scholarly journals Fluorescent Protein-Based Autophagy Biosensors

Materials ◽  
2021 ◽  
Vol 14 (11) ◽  
pp. 3019
Author(s):  
Heejung Kim ◽  
Jihye Seong
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Treatment Strategies ◽  
Resonance Energy ◽  
Live Cells ◽  
Spectral Profile ◽  
Spatiotemporal Resolution ◽  
Future Directions ◽  
Complex Process

Autophagy is an essential cellular process of self-degradation for dysfunctional or unnecessary cytosolic constituents and organelles. Dysregulation of autophagy is thus involved in various diseases such as neurodegenerative diseases. To investigate the complex process of autophagy, various biochemical, chemical assays, and imaging methods have been developed. Here we introduce various methods to study autophagy, in particular focusing on the review of designs, principles, and limitations of the fluorescent protein (FP)-based autophagy biosensors. Different physicochemical properties of FPs, such as pH-sensitivity, stability, brightness, spectral profile, and fluorescence resonance energy transfer (FRET), are considered to design autophagy biosensors. These FP-based biosensors allow for sensitive detection and real-time monitoring of autophagy progression in live cells with high spatiotemporal resolution. We also discuss future directions utilizing an optobiochemical strategy to investigate the in-depth mechanisms of autophagy. These cutting-edge technologies will further help us to develop the treatment strategies of autophagy-related diseases.

Imaging Protein Interactions and Gene Expression in Individual Cells by Fluorescence Resonance Energy Transfer

1999 ◽  
Vol 5 (S2) ◽  
pp. 1036-1037
Author(s):  
R.Y. Tsien ◽  
A. Miyawaki ◽  
R. Kerr ◽  
G. Baird ◽  
B.A. Griffin ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Transgenic Animals ◽  
Dynamic Imaging ◽  
Live Cells ◽  
Fluorescence Resonance

Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway.A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking > 600 nm) and better temporal control of the labeling.

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

scholarly journals Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan
Keyword(s):  
Energy Transfer ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

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