scholarly journals Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells

10.5772/52386 ◽  
2013 ◽  
Author(s):  
Kayo Hibino ◽  
Michio Hiroshima ◽  
Yuki Nakamura ◽  
Yasushi Sako
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Living Cells ◽  
Single Molecule Imaging ◽  
Protein Protein Interactions

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 Proximity-assisted photoactivation (PAPA): Detecting molecular interactions in live-cell single-molecule imaging

2021 ◽  
Author(s):  
Thomas George Wade Graham ◽  
John Joseph Ferrie ◽  
Gina M. Dailey ◽  
Robert Tjian ◽  
Xavier Darzacq
Keyword(s):  
Single Molecule ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Single Molecule Imaging ◽  
Protein Protein Interactions ◽  
Self Association

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the "sender") can reactivate a second fluorophore (the "receiver") from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Forster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging

FEBS Letters ◽  
2001 ◽  
Vol 502 (3) ◽  
pp. 79-83 ◽  
Author(s):  
Junichi Yamaguchi ◽  
Naoto Nemoto ◽  
Toru Sasaki ◽  
Ako Tokumasu ◽  
Yuko Mimori-Kiyosue ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Single Molecule ◽  
Protein Interactions ◽  
Single Molecule Imaging ◽  
Protein Protein Interactions

scholarly journals Fluorescence lifetime imaging microscopy (FLIM) to quantify protein–protein interactions inside cells

2006 ◽  
Vol 34 (5) ◽  
pp. 679-682 ◽  
Author(s):  
R.R. Duncan
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Protein Interactions ◽  
Single Photon ◽  
Imaging Spectroscopy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Recent Developments

Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein–protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

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