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

Abstract

Cilia are hair-like membrane protrusions that emanate from the surface of most vertebrate cells and are classified into motile and primary cilia. Motile cilia move fluid flow or propel cells, while also fulfill sensory functions. Primary cilia are immotile and act as a cellular antenna, translating environmental cues into cellular responses. Ciliary dysfunction leads to severe diseases, commonly termed ciliopathies. The molecular details underlying ciliopathies and ciliary function are, however, not well understood. Since cilia are small subcellular compartments, imaging-based approaches have been used to study them. However, tools to comprehensively analyze images are lacking. Automatic analysis approaches require commercial software and are limited to 2D analysis and only a few parameters. The widely used manual analysis approaches are time consuming, user-biased, and difficult to compare. Here, we present CiliaQ, a package of open-source, freely available, and easy-to-use ImageJ plugins. CiliaQ allows high-throughput analysis of 2D and 3D, static or time-lapse images from fluorescence microscopy of cilia in cell culture or tissues, and outputs a comprehensive list of parameters for ciliary morphology, length, bending, orientation, and fluorescence intensity, making it broadly applicable. We envision CiliaQ as a resource and platform for reproducible and comprehensive analysis of ciliary function in health and disease. Graphic abstract

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits

“…Three-dimensional reconstructions of Z-stacks were performed in Fiji using the 3D Viewer plugin. Quantification of SMO (red) and PKA-C/NbSmo2/Nbβ2AR80 staining was performed using CiliaQ [ 134 ] and reported as a ratio of the green fluorescence in each cilium, normalized to the red fluorescence. For fixed-cell imaging of SMO wild-type and Ala mutants ( S11B Fig ), Smo −/− MEFs were transfected on coverslips with the indicated plasmids (N-terminally tdTomato-tagged SMO657 or SMO657-Ala), grown to confluency, shifted to DMEM + 0.5% FBS + SAG21k overnight, then fixed, permeabilized, and stained as previously described, using an anti-Arl13b antibody to mark cilia.…”

Impaired Ciliary Beat Frequency and Ciliogenesis Alteration during Airway Epithelial Cell Differentiation in COPD

“…The density of multiciliated cells (MCC) was evaluated with the average pixel density of the fluorescence associated to cilia staining as previously described [10]. The percentage of primary ciliated cells and the average cilia length were automatically quantified with CiliaQ as previously described [28].…”

A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility

“…The two channels were aligned using Zen Blue (Zeiss). Cilia were segmented using CiliaQ based on the JF552 channel (Hansen et al, 2021). Per cilium GFP mean fluorescent intensities were then calculated for different concentrations.…”

“…These 4D stacks (XYZT) were projected onto single planes (XYT) by maximum intensity projection in Fiji/ImageJ. Cilia were segmented using CiliaQ (Hansen et al, 2021). The first 10 time points, which often showed significant quenching, were discarded for subsequent analyses.…”