Showing 1 - 25 of 74 results
Endosomal cAMP production broadly impacts the cellular phosphoproteome.
Endosomal signaling downstream of G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that the same amount of cAMP produced from endosomes led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56δ as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.
Optogenetic tools for manipulation of cyclic nucleotides, functionally coupled to CNG-channels.
The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers that regulate numerous biological processes. Malfunctional cNMP signalling is linked to multiple diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in C. elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarising rhodopsins, yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarisers based on K+ -currents.
Light-Induced Change of Arginine Conformation Modulates the Rate of Adenosine Triphosphate to Cyclic Adenosine Monophosphate Conversion in the Optogenetic System Containing Photoactivated Adenylyl Cyclase.
We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to the reaction products, cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi), catalyzed by ACs initiated by excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes that modulate cellular concentrations of signaling messengers. The photoactivated AC from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing a considerable increase in the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 μs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore-binding pocket results in switching of the position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP → cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in the BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild-type bPAC.
Optogenetic Modification of Pseudomonas aeruginosa Enables Controllable Twitching Motility and Host Infection.
Cyclic adenosine monophosphate (cAMP) is an important secondary messenger that controls carbon metabolism, type IVa pili biogenesis, and virulence in Pseudomonas aeruginosa. Precise manipulation of bacterial intracellular cAMP levels may enable tunable control of twitching motility or virulence, and optogenetic tools are attractive because they afford excellent spatiotemporal resolution and are easy to operate. Here, we developed an engineered P. aeruginosa strain (termed pactm) with light-dependent intracellular cAMP levels through introducing a photoactivated adenylate cyclase gene (bPAC) into bacteria. On blue light illumination, pactm displayed a 15-fold increase in the expression of the cAMP responsive promoter and an 8-fold increase in its twitching activity. The skin lesion area of nude mouse in a subcutaneous infection model after 2-day pactm inoculation was increased 14-fold by blue light, making pactm suitable for applications in controllable bacterial host infection. In addition, we achieved directional twitching motility of pactm colonies through localized light illumination, which will facilitate the studies of contact-dependent interactions between microbial species.
Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.
Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
Photoactivated Adenylyl Cyclases: Fundamental Properties and Applications.
Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.
Biphasic Response of Protein Kinase A to Cyclic Adenosine Monophosphate Triggers Distinct Epithelial Phenotypes.
Despite the large diversity of the proteins involved in cellular signaling, many intracellular signaling pathways converge onto one of only dozens of small molecule second messengers. Cyclic adenosine monophosphate (cAMP), one of these second messengers, is known to regulate activity of both Protein Kinase A (PKA) and the Extracellular Regulated Kinase (ERK), among other signaling pathways. In its role as an important cellular signaling hub, intracellular cAMP concentration has long been assumed to monotonically regulate its known effectors. Using an optogenetic tool that can introduce precise amounts of cAMP in MDCKI cells, we identify genes whose expression changes biphasically with monotonically increasing cAMP levels. By examining the behavior of PKA and ERK1/2 in the same dose regime, we find that these kinases also respond biphasically to increasing cAMP levels, with opposite phases. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge to inhibit PKA. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, influencing the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational modeling, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.
The rise and shine of yeast optogenetics.
Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.
Dual Activation of cAMP Production Through Photostimulation or Chemical Stimulation.
cAMP is a crucial mediator of multiple cell signaling pathways. This cyclic nucleotide requires strict spatiotemporal control for effective function. Light-activated proteins have become a powerful tool to study signaling kinetics due to having quick on/off rates and minimal off-target effects. The photoactivated adenylyl cyclase from Beggiatoa (bPAC) produces cAMP rapidly upon stimulation with blue light. However, light delivery is not always feasible, especially in vivo. Hence, we created a luminescence-activated cyclase by fusing bPAC with nanoluciferase (nLuc) to allow chemical activation of cAMP activity. This dual-activated adenylyl cyclase can be stimulated using short bursts of light or long-term chemical activation with furimazine and other related luciferins. Together these can be used to mimic transient, chronic, and oscillating patterns of cAMP signaling. Moreover, when coupled to compartment-specific targeting domains, these reagents provide a new powerful tool for cAMP spatiotemporal dynamic studies. Here, we describe detailed methods for working with bPAC-nLuc in mammalian cells, stimulating cAMP production with light and luciferins, and measuring total cAMP accumulation.
Optogenetic Techniques for Manipulating and Sensing G Protein-Coupled Receptor Signaling.
G protein-coupled receptors (GPCRs) form the largest class of membrane receptors in the mammalian genome with nearly 800 human genes encoding for unique subtypes. Accordingly, GPCR signaling is implicated in nearly all physiological processes. However, GPCRs have been difficult to study due in part to the complexity of their function which can lead to a plethora of converging or diverging downstream effects over different time and length scales. Classic techniques such as pharmacological control, genetic knockout and biochemical assays often lack the precision required to probe the functions of specific GPCR subtypes. Here we describe the rapidly growing set of optogenetic tools, ranging from methods for optical control of the receptor itself to optical sensing and manipulation of downstream effectors. These tools permit the quantitative measurements of GPCRs and their downstream signaling with high specificity and spatiotemporal precision.
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 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 adenylate 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.
