1. Cell Biology
Download icon

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
Tools and Resources
  • Cited 0
  • Views 1,132
  • Annotations
Cite this article as: eLife 2021;10:e63129 doi: 10.7554/eLife.63129

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

Publication 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.

Metrics

  • 1,132
    Page views
  • 160
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cell Biology
    Dillon Jevon et al.
    Research Article

    A developing understanding suggests that spatial compartmentalisation in pancreatic β cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell sub-cellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact β cell structure, and enhances glucose dependent Ca2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase, in regulating β cells. Integrins and focal adhesion kinase are exclusively activated at the β cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose dependent Ca2+ responses and insulin secretion. We conclude that focal adhesion kinase orchestrates the final steps of glucose dependent insulin secretion within the restricted domain where β cells contact the islet capillaries.

    1. Cell Biology
    2. Physics of Living Systems
    Sohyeon Park et al.
    Research Article Updated

    In addition to diffusive signals, cells in tissue also communicate via long, thin cellular protrusions, such as airinemes in zebrafish. Before establishing communication, cellular protrusions must find their target cell. Here, we demonstrate that the shapes of airinemes in zebrafish are consistent with a finite persistent random walk model. The probability of contacting the target cell is maximized for a balance between ballistic search (straight) and diffusive search (highly curved, random). We find that the curvature of airinemes in zebrafish, extracted from live-cell microscopy, is approximately the same value as the optimum in the simple persistent random walk model. We also explore the ability of the target cell to infer direction of the airineme’s source, finding that there is a theoretical trade-off between search optimality and directional information. This provides a framework to characterize the shape, and performance objectives, of non-canonical cellular protrusions in general.