Using GFP in FRET-based applications

Pollok, Brian A.

doi:10.1016/s0962-8924(98)01434-2

Cited by 436 publications

(280 citation statements)

References 11 publications

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“…For comparison, E CY for CFP-YFP pair was 0.42 ( Fig. 1d and Supplementary Table 1 online), which is consistent with previously published data 22 .…”

Section: Cfp-yfp-mrfp 3-fret In Vitrosupporting

confidence: 91%

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Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

2004

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Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer . This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.Many cellular processes are governed by multi-component molecular machineries that rely on dynamic and highly coordinated protein-protein interactions. For example, assembly of protein complexes during signal transduction processes increases the speed of enzymatic reactions, ensures the specificity of signaling and targets signaling molecules to proper intracellular compartments. During recent years, fluorescence resonance energy transfer (FRET) has become a key method for the analysis of proteinprotein interactions during signal transduction in living cells [1][2][3] . FRET studies using proteins tagged with mutant derivatives of the green fluorescent protein (GFP) have demonstrated the formation of complexes of signaling proteins in various intracellular compartments 4-7 . FRET-based genetically encoded biosensors for second messengers, protein phosphorylation and activity of small GTPases have provided insights into the spatial and temporal regulation of signaling processes [8][9][10][11][12] .The combination of enhanced cyan (CFP) and yellow (YFP) fluorescent proteins has proven to be most effective in many FRET studies. Cloning of Anthozoa fluorescent proteins, such as red DsRed 13,14 and far-red HcRed 15 , has expanded the in vivo applications of FRET, but a major limitation of Anthozoa proteins for FRET applications is their obligate oligomerization 16 . Recent generation of a monomerized mutant of DsRed, monomeric red fluorescent protein 1 (mRFP) 17 , provides the opportunity to generate functional monomeric fusion proteins and to use mRFP as a FRET acceptor with proteins fused to GFP or its mutants.Various methods of FRET measurements have been used to visualize protein-protein interactions 18 . The general limitation of these methods is that they only permit the analysis of interactions between two proteins. To analyze multi-component signaling complexes, a method to measure interactions betwe...

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“…For comparison, E CY for CFP-YFP pair was 0.42 ( Fig. 1d and Supplementary Table 1 online), which is consistent with previously published data 22 .…”

Section: Cfp-yfp-mrfp 3-fret In Vitrosupporting

confidence: 91%

“…For comparison, E CY for CFP-YFP pair was 0.42 ( Fig. 1d and Supplementary Table 1 online), which is consistent with previously published data 22 .To examine whether FRET between CFP, YFP and mRFP can be detected simultaneously, we prepared a triple-fusion protein consisting of CFP, YFP and mRFP (Fig. 1a).…”

supporting

confidence: 88%

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

2004

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“…FRET microscopy detects the proximity of appropriately labeled or intrinsically fluorescent lipids and proteins with a resolution of tens of angstroms (Uster and Pagano, 1986;Herman, 1989;Jovin and Arndt-Jovin, 1989;Tsien et al, 1993;Nagy et al, 1998;Ng et al, 1999;Pollok and Heim, 1999). The efficiency of energy transfer E for a donor and acceptor fluorophore separated by distance r is given by the following: E ϭ 1/{1 ϩ (r/R o ) 6 }, where R o is a characteristic of the donor and acceptor pair and is typically 30 -60 Å.…”

Section: Rationale For Using Fret Microscopy To Detect Lipid Raftsmentioning

confidence: 99%

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Kenworthy

Petranova

Edidin

2000

MBoC

406

311

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“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

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“…ECFP and EYFP are the most widely used FRET pairs [21]. Because EYFP has a much stronger fluorescence intensity than ECFP, we made the larger E47 (654 amino acids) and EYFP fusion protein the acceptor fluorophore, and the smaller tissue-specific activators, NeuroD1 (357 amino acids), Mash1 (231 amino acids), Ngn1 (244 amino acids) and Ngn2 (263 amino acids), and ECFP fusion proteins the donor fluorophore to ensure comparative expression levels of donor and acceptor.…”

Section: Construction Of Cfp and Yfp Fusion Proteinsmentioning

confidence: 99%

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

et al. 2006

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Using GFP in FRET-based applications

Cited by 436 publications

References 11 publications

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

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