scholarly journals Retromer forms low order oligomers on supported lipid bilayers

2020 ◽  
Vol 295 (34) ◽  
pp. 12305-12316 ◽  
Author(s):  
Catherine L. Deatherage ◽  
Joerg Nikolaus ◽  
Erdem Karatekin ◽  
Christopher G. Burd
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Supported Lipid Bilayer ◽  
Sorting Nexin ◽  
Cargo Proteins ◽  
Transport Carrier ◽  
Accessory Factors ◽  
Endosomal System ◽  
Low Order

Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)–retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.

scholarly journals Retromer forms low order oligomers on supported lipid bilayers

2020 ◽  
Author(s):  
Catherine L. Deatherage ◽  
Joerg Nikolaus ◽  
Erdem Karatekin ◽  
Christopher G. Burd
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Supported Lipid Bilayers ◽  
Supported Bilayer ◽  
Cargo Protein ◽  
Transport Carrier ◽  
Bulk Membrane ◽  
Accessory Factors ◽  
Endosomal System ◽  
Low Order

AbstractRetromer is a protein sorting device that orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long standing hypotheses regarding the Retromer sorting mechanism posit that oligomeric interactions between Retromer and associated accessory factors on the endosome membrane drives clustering of Retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this hypothesis, we examined interactions between the components of the SNX3-Retromer sorting pathway using quantitative single particle fluorescence microscopy of a reconstituted system comprising a supported bilayer, Retromer, a model cargo protein, the accessory proteins SNX3, RAB7, and the Retromer-binding segment of the WASHC2C subunit of the WASH complex. The predominant species of membrane associated Retromer are low order: monomers (∼18%), dimers (∼35%), trimers (∼24%) and tetramers (∼24%). Unexpectedly, neither cargo nor accessory factors promote Retromer oligomerization on a supported bilayer. The results indicate that Retromer has an intrinsic propensity to form low order oligomers and that neither membrane association nor accessory factors potentiate oligomerization. Hence, Retromer is a minimally concentrative sorting device adapted to bulk membrane trafficking from the endosomal system.

scholarly journals Lipid flip-flop and desorption from supported lipid bilayers is independent of curvature

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244460
Author(s):  
Haoyuan Jing ◽  
Yanbin Wang ◽  
Parth Rakesh Desai ◽  
Kumaran S. Ramamurthi ◽  
Siddhartha Das
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Md Simulations ◽  
Packing Fraction ◽  
Membrane Curvature ◽  
Supported Lipid Bilayer ◽  
Flip Flop ◽  
Lipid Molecule ◽  
Membrane Asymmetry ◽  
Cell Shapes

Flip-flop of lipids of the lipid bilayer (LBL) constituting the plasma membrane (PM) plays a crucial role in a myriad of events ranging from cellular signaling and regulation of cell shapes to cell homeostasis, membrane asymmetry, phagocytosis, and cell apoptosis. While extensive research has been conducted to probe the lipid flip flop of planar lipid bilayers (LBLs), less is known regarding lipid flip-flop for highly curved, nanoscopic LBL systems despite the vast importance of membrane curvature in defining the morphology of cells and organelles and in maintaining a variety of cellular functions, enabling trafficking, and recruiting and localizing shape-responsive proteins. In this paper, we conduct molecular dynamics (MD) simulations to study the energetics, structure, and configuration of a lipid molecule undergoing flip-flop and desorption in a highly curved LBL, represented as a nanoparticle-supported lipid bilayer (NPSLBL) system. We compare our findings against those of a planar substrate supported lipid bilayer (PSSLBL). Our MD simulation results reveal that despite the vast differences in the curvature and other curvature-dictated properties (e.g., lipid packing fraction, difference in the number of lipids between inner and outer leaflets, etc.) between the NPSLBL and the PSSLBL, the energetics of lipid flip-flop and lipid desorption as well as the configuration of the lipid molecule undergoing lipid flip-flop are very similar for the NPSLBL and the PSSLBL. In other words, our results establish that the curvature of the LBL plays an insignificant role in lipid flip-flop and desorption.

Kinetic evolution of DOPC lipid bilayers exposed to α-cyclodextrins

Soft Matter ◽  
2018 ◽  
Vol 14 (28) ◽  
pp. 5800-5810 ◽  
Author(s):  
Monika Kluzek ◽  
Marc Schmutz ◽  
Carlos M. Marques ◽  
Fabrice Thalmann
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Supported Lipid Bilayer ◽  
Microscopy Image

Confocal laser scanning microscopy image of a fluorescent supported lipid bilayer exposed to a 15 mM solution of α-cyclodextrin.

