Fluorophore-Encapsulated Solid-Supported Bilayer Vesicles:  A Method for Studying Membrane Permeation Processes

Williams, Thomas L.; Vareiro, Margarida M.L.M.; Jenkins, A. Toby A.

doi:10.1021/la060853q

Cited by 17 publications

(12 citation statements)

References 18 publications

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“…SPFS (surface plasmon fieldenhanced fluorescence spectroscopy) is a technique that combines the ability of SPR (surface plasmon resonance) to monitor adsorption and desorption events at the air/dielectric interface with the ability to resonantly excite fluorescent molecules within the evanescent field extending 200 nm within the dielectric, and this allows us to simultaneously observe both Aβ-membrane interactions and correlate these with permeation events. SPFS has previously been used to monitor the tethering of intact LUVs (large unilamellar vesicles) to a functionalized gold substrate, and the events following the addition of the membrane-lysing protein phospholipase A 2 [27] and membrane permeation caused by cholera toxin [28]. Permeation is the term we apply in the present study to mean the penetration of the lipid bilayer by Aβ resulting in a non-continuous membrane surface and the emergence of defects/holes or deformations within the bilayer to allow increased diffusion across the membrane bilayer.…”

Section: Introductionmentioning

confidence: 99%

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Williams

Johnson

Urbanc

et al. 2011

Biochemical Journal

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Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.

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Section: Introductionmentioning

confidence: 99%

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Williams

Johnson

Urbanc

et al. 2011

Biochemical Journal

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“…The initial binding of PLA 2 to lipid membranes and delay before lysis is observed has been seen in previous studies which looked at the interaction of PLA 2 with lipid vesicles [Sanchez 2002]. In a study by Williams et al 2006, the permeation of the bilayer was observed via the leakage of encapsulated fluorescent dye from solid supported lipid vesicles. Both studies showed a delay between PLA 2 binding to a lipid membrane and lysis of around 8-10 minutes.…”

Section: Figurementioning

confidence: 68%

Sensing of pathogenic bacteria based on their interaction with supported bilayer membranes studied by impedance spectroscopy and surface plasmon resonance

Tun

Cameron

Jenkins

2011

Biosensors and Bioelectronics

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Pathogenic bacteria secrete various virulence factors, including toxins, lipases and proteases that allow them to infect, breakdown and colonize host tissue. Among various modes of action that the pathogenic bacteria use to damage the host, pore formation (by pore forming toxins (PFTs)) and lipid hydrolysis (by phospholipases) modes are common in damaging the eukaryotic cell membrane. PFTs in their monomeric form are extracellular diffusible and able to form hydrophilic pores in cell membrane while phospholipases cleaves and hydrolyzes the ester bonds of most phospholipids in cell membrane. Both modes of action cause uncontrolled permeation of ions and molecules across cell membrane, leading to cell death by apoptosis or necrosis. In this work, the toxins secreted by two clinically important human pathogens, methicillin susceptible Staphylococcus aureus (MSSA476) and Pseudomonas aeruginosa (PAO1) were studied via their interaction with a planar tethered bilayer lipid membrane (pTBLM) using surface plasmon resonance spectroscopy (SPR) and electrochemical impedance spectroscopy (EIS). Detection and discrimination is based on lipid-loss (lipid hydrolysis by phospholipases) or non lipid-loss (pore formation by PFTs) from pTBLM upon interaction with supernatant of pathogenic bacteria. Using EIS and SPR, it is demonstrated that major toxins of S. auerus are PFTs while most of toxin associated with P. aeruginosa are more lipid damaging lipolytic enzymes. Such a format might have future utility as a simple assay for measuring the presence membrane lytic virulence factors in clinical samples.

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“…As such, the calcein release assay offers a reproducible means to quantify the effect of potentially toxic protein assemblies in the solution on membrane integrity. Using the calcein release assay, Williams et al previously reported that Aβ40 and, even more so, Aβ42 at 0 hours of incubation cause significant membrane disruption. − , The disruption was the largest at 0 h Aβ42 incubation and then steadily diminished as Aβ42 formed amyloid fibrils at longer incubation times.…”

Section: Resultsmentioning

confidence: 99%

Insulin Inhibits Aβ42 Aggregation and Prevents Aβ42-Induced Membrane Disruption

2019

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Alzheimer’s disease (AD) is associated with self-assembly of amyloid β-protein (Aβ) into soluble oligomers. Of the two predominant Aβ alloforms, Aβ40 and Aβ42, the latter is particularly strongly linked to AD. Longitudinal studies revealed a correlation between AD and type 2 diabetes (T2D), characterized by abnormal insulin levels and insulin resistance. Although administration of intranasal insulin is explored as a therapy against AD, the extent to which insulin affects Aβ dynamics and activity is unclear. We here investigate the effect of insulin on Aβ42 self-assembly and characterize the capacity of insulin, Aβ42, and Aβ42 co-incubated with insulin to disrupt the integrity of biomimetic lipid vesicles. We demonstrate that quiescently incubated insulin, which does not form amyloid fibrils, over time develops membrane-disrupting capacity, which we propose to originate in misfolded insulin monomers. These hypothetically toxic misfolded monomers might contribute to the development of insulin resistance in early stages of T2D that are associated with abnormally high insulin levels. We show that in contrast to quiescent incubation, insulin incubated under agitated conditions readily forms amyloid fibrils, which protect against membrane permeation. Insulin quiescently incubated with Aβ42 attenuates both Aβ42 fibril formation and the ability of Aβ42 to disrupt membranes in a concentration-dependent manner. Our findings offer insights into interactions between insulin and Aβ42 that are relevant to understanding the molecular basis of intranasal insulin as a therapy against Aβ-induced AD pathology, thereby elucidating a plausible mechanism underlying the observed correlations between AD and T2D.

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Fluorophore-Encapsulated Solid-Supported Bilayer Vesicles: A Method for Studying Membrane Permeation Processes

Cited by 17 publications

References 18 publications

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Sensing of pathogenic bacteria based on their interaction with supported bilayer membranes studied by impedance spectroscopy and surface plasmon resonance

Insulin Inhibits Aβ42 Aggregation and Prevents Aβ42-Induced Membrane Disruption

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