scholarly journals Quantification of protein interaction in living cells by two-photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed-through

2011 ◽  
Vol 81A (2) ◽  
pp. 112-119 ◽  
Author(s):  
Jiho Kim ◽  
Xiaolan Li ◽  
Moon-Sik Kang ◽  
Kang-Bin Im ◽  
Auguste Genovesio ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interaction ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Protein Fluorescence ◽  
Two Photon ◽  
Bleed Through

Three-dimensional localisation of fluorescence resonance energy transfer in living cells under two-photon excitation

Scanning ◽  
2001 ◽  
Vol 23 (1) ◽  
pp. 9-13 ◽  
Author(s):  
Ming G. Xu ◽  
Blessing Crimeen ◽  
Mandy J. Ludford-Menting ◽  
Xiaosong Gan ◽  
Sarah M. Russell ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Two Photon ◽  
Photon Excitation ◽  
Fluorescence Resonance ◽  
Two Photon Excitation

scholarly journals Homomultimerization of the Coxsackievirus 2B Protein in Living Cells Visualized by Fluorescence Resonance Energy Transfer Microscopy

2002 ◽  
Vol 76 (18) ◽  
pp. 9446-9456 ◽  
Author(s):  
Frank J. M. van Kuppeveld ◽  
Willem J. G. Melchers ◽  
Peter H. G. M. Willems ◽  
Theodorus W. J. Gadella,
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Cell Membrane Permeability ◽  
Molecular Architecture ◽  
2B Protein ◽  
Fluorescence Resonance

ABSTRACT The 2B protein of enteroviruses is the viral membrane-active protein that is responsible for the modifications in host cell membrane permeability that take place in enterovirus-infected cells. The 2B protein shows structural similarities to the group of lytic polypeptides, polypeptides that permeate membranes either by forming multimeric membrane-integral pores or, alternatively, by lying parallel to the lipid bilayer and disturbing the curvature and symmetry of the membrane. Our aim is to gain more insight into the molecular architecture of the 2B protein in vivo. In this study, the possible existence of multimers of the coxsackie B3 virus 2B protein in single living cells was explored by fluorescence resonance energy transfer (FRET) microscopy. FRET between fusion proteins 2B-ECFP and 2B-EYFP (enhanced cyan and yellow fluorescent variants of green fluorescent protein) was monitored by using spectral imaging microscopy (SPIM) and fluorescence lifetime imaging microscopy (FLIM). Both techniques revealed the occurrence of intermolecular FRET between 2B-ECFP and 2B-EYFP, providing evidence for the formation of protein 2B homomultimers. Putative models for the mode of action of the membrane-active 2B protein and the formation of membrane-integral pores by 2B multimers are discussed.

scholarly journals Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy

1998 ◽  
Vol 12 (9) ◽  
pp. 1410-1419 ◽  
Author(s):  
Richard N. Day
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Resonance

Abstract The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 Å. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.

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