Imaging Protein Interactions in Living Cells Using the Fluorescent Proteins

2009 ◽  
pp. 337-357
Author(s):  
Richard N. Day ◽  
Ammasi Periasamy ◽  
Ignacio Demarco
Keyword(s):  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Living Cells

scholarly journals TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shipeng Shao ◽  
Hongchen Zhang ◽  
Yong Zeng ◽  
Yongliang Li ◽  
Chaoying Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Time Course ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Spatiotemporal Resolution ◽  
Dynamic Interactions ◽  
Single Molecule Tracking

AbstractProtein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

scholarly journals Fluorescent proteins for FRET microscopy: Monitoring protein interactions in living cells

BioEssays ◽  
2012 ◽  
Vol 34 (5) ◽  
pp. 341-350 ◽  
Author(s):  
Richard N. Day ◽  
Michael W. Davidson
Keyword(s):  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Living Cells

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

2010 ◽  
Vol 90 (3) ◽  
pp. 1103-1163 ◽  
Author(s):  
Dmitriy M. Chudakov ◽  
Mikhail V. Matz ◽  
Sergey Lukyanov ◽  
Konstantin A. Lukyanov
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Visible Spectrum ◽  
Living Cells ◽  
Alternative Possibilities ◽  
Structure Evolution ◽  
Marine Animals

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

scholarly journals Compartmentalization of androgen receptor protein–protein interactions in living cells

2007 ◽  
Vol 177 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Martin E. van Royen ◽  
Sónia M. Cunha ◽  
Maartje C. Brink ◽  
Karin A. Mattern ◽  
Alex L. Nigg ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Steroid Receptors ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Receptor Protein ◽  
Dependent Manner

Steroid receptors regulate gene expression in a ligand-dependent manner by binding specific DNA sequences. Ligand binding also changes the conformation of the ligand binding domain (LBD), allowing interaction with coregulators via LxxLL motifs. Androgen receptors (ARs) preferentially interact with coregulators containing LxxLL-related FxxLF motifs. The AR is regulated at an extra level by interaction of an FQNLF motif in the N-terminal domain with the C-terminal LBD (N/C interaction). Although it is generally recognized that AR coregulator and N/C interactions are essential for transcription regulation, their spatiotemporal organization is largely unknown. We performed simultaneous fluorescence resonance energy transfer and fluorescence redistribution after photobleaching measurements in living cells expressing ARs double tagged with yellow and cyan fluorescent proteins. We provide evidence that AR N/C interactions occur predominantly when ARs are mobile, possibly to prevent unfavorable or untimely cofactor interactions. N/C interactions are largely lost when AR transiently binds to DNA, predominantly in foci partly overlapping transcription sites. AR coregulator interactions occur preferentially when ARs are bound to DNA.

Imaging Protein Interactions and Modifications in Living Cells

2004 ◽  
Vol 10 (S02) ◽  
pp. 160-161
Author(s):  
Asya V. Grinberg ◽  
Deyu Fang ◽  
Chang-Deng Hu ◽  
Tom K. Kerppola
Keyword(s):  
Protein Interactions ◽  
Living Cells

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

scholarly journals Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells

2010 ◽  
Vol 107 (31) ◽  
pp. 13582-13587 ◽  
Author(s):  
H. E. Rajapakse ◽  
N. Gahlaut ◽  
S. Mohandessi ◽  
D. Yu ◽  
J. R. Turner ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Protein Protein Interactions ◽  
Time Resolved
Sign in / Sign up

Export Citation Format

Share Document