Supported lipid bilayer repair mediated by AH peptide

2016 ◽  
Vol 18 (4) ◽  
pp. 3040-3047 ◽  
Author(s):  
Min Chul Kim ◽  
Anders Gunnarsson ◽  
Seyed R. Tabaei ◽  
Fredrik Höök ◽  
Nam-Joon Cho
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Silicon Oxide ◽  
Supported Lipid Bilayers ◽  
High Quality ◽  
Supported Lipid Bilayer

High quality and complete supported lipid bilayers are formed on silicon oxide by employing an AH peptide mediated repair step.

scholarly journals Membrane attack complex formation on a supported lipid bilayer: initial steps towards a CARPA predictor nanodevice

2015 ◽  
Vol 7 (3) ◽  
Author(s):  
Saziye Yorulmaz ◽  
Seyed R. Tabaei ◽  
Myunghee Kim ◽  
Jeongeun Seo ◽  
Walter Hunziker ◽  
...  
Keyword(s):  
Real Time ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Ambient Medium ◽  
Membrane Attack Complex ◽  
Common Adverse Effect ◽  
Individual Risk ◽  
Supported Lipid Bilayer ◽  
Activation Assay

AbstractThe rapid advance of nanomedicines and biologicals in pharmacotherapy gives increasing importance to a common adverse effect of these modern therapeutics: complement (C) activation-related pseudoallergy (CARPA). CARPA is a relatively frequent and potentially lethal acute immune toxicity of many intravenous drugs that contain nanoparticles or proteins, whose prediction by laboratory or in vivo testing has not yet been solved. Preliminary studies suggest that proneness of the drug to cause C activation in the blood of patients may predict the individual risk of CARPA, thus, a sensitive and rapid bedside assay for individualized assessment of a drug’s C activating potential could alleviate the CARPA problem. The goal of the present study was to lay down the foundations of a novel approach for real-time sensing of C activation on a supported lipid bilayer platform. We utilized the quartz crystal microbalance with dissipation (QCM-D) monitoring technique to measure the self-assembly of C terminal complex (or membrane attack complex [MAC]) on supported lipid bilayers rapidly assembled by the solvent-assisted lipid bilayer (SALB) formation method, as an immediate measure of C activation. By measuring the changes in frequency and energy dissipation of deposited protein, the technique allows extremely sensitive real-time quantification of the sequential assembly of MAC from its molecular components (C5b-6, C7, C8 and C9) and hence, measure C activation in the ambient medium. The present paper delineates the technique and our initial evidence with purified C proteins that the approach enables sensitive and rapid (real-time) quantification of MAC formation on a silicon-supported planar (phospho) lipid bilayer, which can be used as an endpoint in a clinically useful bedside C activation assay.

Microcavity array supported lipid bilayer models of ganglioside – influenza hemagglutinin1 binding

2020 ◽  
Vol 56 (76) ◽  
pp. 11251-11254 ◽  
Author(s):  
Guilherme B. Berselli ◽  
Nirod Kumar Sarangi ◽  
Aurélien V. Gimenez ◽  
Paul V. Murphy ◽  
Tia E. Keyes
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Supported Lipid Bilayers ◽  
Supported Lipid Bilayer

The binding of influenza receptor (HA1) to membranes containing different glycosphingolipid receptors was investigated at Microcavity Supported Lipid Bilayers (MSLBs).

scholarly journals Nanoarchitectured air-stable supported lipid bilayer incorporating sucrose–bicelle complex system

2022 ◽  
Vol 9 (1) ◽  
Author(s):  
Hyunhyuk Tae ◽  
Soohyun Park ◽  
Gamaliel Junren Ma ◽  
Nam-Joon Cho
Keyword(s):  
Complex System ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Supported Lipid Bilayers ◽  
Air Exposure ◽  
Supported Lipid Bilayer ◽  
Wide Range ◽  
Aqueous Environments ◽  
One Step ◽  
Self Assembled Layer

AbstractCell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose–bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration–rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose–bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications.

scholarly journals Stripping Material from a Supported Lipid Bilayer with High Speed Buffer Flow

2020 ◽  
Vol 17 (3) ◽  
pp. 51-59
Author(s):  
Michael Ornstead ◽  
Ruth Hunter ◽  
Mason Valentine ◽  
Cameron Cooper ◽  
Stephen Smith ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Microfluidic Device ◽  
High Speed ◽  
Membrane Vesicle ◽  
Microfluidic Channel ◽  
Lipid Vesicles ◽  
Membrane Properties ◽  
Supported Lipid Bilayer ◽  
Glass Coverslip

