@phdthesis{Bhat2021Fluor-53124,
  year={2021},
  title={Live Cell Fluorescence Imaging of Nucleotide Dynamics : ATP Hydrolysis and DNA Damage Response},
  author={Bhat, Anayat},
  address={Konstanz},
  school={Universität Konstanz}
}

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    <dcterms:title>Live Cell Fluorescence Imaging of Nucleotide Dynamics : ATP Hydrolysis and DNA Damage Response</dcterms:title>
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    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2021-03-10T11:37:49Z</dc:date>
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    <dc:creator>Bhat, Anayat</dc:creator>
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    <dc:contributor>Bhat, Anayat</dc:contributor>
    <dcterms:abstract xml:lang="eng">Nucleotides are biomolecules that not only act as a cellular constituent but are also crucial for cellular functioning and homeostasis. A nucleotide consists of a nitrogenous base, sugar, and phosphate groups. Nucleotides play a critical role in regulatory processes including cellular signaling, metabolism, and energy transfer. Adenosine triphosphate (ATP) is a mononucleotide that acts as an intermediate energy source. Nicotinamide adenine dinucleotide (NAD+) is a dinucleotide central to metabolism and is involved in electron transfer in various redox reactions. ATP and NAD+ are, however, also involved in posttranslational protein modifications (PTM). The present work involves the study of two prominent nucleotides and their dynamics in living cells using fluorescence microscopy. We have used the Förster resonance energy transfer (FRET) microscopy based on fluorescence lifetime imaging (FLIM) to monitor the activity and kinetics of two nucleotide analogs of ATP and NAD+. The activity of ATP hydrolysis was visualized by using fluorescence microscopy in combination with fluorescent ATP analogs. The ATP analog adenosine tetraphosphate (Ap4) was modified with the rhodamine derivative Atto-488 at the terminal phosphate (donar) and a non-fluorescent quencher group at the adenosine base (acceptor). In the intact molecule, the fluorescence of the dye is quenched due to the Förster resonance energy transfer (FRET) because of the spatial proximity between the donor and acceptor. Enzymatic cleavage results in an increase in the fluorescence intensity as well as an increase in the fluorescence lifetime of the dye. This principle was used to quantify the hydrolysis of analogs in vitro as well as in living cells by using fluorescence lifetime imaging (FLIM) microscopy and confocal scanning microscopy. Our experiments revealed that the hydrolysis of Ap4 analogs largely takes place in lysosomes. It was observed that the analog is hydrolyzed and is used as a potential energy source in lysosomes and autolysosomes during the process of autophagy. We thus have been able to successfully monitor the hydrolysis of a nucleotide in living cells. Protein Poly(ADP-ribosyl)ation (PARylation) is a primary step in DNA damage response. NAD+ is used as a donor for the process of protein PARylation. A novel fluorescent analog of NAD+, TMR-NAD, was synthesized with tetramethylrhodamine dye attached to it. The analog was used in combination with EGFP fusion proteins to monitor the PARylation of ARTD1 which is primarily involved in PARylation. With the application of FLIM-FRET imaging, the real-time visualization of PARylation in living cells using this analog was achieved. The decrease in the fluorescence lifetime of the donor fluorophore is measured to visualize and quantify protein PARylation and the associated interactions. The kinetics of the recruitment and PARylation of two proteins i.e. ARTD1 and mH2A were determined in DNA damage response. To induce the DNA damage, ultra-short laser pulses from the near-infrared (NIR) laser were used for the micro-irradiation of the nucleus in a well-defined region. Using this analog approach, it is conceivable to monitor the dynamics of molecular events involved in the DNA damage response with incredible spatial and temporal resolution.</dcterms:abstract>
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