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Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells.

ABSTRACT: The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1-4]⁺ have been synthesized and their binding capabilities towards nucleoside polyphosphate anions examined in aqueous solution at physiological pH. Complexes [Eu.1]⁺ and [Eu.3]⁺ bearing hydrogen bond donor groups in the pendant arms showed excellent discrimination between ATP, ADP and monophosphate species. Complex [Eu.3]⁺ showed relatively strong binding to ATP (logK_a =5.8), providing a rapid, long-lived luminescent signal that enabled its detection in a highly competitive aqueous medium containing biologically relevant concentrations of Mg²⁺ , ADP, GTP, UTP and human serum albumin. This Eu^III complex responds linearly to ATP within the physiological concentration range (1-5?mm), and was used to continuously monitor the apyrase-catalyzed hydrolysis of ATP to ADP in vitro. We demonstrate that [Eu.3]⁺ can permeate mammalian (NIH-3T3) cells efficiently and localize to the mitochondria selectively, permitting real-time visualization of elevated mitochondrial ATP levels following treatment with a broad spectrum kinase inhibitor, staurosporine, as well as depleted ATP levels upon treatment with potassium cyanide under glucose starvation conditions.

SUBMITTER: Mailhot R

PROVIDER: S-EPMC6175470 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells.

Mailhot Romain R Traviss-Pollard Thomas T Pal Robert R Butler Stephen J SJ

Chemistry (Weinheim an der Bergstrasse, Germany) 20180628 42

The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1-4]<sup>+</sup> have been synthesized ...[more]

PMID: 29761917

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Project description:The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.

Dataset Information

Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells.

Publications

Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells.

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