Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Abstract

<jats:p>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.</jats:p>

CiliaQ – a simple, open-source software for automated quantification of ciliary morphology and fluorescence in 2D, 3D, and 4D images

“…In addition, we observe that cilia labeled by overexpressing a cilium-localized fluorescent protein appeared longer than antibody-labeled cilia. In line with previous reports [19,30,31], this highlights that the amount of overexpressed ciliary protein needs to be carefully titrated to avoid biasing ciliary morphology.…”

“…For such standard applications of CiliaQ, i.e. stained cells in culture, we show that CiliaQ provides a precise, fully-automatized 3D (and also time-lapse 3D [19]) analysis, allowing its application even in high-throughput image analysis.…”

“…However, 3D analysis has been challenging for a standard biology lab, whereby many labs preferred to perform a manual 2D analysis. Hence, CiliaQ was developed to handle the full range of simple 2D imaging settings up to sophisticated time-lapse 3D spinning-disk microscopy [19].…”

“…Although not presented in this paper, CiliaQ can also be employed to analyze time-lapse 3D images. A CiliaQ analysis using data from a spinning-disk confocal microscope has recently been published [19].…”

“…6A). Again, we excluded cilia, this time at the ventricle, where we could not clearly separate adjacent cilia belonging to highly polarized neuroprogenitors [19,[32][33][34][35]. CiliaQ allowed to precisely determine the orientation of the cilia in relation to the ventricle (midline) (Fig.…”