Aileen Weide ◽  
Theresia Gutmann ◽  
Mihaela Delcea
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Conformational Dynamics ◽  
Membrane System ◽  
Supported Lipid Bilayers ◽  
Label Free ◽  
Membrane Composition ◽  
Integrin Αiibβ3 ◽  
Membrane Mimetic ◽  
Platelet Receptor

The study of the platelet receptor integrin αIIbβ3 in a membrane-mimetic environment without interfering signalling pathways is crucial to understand protein structure and dynamics. Our understanding of this receptor and its sequential activation steps has been tremendously progressing using structural and reconstitution approaches in model membranes, such as liposomes or supported-lipid bilayers. For most αIIbβ3 reconstitution approaches, saturated short-chain lipids have been used, which is not reflecting the native platelet cell membrane composition. We report here on the reconstitution of label-free full-length αIIbβ3 in liposomes containing cholesterol, sphingomyelin, and unsaturated phosphatidylcholine mimicking the plasma membrane that formed supported-lipid bilayers for quartz-crystal microbalance with dissipation (QCM-D) experiments. We demonstrate the relevance of the lipid environment and its resulting physicochemical properties on integrin reconstitution efficiency and its conformational dynamics. We present here an approach to investigate αIIbβ3 in a biomimetic membrane system as a useful platform do dissect disease-relevant integrin mutations and effects on ligand binding in a lipid-specific context, which might be applicable for drug screening.

scholarly journals Integration of Plasma Membrane in Supported Lipid Bilayers

2009 ◽  
Vol 96 (3) ◽  
pp. 329a
Author(s):  
Nicholas Hagerty ◽  
Edwin Li ◽  
Kalina Hristova
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Supported Lipid Bilayers

scholarly journals Structural characterization of the antimicrobial peptides myxinidin and WMR in bacterial membrane mimetic micelles and bicelles

2021 ◽  
Author(s):  
Yevhen K. Cherniavskyi ◽  
Rosario Oliva ◽  
Marco Stellato ◽  
Pompea Del Vecchio ◽  
Stefania Galdiero ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Antimicrobial Peptides ◽  
Molecular Dynamics Simulations ◽  
Lipid Bilayers ◽  
Structural Characterization ◽  
Bacterial Membrane ◽  
Membrane Composition ◽  
Membrane Mimetic ◽  
Dynamics Simulations

Antimicrobial peptides are a promising class of alternative antibiotics that interact selectively with negatively charged lipid bilayers. This paper presents the structural characterization of the antimicrobial peptides myxinidin and WMR associated with bacterial membrane mimetic micelles and bicelles by NMR, CD spectroscopy, and Molecular Dynamics simulations. Both peptides adopt a different conformation in the lipidic environment than in aqueous solution. The location of peptides in micelles and bicelles has been studied by paramagnetic relaxation enhancement experiments with paramagnetic tagged 5- and 16-doxyl stearic acid (5-/16-SASL). Multi-microsecond long molecular dynamics simulations of multiple copies of the peptides were used to gain an atomic level of detail on membrane-peptide and peptide-peptide interactions. Our results highlight an essential role of the negatively charged membrane mimetic in the structural stability of both myxinidin and WMR. The peptides localize predominantly in the membrane's headgroup region and have a noticeable membrane thinning effect on the overall bilayer structure. Myxinidin and WMR show different tendency to self-aggregate, which is also influenced by the membrane composition (DOPE/DOPG versus DOPE/DOPG/CL) and can be related to the previously observed difference in the ability of the peptides to disrupt different types of model membranes.

scholarly journals Phospholipid membranes promote the early stage assembly of α-synuclein aggregates

2018 ◽  
Author(s):  
Zhengjian Lv ◽  
Mohtadin Hashemi ◽  
Siddhartha Banerjee ◽  
Karen Zagorski ◽  
Jean-Christophe Rochet ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Self Assembly ◽  
Early Stage ◽  
Time Lapse ◽  
Supported Lipid Bilayers ◽  
Membrane Composition ◽  
Force Microscopy ◽  
Atomic Force ◽  
Nanomolar Concentration ◽  
Surface Changes