Optogenetic Manipulation of Postsynaptic cAMP Using a Novel Transgenic Mouse Line Enables Synaptic Plasticity and Enhances Depolarization Following Tetanic Stimulation in the Hippocampal Dentate Gyrus.
cAMP is a positive regulator tightly involved in certain types of synaptic plasticity and related memory functions. However, its spatiotemporal roles at the synaptic and neural circuit levels remain elusive. Using a combination of a cAMP optogenetics approach and voltage-sensitive dye (VSD) imaging with electrophysiological recording, we define a novel capacity of postsynaptic cAMP in enabling dentate gyrus long-term potentiation (LTP) and depolarization in acutely prepared murine hippocampal slices. To manipulate cAMP levels at medial perforant path to granule neuron (MPP-DG) synapses by light, we generated transgenic (Tg) mice expressing photoactivatable adenylyl cyclase (PAC) in DG granule neurons. Using these Tg(CMV-Camk2a-RFP/bPAC)3Koka mice, we recorded field excitatory postsynaptic potentials (fEPSPs) from MPP-DG synapses and found that photoactivation of PAC during tetanic stimulation enabled synaptic potentiation that persisted for at least 30 min. This form of LTP was induced without the need for GABA receptor blockade that is typically required for inducing DG plasticity. The paired-pulse ratio (PPR) remained unchanged, indicating the cAMP-dependent LTP was likely postsynaptic. By employing fast fluorescent voltage-sensitive dye (VSD: di-4-ANEPPS) and fluorescence imaging, we found that photoactivation of the PAC actuator enhanced the intensity and extent of dentate gyrus depolarization triggered following tetanic stimulation. These results demonstrate that the elevation of cAMP in granule neurons is capable of rapidly enhancing synaptic strength and neuronal depolarization. The powerful actions of cAMP are consistent with this second messenger having a critical role in the regulation of synaptic function.
SynaptoPAC, an Optogenetic Tool for Induction of Presynaptic Plasticity.
Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long-term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood. In order to establish optogenetic control of presynaptic cAMP levels and thereby presynaptic potentiation, we developed synaptoPAC, a presynaptically targeted version of the photoactivated adenylyl cyclase bPAC. In cultures of hippocampal granule cells, activation of synaptoPAC with blue light increases action potential-evoked transmission, an effect not seen in hippocampal cultures of non-granule cells. In acute brain slices, synaptoPAC activation immediately triggers a strong presynaptic potentiation at mossy fiber terminals in CA3, but not at Schaffer collateral synapse in CA1. Following light-triggered potentiation, mossy fiber transmission decreases within 20 minutes, but remains enhanced still after 30 min. Optogenetic potentiation alters the short-term plasticity dynamics of release, reminiscent of presynaptic LTP. SynaptoPAC is the first optogenetic tool that allows acute light-controlled potentiation of transmitter release at specific synapses of the brain, and will enable to investigate the role of presynaptic potentiation in network function and the animal’s behavior in an unprecedented manner.
Booster, a Red-Shifted Genetically Encoded Förster Resonance Energy Transfer (FRET) Biosensor Compatible with Cyan Fluorescent Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue Light-Responsive Optogenetic Tools.
Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOκ and mKate2 as the favorable donor and acceptor pair by calculating the Förster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named "Booster". The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation by Beggiatoa photoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.
An optogenetic tool for induced protein stabilization based on the Phaeodactylum tricornutum aureochrome 1a LOV domain.
Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light-oxygen-voltage domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254 and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp.. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic AMP production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.
Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events.
Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
Role of cyclic nucleotides and their downstream signaling cascades in memory function: being at the right time at the right spot.
A plethora of studies indicate the important role of cAMP and cGMP cascades in neuronal plasticity and memory function. As a result, altered cyclic nucleotide signaling has been implicated in the pathophysiology of mnemonic dysfunction encountered in several diseases. In the present review we provide a wide overview of studies regarding the involvement of cyclic nucleotides, as well as their upstream and downstream molecules, in physiological and pathological mnemonic processes. Next, we discuss the regulation of the intracellular concentration of cyclic nucleotides via phosphodiesterases, the enzymes that degrade cAMP and/or cGMP, and via A-kinase-anchoring proteins that refine signal compartmentalization of cAMP signaling. We also provide an overview of the available data pointing to the existence of specific time windows in cyclic nucleotide signaling during neuroplasticity and memory formation and the significance to target these specific time phases for improving memory formation. Finally, we highlight the importance of emerging imaging tools like Förster resonance energy transfer imaging and optogenetics in detecting, measuring and manipulating the action of cyclic nucleotide signaling cascades.
Photoreaction Mechanisms of Flavoprotein Photoreceptors and Their Applications.
Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.
The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata.
Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.
Primary Cilia Signaling Promotes Axonal Tract Development and Is Disrupted in Joubert Syndrome-Related Disorders Models.
Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.
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.
Amelioration of Diabetes in a Murine Model upon Transplantation of Pancreatic β-Cells with Optogenetic Control of Cyclic Adenosine Monophosphate.
Pharmacological augmentation of glucose-stimulated insulin secretion (GSIS), for example, to overcome insulin resistance in type 2 diabetes is linked to suboptimal regulation of blood sugar. Cultured β-cells and islets expressing a photoactivatable adenylyl cyclase (PAC) are amenable to GSIS potentiation with light. However, whether PAC-mediated enhancement of GSIS can improve the diabetic state remains unknown. To this end, β-cells were engineered with stable PAC expression that led to over 2-fold greater GSIS upon exposure to blue light while there were no changes in the absence of glucose. Moreover, the rate of oxygen consumption was unaltered despite the photoinduced elevation of GSIS. Transplantation of these cells into streptozotocin-treated mice resulted in improved glucose tolerance, lower hyperglycemia, and higher plasma insulin when subjected to illumination. Embedding optogenetic networks in β-cells for physiologically relevant control of GSIS will enable novel solutions potentially overcoming the shortcomings of current treatments for diabetes.
Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.
Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.
Using a Robust and Sensitive GFP-Based cGMP Sensor for Real Time Imaging in Intact Caenorhabditis elegans.
cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.
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.