A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation.

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.

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

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.

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

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.

Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors.

The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.

Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases.

As diffusible second messengers, cyclic nucleoside monophosphates (cNMPs) relay and amplify molecular signals in myriad cellular pathways. The triggering of downstream physiological responses often requires defined cNMP gradients in time and space, generated through the concerted action of nucleotidyl cyclases and phosphodiesterases (PDEs). In an approach denoted optogenetics, sensory photoreceptors serve as genetically encoded, light-responsive actuators to enable the noninvasive, reversible, and spatiotemporally precise control of manifold cellular processes, including cNMP metabolism. Although nature provides efficient photoactivated nucleotidyl cyclases, light-responsive PDEs are scarce. Through modular recombination of a bacteriophytochrome photosensor and the effector of human PDE2A, we previously generated the light-activated, cNMP-specific PDE LAPD. By pursuing parallel design strategies, we here report a suite of derivative PDEs with enhanced amplitude and reversibility of photoactivation. Opposite to LAPD, far-red light completely reverts prior activation by red light in several PDEs. These improved PDEs thus complement photoactivated nucleotidyl cyclases and extend the sensitivity of optogenetics to red and far-red light. More generally, our study informs future efforts directed at designing bacteriophytochrome photoreceptors.

Cyclic Nucleotide-Specific Optogenetics Highlights Compartmentalization of the Sperm Flagellum into cAMP Microdomains.

Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Characterization and engineering of photoactivated adenylyl cyclases.

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.

Primer-Aided Truncation for the Creation of Hybrid Proteins.

Proteins frequently display modular architecture with several domains and segments connected by linkers. Proper protein functionality hinges on finely orchestrated interactions among these constituent elements. The underlying modularity lends itself to the engineering of hybrid proteins via modular rewiring; novel properties can thus be obtained, provided the linkers connecting the individual elements are conducive to productive interactions. As a corollary, the process of protein engineering often encompasses the generation and screening of multiple linker variants. To aid these steps, we devised the PATCHY method (primer-aided truncation for the creation of hybrid proteins) to readily generate hybrid gene libraries of predefined composition. We applied PATCHY to the mechanistic characterization of hybrid receptors that possess blue-light-regulated histidine kinase activity. Comprehensive sampling of linker composition revealed that catalytic activity and response to light are primarily functions of linker length. Variants with linkers of 7n residues mostly have light-repressed activity but those with 7n + 1 residues mostly have inverted, light-induced activity. We further probed linker length in the context of single residue exchanges that also lead to an inversion of the signal response. As in the original context, activity is only observed for certain periodic linker lengths. Taken together, these results provide mechanistic insight into signaling strategies employed by sensory photoreceptors and sensor histidine kinases. PATCHY represents an adequate and facile method to efficiently generate and probe hybrid gene libraries and to thereby identify key determinants for proper function.