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

Abstract

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. Primary cilia constitute a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to manipulate and analyze ciliary signaling in living cells. Here, we describe a nanobodybased targeting approach for optogenetic tools that allows to specifically analyze ciliary signaling and function, and that is applicable in vitro and in vivo. We overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence, and functionally localize the photo-activated adenylate cyclase bPAC, the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we unravel 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.

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

“…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 [34]) 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 [34].…”

“…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 [34].…”

“…It has been reported that overexpression of a ciliary proteins increases cilia length [34,[44][45][46].…”

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

“…Although not presented in this paper, CiliaQ can also be employed to analyze time-lapse 3D images. A CiliaQ analysis using data from a spinningdisk confocal microscope has recently been published [34].…”