Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution

Söhnel, Anna-Carina; Kohl, Wladislaw; Gregor, Ingo; Enderlein, Jörg; Rieger, Bettina; Busch, Karin B.

doi:10.1016/j.bbabio.2016.04.271

Cited by 8 publications

(12 citation statements)

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“…Reduced NADH, and its phosphorylated form, NADPH are autofluorescent electron donors with identical fluorescent spectra, and is referred to as NAD(P)H due to the uncertain origin of the signal 33 . Changes in the lifetime of NAD(P)H are determined by fitting an exponential function to the observed time-resolved fluorescence decay, and have been used to distinguish between mitochondrial protein localisation 43 , 44 , redox ratio 30 , 45 and glucose carbon diversion 32 . It has been suggested that the protein-bound form of NADH localizes mainly within the mitochondria whilst free NADH is primarily located within the cytosol 30 , 46 .…”

Section: Discussionmentioning

confidence: 99%

Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation

Blignaut

Loos

Botchway

et al. 2019

Sci Rep

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The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute ex vivo inhibition of ATM protein kinase significantly decreased mitochondrial electron transfer chain complex I-mediated oxidative phosphorylation rate but did not decrease coupling efficiency or oxygen consumption rate during β-oxidation. Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.

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Section: Discussionmentioning

confidence: 99%

Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation

Blignaut

Loos

Botchway

et al. 2019

Sci Rep

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“…The decay curves were fitted with a tailored bi-exponential fit. For more details concerning fluorescence lifetime imaging see our previous study (Sohnel et al, 2016). For practical reasons, mCitrine fused to Tom7 (Int C -mCitrine-Tom7) was used as a donor when testing full length inteins, and mEGFP fused to Tom20 (Tom20-mEGFP-Int N ) when truncated inteins were used.…”

Section: Co-expression and Crosslinking Of Tom20 And Tom 7 Results In Increased Physical Tom20-tom7 Interactionmentioning

confidence: 99%

“…The procedure of fluorescence lifetime imaging microscopy is described in (Sohnel et al, 2016). Time-resolved fluorescence measurements were performed using a confocal laser scanning microscope (FluoView FV1000, Olympus) equipped with a TCSPC extension module (PicoQuant GmbH).…”

Section: Fret/flimmentioning

confidence: 99%

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells

Bhagawati

Arroum²,

Webeling³

et al. 2021

MBoC

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The outer membrane translocase (TOM) is the import channel for nuclear-encoded mitochondrial proteins. The general import pore contains Tom40, Tom22, Tom5, Tom6 and Tom7. Precursor proteins are bound by the peripheral receptor proteins Tom20, Tom22 and Tom70 before being imported by the TOM complex. Here we investigated the association of the receptor Tom20 with the TOM complex. Tom20 was found in the TOM complex, but not in a smaller subcomplex. In addition, a subcomplex was found without Tom40 and Tom7 but with Tom20. Using single particle tracking of labeled Tom20 in overexpressing human cells, we show that Tom20 has, on average, higher lateral mobility in the membrane than Tom7/TOM. After ligation of Tom20 with the TOM complex by post-tranlational protein trans-splicing using the trackless, ultra-fast cleaved Gp41-1 integrin system, a significant decrease in the mean diffusion coefficient of Tom20 was observed in the resulting Tom20-Tom7 fusion protein. Exposure of Tom20 to high substrate loading also resulted in reduced mobility. Taken together, our data show that the receptor subunit Tom20 interacts dynamically with the TOM core complex. We suggest that the TOM complex containing Tom20 is the active import pore and that Tom20 is associated when substrate is available.

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“…For instance, improved mutant of YFP, mCitrine protein, was used as a lifetime-based sensor for probing protein localization as well as cytosolic protein PINK1 displacement between the mitochondrial microcompartments. 35 Remarkably, because of small diameter of mitochondria, mapping of protein localization in its microcompartments formerly was not achievable by conventional°uorescence microscopy. However, it was found that localization in inner and outer membrane can be identi¯ed by the di®erence in lifetime of mCitrine tag.…”

Section: High Resolution Protein Tra±ckingmentioning

confidence: 99%

“…Moreover, it was demonstrated that FLIM approach is sensitive enough even for protein tra±cking within mitochondrial microcompartments. 35 Furthermore, the potential of FLIM techniques for multiplex imaging was demonstrated. 37 The algorithm for signal di®erentiation from up to¯ve°uor-ophores with similar°uorescence emission spectra was introduced recently.…”

Section: High Resolution Protein Tra±ckingmentioning

confidence: 99%

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Levchenko

Pliss

2017

J. Innov. Opt. Health Sci.

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Fluorescence lifetime imaging (FLIM) is an e®ective noninvasive bioanalytical tool based on measuring°uorescent lifetime of°uorophores. A growing number of FLIM studies utilizes genetically engineered°uorescent proteins targeted to speci¯c subcellular structures to probe local molecular environment, which opens new directions in cell science. This paper highlights the unconventional applications of FLIM for studies of molecular processes in diverse organelles of live cultured cells.

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Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution

Cited by 8 publications

References 0 publications

Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation

Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

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