Fluorescence correlation and lifetime correlation spectroscopy applied to the study of supported lipid bilayer models of the cell membrane

Basit, Hajra; Lopez, Sergio G.; Keyes, Tia E.

doi:10.1016/j.ymeth.2014.02.005

Cited by 22 publications

(20 citation statements)

References 86 publications

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“…15,16,21 An important disadvantage of SLBs is that non-specific interactions that can occur between the underlying substrate and the protein extracellular or cytoplasmic domains due to inadequate space (a 10-20 Å thick water layer) between substrate and lower bilayer leaflet. 27 In the ''nature's own'' GUVs, a lipid diffusion co-efficient of 7.89 AE 0.42 mm 2 s À1 was recorded for labelled DOPE. 48 The use of GUVs resolves this problem.…”

Section: Discussionmentioning

confidence: 98%

“…27 FLCS measurements were performed using a MicroTime 200 confocal fluorescence lifetime microscope system. The autocorrelation functions obtained using FLCS are less prone to noise and distortion caused by scattered excitation light, detector thermal noise and detector afterpulsing than conventional FCS.…”

Section: Fluorescence Lifetime Correlation Spectroscopy (Flcs)mentioning

confidence: 99%

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The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs

et al. 2015

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Platelet integrin αIIbβ3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbβ3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIbβ3 behaviour away from the complexity of the platelet machinery. Here, a novel method of αIIbβ3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. αIIbβ3 was inserted into two GUV lipid compositions that seek to better mimic the platelet membrane. First, "nature's own", comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn(2+) or DTT does not influence the diffusion coefficient of αIIbβ3. Similarly, exposure to αIIbβ3's primary ligand fibrinogen (Fg) alone does not affect αIIbβ3's diffusion coefficient. However, addition of Fg with either activator reduces the integrin diffusion coefficient from 2.52 ± 0.29 to μm(2) s(-1) to 1.56 ± 0.26 (Mn(2+)) or 1.49 ± 0.41 μm(2) s(-1) (DTT) which is consistent with aggregation of activated αIIbβ3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin-Fg complex diffuses through the confocal volume in clusters. Using the Saffman-Delbrück model as a first approximation, the diffusion coefficient of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIbβ3 was also reconstituted into a "raft forming" GUV with well defined liquid disordered (Ld) and liquid ordered (Lo) phases. Using confocal microscopy and lipid partitioning dyes, αIIbβ3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIbβ3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbβ3 into cholesterol rich domains is not responsible for the integrin clustering observed.

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Section: Discussionmentioning

confidence: 98%

Section: Fluorescence Lifetime Correlation Spectroscopy (Flcs)mentioning

confidence: 99%

The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs

et al. 2015

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“…FCS is widely used to study the diffusion of fluorescently labelled proteins in the cellular membrane, cytosol, nucleus in living cells and in synthetic membranes. [30][31][32][33] It has also been applied to study the aggregation state and critical micelle concentration of peptides and polymers in solution. 33,34 Here, changes in lateral diffusion of lipids in a cavity supported DOPC bilayer were studied by fluorescent lifetime correlation spectroscopy (FLCS), an adaptation of FCS in which the FCS is time gated permitting removal of background noise, after-pulse from the detectors.…”

Section: Introductionmentioning

confidence: 99%

“…This approach provides FCS curves along with fluorescent lifetime data of the probes. 22,30,35 The lipid diffusivity studies are supported by electrochemical impedance measurements to investigate changes to the permeability of the membrane. 26,36 Electrochemical impedance spectroscopy (EIS) is used to measure the complex impedance properties of a material and has been widely used to study interfacial interactions, such as antigen-antibody interaction, and cancer cell capture as well as for evaluation of biomembranes.…”

Section: Introductionmentioning

confidence: 99%

Dynamic studies of the interaction of a pH responsive, amphiphilic polymer with a DOPC lipid membrane

et al. 2017

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Deeper understanding of the molecular interactions between polymeric materials and the lipid membrane is important across a range of applications from permeation for drug delivery to encapsulation for immuno-evasion. Using highly fluidic microcavity supported lipid bilayers, we studied the interactions between amphiphilic polymer PP50 and a DOPC lipid bilayer. As the PP50 polymer is pH responsive the studies were carried out at pH 6.5, 7.05 and 7.5, corresponding to fully, partly protonated (pH = pK = 7.05) and fully ionized states of the polymer, respectively. Fluorescence correlation spectroscopy (FCS) using both labelled lipid and polymer revealed the PP50 associates with the bilayer interface across all pHs where its diffusion along the interface is impeded. Both FCS and electrochemical impedance spectroscopy (EIS) data indicate that the PP50 does not penetrate fully into the bilayer core but rather forms a layer at the bilayer aqueous interface reflected in increased resistance and decreased capacitance of the bilayer on PP50 binding. The extent of these effects and the dynamics of binding are influenced by pH, increasing with decreasing pH. These experimental trends concurred with coarse grained Monte Carlo simulations of polymer-bilayer interactions wherein a model hydrophilic polymer backbone grafted with side chains of varying hydrophobicity, to mimic the effect of varying pH, was simulated based on the bond fluctuation model with explicit solvent. Simulation results showed that with increasing hydrophobicity, the polymer penetrated deeper into the contacting bilayer leaflet of the membrane suppressing, consistent with EIS data, solvent permeation and that a full insertion of the polymer into the bilayer core is not necessary for suppression of permeability.

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“…Recent FFS studies have investigated membrane proteins to measure signaling, disassociation constants, and clustering (24)(25)(26)(27)(28). In this work, we used two-color FFS to examine the specific activation of dopamine receptor subtypes in a pancreatic b-cell model.…”

Section: Introductionmentioning

confidence: 99%

Dopamine Receptor Signaling in MIN6 β -Cells Revealed by Fluorescence Fluctuation Spectroscopy

Caldwell

Ustione

Piston

2016

Biophysical Journal

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Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release.

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Fluorescence correlation and lifetime correlation spectroscopy applied to the study of supported lipid bilayer models of the cell membrane

Cited by 22 publications

References 86 publications

The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs

The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs

Dynamic studies of the interaction of a pH responsive, amphiphilic polymer with a DOPC lipid membrane

Dopamine Receptor Signaling in MIN6 β -Cells Revealed by Fluorescence Fluctuation Spectroscopy

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Fluorescence correlation and lifetime correlation spectroscopy applied to the study of supported lipid bilayer models of the cell membrane

Cited by 22 publications

References 86 publications

The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbβ3reconstituted into physiologically mimetic GUVs

The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbβ3reconstituted into physiologically mimetic GUVs

Dynamic studies of the interaction of a pH responsive, amphiphilic polymer with a DOPC lipid membrane

Dopamine Receptor Signaling in MIN6 β -Cells Revealed by Fluorescence Fluctuation Spectroscopy

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The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs

The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs