1. Cell Biology
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Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

  1. Jan Niklas Hansen
  2. Fabian Kaiser
  3. Christina Klausen
  4. Birthe Stüven
  5. Raymond Chong
  6. Wolfgang Bönigk
  7. David U Mick
  8. Andreas Möglich
  9. Nathalie Jurisch-Yaksi
  10. Florian I Schmidt  Is a corresponding author
  11. Dagmar Wachten  Is a corresponding author
  1. University of Bonn, Institute of Innate Immunity, Medical Faculty, Germany
  2. Center of Advanced European Studies and Research, Germany
  3. Saarland University School of Medicine, Germany
  4. Universität Bayreuth, Germany
  5. Norwegian University of Science and Technology, Norway
  6. University of Bonn, Medical Faculty, Germany
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Cite this article as: eLife 2020;9:e57907 doi: 10.7554/eLife.57907

Abstract

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

Data availability

All data generated or analysed during this study are included in the manuscript and are available through the following doi 10.6084/m9.figshare.c.4792248. The analysis workflow to study cilia length and fluorescence signal with its custom-written ImageJ plug-ins ('CiliaQ') is available through the following link https://github.com/hansenjn/CiliaQ.

The following data sets were generated

Article and author information

Author details

  1. Jan Niklas Hansen

    University of Bonn, Institute of Innate Immunity, Medical Faculty, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0489-7535
  2. Fabian Kaiser

    University of Bonn, Institute of Innate Immunity, Medical Faculty, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Christina Klausen

    University of Bonn, Institute of Innate Immunity, Medical Faculty, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Birthe Stüven

    University of Bonn, Institute of Innate Immunity, Medical Faculty, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Raymond Chong

    University of Bonn, Institute of Innate Immunity, Medical Faculty, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Wolfgang Bönigk

    Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. David U Mick

    Center of Human and Molecular Biology, Saarland University School of Medicine, Homburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1427-9412
  8. Andreas Möglich

    Bayreuth Center for Biochemistry & Molecular Biology, Universität Bayreuth, Bayreuth, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Nathalie Jurisch-Yaksi

    Kavli Institute for Systems Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8767-6120
  10. Florian I Schmidt

    Institute of Innate Immunity, University of Bonn, Medical Faculty, Bonn, Germany
    For correspondence
    fschmidt@uni-bonn.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9979-9769
  11. Dagmar Wachten

    University of Bonn, Institute of Innate Immunity, Medical Faculty, Bonn, Germany
    For correspondence
    dwachten@uni-bonn.de
    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

Funding

Deutsche Forschungsgemeinschaft (SPP 1926)

  • Andreas Möglich
  • Dagmar Wachten

Deutsche Forschungsgemeinschaft (SPP1726)

  • Dagmar Wachten

Deutsche Forschungsgemeinschaft (TRR83/SFB)

  • Dagmar Wachten

Deutsche Forschungsgemeinschaftgemeinschaft (Germany's Excellence Strategy - EXC2151 - 390873048)

  • Florian I Schmidt
  • Dagmar Wachten

Deutsche Forschungsgemeinschaft (FOR2743)

  • Dagmar Wachten

Deutsche Forschungsgemeinschaft (Emmy Noether)

  • Florian I Schmidt

Boehringer Ingelheim Fonds (PhD fellowship)

  • Jan Niklas Hansen

Samarbeidsorganet Helse Midt-Norge (grant)

  • Nathalie Jurisch-Yaksi

Deutsche Forschungsgemeinschaft (SFB894/TP-A22)

  • David U Mick

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

Ethics

Animal experimentation: The animal facilities and maintenance of the zebrafish, Danio rerio, were approved by the Norwegian Food Safety Authority (19/175222).

Reviewing Editor

  1. Junmin Pan, Tsinghua University, China

Publication history

  1. Received: April 15, 2020
  2. Accepted: June 24, 2020
  3. Accepted Manuscript published: June 24, 2020 (version 1)
  4. Version of Record published: July 6, 2020 (version 2)

Copyright

© 2020, Hansen 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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