Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Homburg, Germany
Andreas Möglich
Lehrstuhl für Biochemie, Universität Bayreuth, Bayreuth, Germany; Research Center for Bio-Macromolecules, Universität Bayreuth, Bayreuth, Germany; Bayreuth Center for Biochemistry & Molecular Biology, Universität Bayreuth, Bayreuth, Germany
Kavli Institute for Systems Neuroscience and Centre for Neural Computation, The Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway; Department of Neurology and Clinical Neurophysiology, St. Olavs University Hospital, Trondheim, Norway
Institute of Innate Immunity, Emmy Noether research group, Medical Faculty, University of Bonn, Bonn, Germany; Core Facility Nanobodies, University of Bonn, Bonn, Germany
Institute of Innate Immunity, Biophysical Imaging, Medical Faculty, University of Bonn, Bonn, Germany; Research Group Molecular Physiology, Center of Advanced European Studies and Research (caesar), Bonn, Germany
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 to 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 adenylyl 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.
