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Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling.

ABSTRACT: B cell responses are initiated by the clustering of the B cell receptor (BCR) by the binding of multivalent antigens. Clustering leads to phosphorylation of tyrosines in the cytoplasmic domains of the BCR by the inner plasma membrane leaflet-associated Src-family kinase Lyn. At present, little is known about the earliest events after BCR clustering that precede the BCR's phosphorylation by Lyn. Here we use fluorescence resonance energy transfer (FRET) in living cells to detect the interaction of the BCR with a Lyn-based membrane-targeted reporter in the first several seconds after BCR clustering. The results showed that, within seconds of antigen binding, the BCR selectively and transiently associated with the Lyn construct and that this association preceded by several seconds the triggering of Ca2+ fluxes and could be prolonged by the engagement of the B cell coreceptor complex, CD19/CD21. Thus, FRET measurements in living B cells revealed highly dynamic and regulated antigen-induced changes in the plasma membrane, allowing association of the BCR with the earliest components of its signaling cascade.

SUBMITTER: Sohn HW

PROVIDER: S-EPMC1472443 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling.

Sohn Hae Won HW Tolar Pavel P Jin Tian T Pierce Susan K SK

Proceedings of the National Academy of Sciences of the United States of America 20060511 21

B cell responses are initiated by the clustering of the B cell receptor (BCR) by the binding of multivalent antigens. Clustering leads to phosphorylation of tyrosines in the cytoplasmic domains of the BCR by the inner plasma membrane leaflet-associated Src-family kinase Lyn. At present, little is known about the earliest events after BCR clustering that precede the BCR's phosphorylation by Lyn. Here we use fluorescence resonance energy transfer (FRET) in living cells to detect the interaction of ...[more]

PMID: 16690746

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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.

Dataset Information

Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling.

Publications

Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling.

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