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Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer.

ABSTRACT: Nanometer-scale intermembrane contact areas (CAs) formed between single small unilamellar lipid vesicles (SUVs) and planar supported lipid bilayers are quantified by measuring fluorescence resonance energy transfer (FRET) between a homogenous layer of donor fluorophores labeling the supported bilayer and acceptor fluorophores labeling the SUVs. The smallest CAs detected in our setup between biotinylated SUVs and dense monolayers of streptavidin were approximately 20 nm in radius. Deformation of SUVs is revealed by comparing the quenching of the donors to calculations of FRET between a perfectly spherical shell and a flat surface containing complementary fluorophores. These results confirmed the theoretical prediction that the degree of deformation scales with the SUV diameter. The size of the CA can be controlled experimentally by conjugating polyethylene glycol polymers to the SUV or the surface and thereby modulating the interfacial energy of adhesion. In this manner, we could achieve secure immobilization of SUVs under conditions of minimal deformation. Finally, we demonstrate that kinetic measurements of CA, at constant adhesion, can be used to record in real-time quantitative changes in the bilayer tension of a nano-scale lipid membrane system.

SUBMITTER: Bendix PM

PROVIDER: S-EPMC2709668 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

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Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer.

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