AbstractDevelopment of Parkinson’s disease is associated with spontaneous self-assembly of α-synuclein (α-syn). Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. We report a novel effect of phospholipid bilayers on the catalysis of α-syn aggregation from monomers. We directly visualized α-syn aggregation on supported lipid bilayers using time-lapse atomic force microscopy. We discovered that α-syn assemble in aggregates on bilayer surfaces even at the nanomolar concentration of monomers in solution. The efficiency of the aggregation process depends on the membrane composition, being highest for a negatively charged bilayer. Furthermore, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation can be a mechanism by which pathological aggregates are produced. Computational modeling revealed that interaction of α-syn with bilayer surface changes the protein conformation and its affinity to assemble into dimers, and these properties depend on the bilayer composition. A model of the membrane-mediated aggregation triggering the assembly of neurotoxic aggregates is proposed.

scholarly journals Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

2016 ◽  
Vol 213 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Andrew A. Bridges ◽  
Maximilian S. Jentzsch ◽  
Patrick W. Oakes ◽  
Patricia Occhipinti ◽  
Amy S. Gladfelter
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Fundamental Property ◽  
Membrane Curvature ◽  
Supported Lipid Bilayers ◽  
Nanometer Scale ◽  
Local Shape ◽  
A Cell ◽  
Developmental Conditions ◽  
In Cells

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape.

scholarly journals Single-lipid dynamics in phase-separated supported lipid bilayers

2020 ◽  
Author(s):  
Xinxin Woodward ◽  
Christopher V. Kelly
Keyword(s):  
Lipid Bilayers ◽  
Fluorescence Correlation Spectroscopy ◽  
Cholesterol Content ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Supported Lipid Bilayers ◽  
Membrane Composition ◽  
Fluorescence Correlation ◽  
Diffusion Rates ◽  
Lipid Diffusion

ABSTRACTPhase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases.HIGHLIGHTSLipid diffusion varies with the lipid phases, temperature, and aggregationAggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopyMembrane with higher cholesterol content or at low temperature have more aggregatesA more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content

scholarly journals A combination of electrochemistry and mass spectrometry to monitor the interaction of reactive species with supported lipid bilayers

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
M. Ravandeh ◽  
H. Kahlert ◽  
H. Jablonowski ◽  
J.-W. Lackmann ◽  
J. Striesow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Reactive Species ◽  
Oxidation Products ◽  
Supported Lipid Bilayers ◽  
Resonance Spectroscopy ◽  
Membrane Composition ◽  
Physical Plasma ◽  
The Impact

Abstract Reactive oxygen and nitrogen species (RONS), e.g. generated by cold physical plasma (CPP) or photodynamic therapy, interfere with redox signaling pathways of mammalian cells, inducing downstream consequences spanning from migratory impairment to apoptotic cell death. However, the more austere impact of RONS on cancer cells remains yet to be clarified. In the present study, a combination of electrochemistry and high-resolution mass spectrometry was developed to investigate the resilience of solid-supported lipid bilayers towards plasma-derived reactive species in dependence of their composition. A 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer was undisturbed by 200 µM H2O2 (control) but showed full permeability after CPP treatment and space-occupying oxidation products such as PoxnoPC, PAzePC, and POPC hydroperoxide were found. Electron paramagnetic resonance spectroscopy demonstrated the presence of hydroxyl radicals and superoxide anion/hydroperoxyl radicals during the treatment. In contrast, small amounts of the intramembrane antioxidant coenzyme Q10 protected the bilayer to 50% and LysoPC was the only POPC derivative found, confirming the membrane protective effect of Q10. Such, the lipid membrane composition including the presence of antioxidants determines the impact of pro-oxidant signals. Given the differences in membrane composition of cancer and healthy cells, this supports the application of cold physical plasma for cancer treatment. In addition, the developed model using the combination of electrochemistry and mass spectrometry could be a promising method to study the effect of reactive species or mixes thereof generated by chemical or physical sources.

scholarly journals Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers

2021 ◽  
Vol 18 (10) ◽  
pp. 1247-1252 ◽  
Author(s):  
Eric D. B. Foley ◽  
Manish S. Kushwah ◽  
Gavin Young ◽  
Philipp Kukura
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
State Of The Art ◽  
Mass Measurement ◽  
Integral Membrane Proteins ◽  
Supported Lipid Bilayers ◽  
Label Free ◽  
Cellular Processes ◽  
Dynamic Mass ◽  
Associated Proteins

AbstractThe quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg
Keyword(s):  
Lipid Bilayers ◽  
Binding Kinetics ◽  
Supported Lipid Bilayers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Supported Lipid Bilayer ◽  
Head Groups ◽  
Lipid Diffusion ◽  
Kinetics Of ◽  
Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

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