pH-Dependent Fluorescence of a Heterologously ExpressedAequoreaGreen Fluorescent Protein Mutant: In Situ Spectral Characteristics and Applicability to Intracellular pH Estimation†,‡

Biochemistry ◽  
1998 ◽  
Vol 37 (28) ◽  
pp. 9894-9901 ◽  
Author(s):  
R. Brooks Robey ◽  
Ofelia Ruiz ◽  
Anna V. P. Santos ◽  
Jianfei Ma ◽  
Fely Kear ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescent Protein ◽  
Spectral Characteristics ◽  
Ph Dependent

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.

scholarly journals Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator

1998 ◽  
Vol 74 (3) ◽  
pp. 1591-1599 ◽  
Author(s):  
Malea Kneen ◽  
Javier Farinas ◽  
Yuxin Li ◽  
A.S. Verkman
Keyword(s):  
Green Fluorescent Protein ◽  
Intracellular Ph ◽  
Fluorescent Protein ◽  
Ph Indicator ◽  
Green Fluorescent

scholarly journals Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms

2013 ◽  
Vol 11 (4) ◽  
pp. 629-635 ◽  
Author(s):  
L. M. Schaefer ◽  
V. S. Brözel ◽  
S. N. Venter
Keyword(s):  
Drinking Water ◽  
Green Fluorescent Protein ◽  
Health Risk ◽  
Salmonella Typhimurium ◽  
Fluorescent Protein ◽  
Laboratory Scale ◽  
Green Fluorescent ◽  
In Situ Detection

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.

H+-linked transport of salicylic acid, an NSAID, in the human trophoblast cell line BeWo

2002 ◽  
Vol 282 (5) ◽  
pp. C1064-C1075 ◽  
Author(s):  
Akiko Emoto ◽  
Fumihiko Ushigome ◽  
Noriko Koyabu ◽  
Hiroshi Kajiya ◽  
Koji Okabe ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Lactic Acid ◽  
Cell Line ◽  
Intracellular Ph ◽  
Trophoblast Cell ◽  
Monocarboxylate Transporter ◽  
Human Trophoblast ◽  
Inflammatory Drugs ◽  
Ph Dependent ◽  
Trophoblast Cell Line

We investigated the transport of salicylic acid and l-lactic acid across the placenta using the human trophoblast cell line BeWo. We performed uptake experiments and measured the change in intracellular pH (pHi). The uptakes of [14C]salicylic acid andl-[14C]lactic acid were temperature- and extracellular pH-dependent and saturable at higher concentrations. Both uptakes were also reduced by FCCP, nigericin, and NaN3. Various nonsteroidal anti-inflammatory drugs (NSAIDs) strongly inhibited the uptake of l-[14C]lactic acid. Salicylic acid and ibuprofen noncompetitively inhibited the uptake ofl-[14C]lactic acid. α-Cyano-4-hydroxycinnamate (CHC), a monocarboxylate transporter inhibitor, suppressed the uptake ofl-[14C]lactic acid but not that of [14C]salicylic acid. CHC also suppressed the decrease of pHi induced by l-lactic acid but had little effect on that induced by salicylic acid or diclofenac. These results suggest that NSAIDs are potent inhibitors of lactate transporters, although they are transported mainly by a transport system distinct from that for l-lactic acid.

scholarly journals β-Catenin is a pH sensor with decreased stability at higher intracellular pH

2018 ◽  
Vol 217 (11) ◽  
pp. 3965-3976 ◽  
Author(s):  
Katharine A. White ◽  
Bree K. Grillo-Hill ◽  
Mario Esquivel ◽  
Jobelle Peralta ◽  
Vivian N. Bui ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Ph Sensor ◽  
Adherens Junction ◽  
Protein Protein Interactions ◽  
Adherens Junction Protein ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Ph Dependent

β-Catenin functions as an adherens junction protein for cell–cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein–protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster. β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R–β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation.

scholarly journals Nuclear pores as versatile reference standards for quantitative superresolution microscopy

2019 ◽  
Author(s):  
Jervis Vermal Thevathasan ◽  
Maurice Kahnwald ◽  
Konstanty Cieśliński ◽  
Philipp Hoess ◽  
Sudheer Kumar Peneti ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fluorescent Protein ◽  
Reference Standards ◽  
Nuclear Pore ◽  
Biologically Relevant ◽  
Superresolution Microscopy ◽  
Absolute Measurements ◽  
Quantitative Fluorescence

AbstractQuantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

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