scholarly journals Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells

Biology Open ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. bio048975
Author(s):  
Koichi Ojima ◽  
Shoji Hata ◽  
Fumiko Shinkai-Ouchi ◽  
Mika Oe ◽  
Susumu Muroya ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protease Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Spectral Studies ◽  
Partial Amino Acid Sequence ◽  
Promising Tool ◽  
Calpain 3 ◽  
Sensor Probe

ABSTRACTCalpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Förster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.

scholarly journals A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells

ACS Omega ◽  
2020 ◽  
Vol 5 (46) ◽  
pp. 30306-30314
Author(s):  
Urooj Fatima ◽  
Fuad Ameen ◽  
Neha Soleja ◽  
Parvez Khan ◽  
Abobakr Almansob ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Analytical Tool ◽  
Fluorescence Resonance

scholarly journals Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

2008 ◽  
Vol 363 (1500) ◽  
pp. 2143-2151 ◽  
Author(s):  
Kazuhiro Aoki ◽  
Etsuko Kiyokawa ◽  
Takeshi Nakamura ◽  
Michiyuki Matsuda
Keyword(s):  
Signal Transduction ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Small Gtpases ◽  
Forster Resonance Energy Transfer ◽  
Fluorescence Probes ◽  
Förster Resonance Energy Transfer ◽  
Förster Resonance

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

scholarly journals Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

2009 ◽  
Vol 106 (37) ◽  
pp. 15651-15656 ◽  
Author(s):  
Hiromi Imamura ◽  
Kim P. Huynh Nhat ◽  
Hiroko Togawa ◽  
Kenta Saito ◽  
Ryota Iino ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Atp Synthase ◽  
Hela Cells ◽  
Dissociation Constants ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Atp Levels ◽  
Fluorescence Resonance

Adenosine 5′-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

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