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
Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3′, 5′-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.
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