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 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.
Highlights
Primary cilia are membrane protrusions that extend from the surface of almost all vertebrate cells
To utilize the optogenetic tools bPAC- or LAPD-mCherry in a cilium-specific manner, we first tested whether N-terminal fusion of mNphp3(201) is sufficient for targeting to the primary cilium
To measure LAPD activity, HEK-TM cells were pre-stimulated with NKH477, a water-soluble forskolin analog that activates transmembrane adenylyl cyclases and, thereby, increases cyclic adenosine monophosphate (cAMP) levels, leading to a Ca2+ influx
Summary
Primary cilia are membrane protrusions that extend from the surface of almost all vertebrate cells. Primary cilia function as antennae that translate sensory information into a cellular response. The sensory function is governed by a subset of receptors and downstream signaling components that are targeted to the cilium. This allows to orchestrate rapid signal transduction in a minuscule reaction volume, independent of the cell body. A central component of ciliary signaling is the second
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