Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes

  1. Michelina Kierzek
  2. Parker E Deal
  3. Evan W Miller
  4. Shatanik Mukherjee
  5. Dagmar Wachten
  6. Arnd Baumann
  7. U Benjamin Kaupp
  8. Timo Strünker  Is a corresponding author
  9. Christoph Brenker  Is a corresponding author
  1. University of Münster, Germany
  2. University of California, Berkeley, United States
  3. Center of Advanced European Studies and Research, Germany
  4. University of Bonn, Germany
  5. Research Center Jülich, Germany

Abstract

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically-encoded FRET biosensors. Moreover, FASTM is compatible with opto-chemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source Data files have been provided for Figures 3, 5, 7, 9 and 11.

Article and author information

Author details

  1. Michelina Kierzek

    Centre of Reproductive Medicine and Andrology, University of Münster, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Parker E Deal

    Department of Chemistry, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Evan W Miller

    Department of Chemistry, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6556-7679
  4. Shatanik Mukherjee

    Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7359-9339
  5. Dagmar Wachten

    Institute of Innate Immunity, Department of Biophysical Imaging, Medical Faculty, University of Bonn, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4800-6332
  6. Arnd Baumann

    Institute of Biological Information Processing (IBI-1), Research Center Jülich, Jülich, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. U Benjamin Kaupp

    Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Timo Strünker

    Center of Reproductive Medicine and Andrology, University of Münster, Münster, Germany
    For correspondence
    timo.struenker@ukmuenster.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0812-1547
  9. Christoph Brenker

    Centre of Reproductive Medicine and Andrology, University of Münster, Münster, Germany
    For correspondence
    christoph.brenker@ukmuenster.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4230-2571

Funding

Deutsche Forschungsgemeinschaft (STR 1342/3-1)

  • Timo Strünker

Deutsche Forschungsgemeinschaft (CRU326)

  • Timo Strünker
  • Christoph Brenker

Deutsche Forschungsgemeinschaft (EXC2151 - 390873048)

  • Dagmar Wachten

Innovative Medical Research of the University of Muenster Medical School (BR 1 2 15 07)

  • Christoph Brenker

Center for Clinical Research, Münster (Str/014/21)

  • Timo Strünker

National Institute of General Medical Sciences (R35GM119855)

  • Evan W Miller

Deutsche Forschungsgemeinschaft (GRK2515)

  • Timo Strünker

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Richard S Lewis, Stanford University School of Medicine, United States

Version history

  1. Received: September 15, 2020
  2. Accepted: December 1, 2021
  3. Accepted Manuscript published: December 3, 2021 (version 1)
  4. Version of Record published: December 23, 2021 (version 2)

Copyright

© 2021, Kierzek et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Michelina Kierzek
  2. Parker E Deal
  3. Evan W Miller
  4. Shatanik Mukherjee
  5. Dagmar Wachten
  6. Arnd Baumann
  7. U Benjamin Kaupp
  8. Timo Strünker
  9. Christoph Brenker
(2021)
Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes
eLife 10:e63129.
https://doi.org/10.7554/eLife.63129

Share this article

https://doi.org/10.7554/eLife.63129

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