1. Cell Biology

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
https://doi.org/10.7554/eLife.57907
Share this article
Cite this article
  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
  11. Dagmar Wachten
(2020)
Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium
eLife 9:e57907.
https://doi.org/10.7554/eLife.57907
  2. Download BibTeX
  3. Download .RIS

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.

Metrics

  • 4,092
    Page views
  • 707
    Downloads
  • 24
    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. 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
  11. Dagmar Wachten
(2020)
Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium
eLife 9:e57907.
https://doi.org/10.7554/eLife.57907

Categories and tags

Research organism

Further reading

    1. Cell Biology
    2. Neuroscience

    APOE expression and secretion are modulated by mitochondrial dysfunction

    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

    The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain

    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.

    1. Cell Biology

    Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture

    Sandipan Dasgupta, Daniella Y Dayagi ... Jeffrey E Gerst
    Research Article

    Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically, <1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.