Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium
- University of Bonn, Institute of Innate Immunity, Medical Faculty, Germany
- Center of Advanced European Studies and Research, Germany
- Saarland University School of Medicine, Germany
- Universität Bayreuth, Germany
- Norwegian University of Science and Technology, Norway
- University of Bonn, Medical Faculty, Germany
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.
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Nanobody-directed targeting of optogenetic tools to study signaling in the primary ciliumfigshare doi 10.6084/m9.figshare.c.4792248.v1.
Article and author information
Author details
Nathalie Jurisch-Yaksi
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).
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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Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium
eLife 9:e57907.
https://doi.org/10.7554/eLife.57907
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