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Live Cell Imaging of Enzymatic Turnover of an Adenosine 5′-Tetraphosphate Analog

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2021

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International Journal of Molecular Sciences (IJMS). MDPI. 2021, 22(16), 8616. ISSN 1661-6596. eISSN 1422-0067. Available under: doi: 10.3390/ijms22168616

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The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5′-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.

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ISO 690BHAT, Anayat, Shuang LI, Daniel HAMMLER, Martin WINTERHALDER, Andreas MARX, Andreas ZUMBUSCH, 2021. Live Cell Imaging of Enzymatic Turnover of an Adenosine 5′-Tetraphosphate Analog. In: International Journal of Molecular Sciences (IJMS). MDPI. 2021, 22(16), 8616. ISSN 1661-6596. eISSN 1422-0067. Available under: doi: 10.3390/ijms22168616
BibTex
@article{Bhat2021-08-10Imagi-54850,
  year={2021},
  doi={10.3390/ijms22168616},
  title={Live Cell Imaging of Enzymatic Turnover of an Adenosine 5′-Tetraphosphate Analog},
  number={16},
  volume={22},
  issn={1661-6596},
  journal={International Journal of Molecular Sciences (IJMS)},
  author={Bhat, Anayat and Li, Shuang and Hammler, Daniel and Winterhalder, Martin and Marx, Andreas and Zumbusch, Andreas},
  note={Article Number: 8616}
}
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