A microfluidic device was created and used to demonstrate that supported lipid bilayers can be deposited on clean glass slides and removed using high velocity buffer flow (1-4 m/s linear velocity). This was accomplished by forcing the flow through a microfluidic channel covering an annealed glass coverslip bearing a supported lipid bilayer (SLB). The removal of bilayer material was monitored via fluorescence microscopy, and two basic regimes were observed: at 1-2 m/s smaller areas were stripped, while at 3-4 m/s larger areas were stripped. SLB removal was verified by two means. First, lipid vesicles labeled with a different fluorescent dye were added to the device and filled in holes left by the removal of the original SLB, allowing stripping to be verified visually. Second, the solutions obtained from stripping were concentrated and the fluorescence in the concentrates was measured. The ability to strip SLB from glass provides a relatively gentle method of creating spatially inhomogeneous SLB, which could be a useful tool in the continued investigation of membrane properties and components. KEYWORDS: Supported Lipid Bilayer; Membrane Vesicle; Microfluidic Device

The small GTPase Arf6 functions as a membrane tether in a chemically-defined reconstitution system

2020 ◽  
Author(s):  
Kana Fujibayashi ◽  
Joji Mima
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Initial Contact ◽  
Experimental Conditions ◽  
Membrane Tethering ◽  
Transport Carrier ◽  
Membrane Tether

AbstractArf-family small GTPases are essential protein components for membrane trafficking in all eukaryotic endomembrane systems, particularly during the formation of membrane-bound, coat protein complex-coated transport carriers. In addition to their roles in the transport carrier formation, a number of Arf-family GTPases have been reported to physically associate with coiled-coil tethering proteins and multisubunit tethering complexes, which are responsible for membrane tethering, a process of the initial contact between transport carriers and their target subcellular compartments. Nevertheless, whether and how indeed Arf GTPases are involved in the tethering process remain unclear. Here, using a chemically-defined reconstitution approach with purified proteins of two representative Arf isoforms in humans (Arf1, Arf6) and synthetic liposomes for model membranes, we discovered that Arf6 can function as a bona fide membrane tether, directly and physically linking two distinct lipid bilayers even in the absence of any other tethering factors, whereas Arf1 retained little potency to trigger membrane tethering under the current experimental conditions. Arf6-mediated membrane tethering reactions require trans-assembly of membrane-anchored Arf6 proteins and can be reversibly controlled by the membrane attachment and detachment cycle of Arf6. The intrinsic membrane tethering activity of Arf6 was further found to be significantly inhibited by the presence of membrane-anchored Arf1, suggesting that the tethering-competent Arf6-Arf6 assembly in trans can be prevented by the heterotypic Arf1-Arf6 association in a cis configuration. Taken together, these findings lead us to postulate that self-assemblies of Arf-family small GTPases on lipid bilayers contribute to driving and regulating the tethering events of intracellular membrane trafficking.

scholarly journals The Small GTPase Arf6 Functions as a Membrane Tether in a Chemically-Defined Reconstitution System

2021 ◽  
Vol 9 ◽  
Author(s):  
Kana Fujibayashi ◽  
Joji Mima
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Initial Contact ◽  
Experimental Conditions ◽  
Membrane Tethering ◽  
Transport Carrier ◽  
Membrane Tether

Arf-family small GTPases are essential protein components for membrane trafficking in all eukaryotic endomembrane systems, particularly during the formation of membrane-bound, coat protein complex-coated transport carriers. In addition to their roles in the transport carrier formation, a number of Arf-family GTPases have been reported to physically associate with coiled-coil tethering proteins and multisubunit tethering complexes, which are responsible for membrane tethering, a process of the initial contact between transport carriers and their target subcellular compartments. Nevertheless, whether and how indeed Arf GTPases are involved in the tethering process remain unclear. Here, using a chemically-defined reconstitution approach with purified proteins of two representative Arf isoforms in humans (Arf1, Arf6) and synthetic liposomes for model membranes, we discovered that Arf6 can function as a bona fide membrane tether, directly and physically linking two distinct lipid bilayers even in the absence of any other tethering factors, whereas Arf1 retained little potency to trigger membrane tethering under the current experimental conditions. Arf6-mediated membrane tethering reactions require trans-assembly of membrane-anchored Arf6 proteins and can be reversibly controlled by the membrane attachment and detachment cycle of Arf6. The intrinsic membrane tethering activity of Arf6 was further found to be significantly inhibited by the presence of membrane-anchored Arf1, suggesting that the tethering-competent Arf6-Arf6 assembly in trans can be prevented by the heterotypic Arf1-Arf6 association in a cis configuration. Taken together, these findings lead us to postulate that self-assemblies of Arf-family small GTPases on lipid bilayers contribute to driving and regulating the tethering events of intracellular membrane trafficking.

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