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

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

  • 1,457
    Page views
  • 212
    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)

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

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

  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

Further reading

    1. Cell Biology
    2. Neuroscience
    Meghan E Wynne, Oluwaseun Ogunbona ... Victor Faundez
    Research Article Updated

    Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer’s disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

    1. Cell Biology
    Ignacio Bravo-Plaza, Victor G Tagua ... Miguel A Peñalva
    Research Article

    Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.