The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide ‘flashes’

2011 ◽  
Vol 437 (3) ◽  
pp. 381-387 ◽  
Author(s):  
Markus Schwarzländer ◽  
David C. Logan ◽  
Mark D. Fricker ◽  
Lee J. Sweetlove
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Ph Sensitivity ◽  
Detectable Effect ◽  
High Ph ◽  
Pharmacological Manipulation ◽  
The Matrix

The properties of a cpYFP [circularly permuted YFP (yellow fluorescent protein)] reported to act as a superoxide sensor have been re-examined in Arabidopsis mitochondria. We have found that the probe has high pH sensitivity and that dynamics in the cpYFP signal disappeared when the matrix pH was clamped by nigericin. In contrast, genetic and pharmacological manipulation of matrix superoxide had no detectable effect on the cpYFP signal. These findings question the existence of superoxide flashes in mitochondria.

Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria

2008 ◽  
Vol 294 (5) ◽  
pp. C1124-C1132 ◽  
Author(s):  
Werner J. H. Koopman ◽  
Felix Distelmaier ◽  
Mark A. Hink ◽  
Sjoerd Verkaart ◽  
Mietske Wijers ◽  
...  
Keyword(s):  
Complex I ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Protein Diffusion ◽  
Enhanced Yellow Fluorescent Protein ◽  
The Matrix ◽  
Matrix Configuration

Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study, we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility, and matrix protein diffusion. To this end, we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity, and the rate of matrix-targeted enhanced yellow fluorescent protein (mitoEYFP) diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria were two to three times higher in patient cells than in control cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI-deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI-deficient patients become more glycolytic, allowing a higher mitochondrial velocity.

scholarly journals Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

2014 ◽  
Vol 81 (5) ◽  
pp. 1847-1858 ◽  
Author(s):  
Anna Sznajder ◽  
Daniel Pfeiffer ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Proteome Analysis ◽  
Detectable Effect ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Associated Proteins ◽  
Phb Synthase

ABSTRACTIdentification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated fromRalstonia eutrophabut absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon withphaB2(acetoacetyl-coenzyme A [CoA] reductase) andphaC2(PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmedin vivoby fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

scholarly journals The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3024
Author(s):  
Martin Fogtmann Berthelsen ◽  
Maria Riedel ◽  
Huiqiang Cai ◽  
Søren H. Skaarup ◽  
Aage K. O. Alstrup ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Somatic Cells ◽  
Genetic Alterations ◽  
Lung Epithelial Cells ◽  
Target Cells ◽  
Multiple Gene ◽  
Conditional Expression ◽  
Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

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 The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

1998 ◽  
Vol 142 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Koji Okamoto ◽  
Philip S. Perlman ◽  
Ronald A. Butow
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Mitochondrial Matrix ◽  
Outer Membranes ◽  
Marker Proteins ◽  
The Matrix ◽  
Green Fluorescent ◽  
Mitochondrial Reticulum

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

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