Melanopsin (Opn4) utilizes Gαi and Gßγ as major signal transducers.

Melanopsin (Opn4), a ubiquitously expressed photoreceptor in all classes of vertebrates, is crucial for both visual and non-visual signaling. Opn4 supports visual functions of the eye by sensing radiance levels and discriminating contrast and brightness. Non-image-forming functions of Opn4 not only regulate circadian behavior, but also control growth and development processes of the retina. It is unclear how a single photoreceptor could govern such a diverse range of physiological functions; a role in genetic hardwiring could be one explanation, but molecular and mechanistic evidence is lacking. In addition to its role in canonical Gq pathway activation, here we demonstrate that Opn4 efficiently activates Gi heterotrimers and signals through the G protein ßγ. Compared with the low levels of Gi pathway activation observed for several Gq-coupled receptors, the robust Gαi and Gßγ signaling of Opn4 led to both generation of PIP3 and directional migration of RAW264.7 macrophages. We propose that the ability of Opn4 to signal through Gαi and Gßγ subunits is a major contributor to its functional diversity.

Microcystins: Biogenesis, Toxicity, Analysis, and Control.

Microcystins are cyclic peptide toxins formed by cyanobacteria. These toxins are recognized for their association with algal blooms, posing a significant threat to ecosystems and drinking water quality. Due to the growing environmental concerns they raise, a comprehensive review on microcystins' genesis, toxicity, and analytical methods for their quantitative determination is outlined. Genes, including the mcyABC cluster, regulate microcystin biogenesis. Bioanalytical experiments have identified key environmental factors, such as temperature and nitrogen availability, that promote microcystin production. Microcystin toxicity is explored based on its modulatory effects on protein phosphatases 1 and 2A in specific tissues and organs. Additionally, biochemical mechanisms of chelation, transportation, resultant oxidative stress, and tumor promotion abilities of microcystins are also discussed. Various analytical methods to separate, detect, and quantify microcystins, including the quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy, and chromatographic platforms-linked tandem mass spectrometry (LC-MS) for unequivocal structural identification, are also reviewed. Since control of microcystins in water is of great necessity, both water treatment and mechanisms of abiotic transformation and microbial degradation are also discussed.

Optical approaches for single-cell and subcellular analysis of GPCR-G protein signaling.

G protein-coupled receptors (GPCRs), G proteins, and their signaling associates are major signal transducers that control the majority of cellular signaling and regulate key biological functions including immune, neurological, cardiovascular, and metabolic processes. These pathways are targeted by over one-third of drugs on the market; however, the current understanding of their function is limited and primarily derived from cell-destructive approaches providing an ensemble of static, multi-cell information about the status and composition of molecules. Spatiotemporal behavior of molecules involved is crucial to understanding in vivo cell behaviors both in health and disease, and the advent of genetically encoded fluorescence proteins and small fluorophore-based biosensors has facilitated the mapping of dynamic signaling in cells with subcellular acuity. Since we and others have developed optogenetic methods to regulate GPCR-G protein signaling in single cells and subcellular regions using dedicated wavelengths, the desire to develop and adopt optogenetically amenable assays to measure signaling has motivated us to take a broader look at the available optical tools and approaches compatible with measuring single-cell and subcellular GPCR-G protein signaling. Here we review such key optical approaches enabling the examination of GPCR, G protein, secondary messenger, and downstream molecules such as kinase and lipid signaling in living cells. The methods reviewed employ both fluorescence and bioluminescence detection. We not only further elaborate the underlying principles of these sensors but also discuss the experimental criteria and limitations to be considered during their use in single-cell and subcellular signal mapping.

Two independent but synchronized Gßγ subunit-controlled pathways are essential for trailing-edge retraction during macrophage migration.

Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (Gi)-coupled receptors (GPCRs), G protein ßγ (Gßγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα12/13 pathway. However, it is not clear how the activation of Gi-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the Gi pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gßγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cß and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the Gi-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.

G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gßγ.

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gßγ. Since we have shown that signaling efficacy of Gßγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gßγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gßγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization.

Reversible G Protein ßγ9 Distribution-Based Assay Reveals Molecular Underpinnings in Subcellular, Single-Cell, and Multicellular GPCR and G Protein Activity.

Current assays to measure the activation of G protein coupled receptors (GPCRs) and G proteins are time-consuming, indirect, and expensive. Therefore, an efficient method which directly measures the ability of a ligand to govern GPCR-G protein interactions can help to understand the molecular underpinnings of the associated signaling. A live cell imaging-based approach is presented here to directly measure ligand-induced GPCR and G protein activity in real time. The number of active GPCRs governs G protein heterotrimer (αßγ) dissociation, thereby controlling the concentration of free ßγ subunits. The described γ9 assay measures the GPCR activation-induced extent of the reversible ßγ9 subunit exchange between the plasma membrane (PM) and internal membranes (IMs). Confocal microscopy-based γ9 assay quantitatively determines the concentration dependency of ligands on GPCR activation. Demonstrating the high-throughput screening (HTS) adaptability, the γ9 assay performed using an imaging plate reader measures the ligand-induced GPCR activation. This suggests that the γ9 assay can be employed to screen libraries of compounds for their ability to activate GPCRs. Together with subcellular optogenetics, the spatiotemporal sensitivity of the γ9 assay permits experimental determination of the limits of spatially restricted activation of GPCRs and G proteins in subcellular regions of single cells. This assay works effectively for GPCRs coupled to αi/o and αs heterotrimers, including light-sensitive GPCRs. In addition, computational modeling of experimental data from the assay is used to decipher intricate molecular details of the GPCR-G protein activation process. Overall, the γ9 assay provides a robust strategy for quantitative as well as qualitative determination of GPCR and G protein function on a single-cell, multicell, and subcellular level. This assay not only provides information about the inner workings of the signaling pathway, but it also strengthens GPCR deorphanization as well as drug discovery efforts.

The spatial distribution of GPCR and Gßγ activity across a cell dictates PIP3 dynamics.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.

Subtype-dependent regulation of Gßγ signalling.

G protein-coupled receptors (GPCRs) transmit information to the cell interior by transducing external signals to heterotrimeric G protein subunits, Gα and Gßγ subunits, localized on the inner leaflet of the plasma membrane. Though the initial focus was mainly on Gα-mediated events, Gßγ subunits were later identified as major contributors to GPCR-G protein signalling. A broad functional array of Gßγ signalling has recently been attributed to Gß and Gγ subtype diversity, comprising 5 Gß and 12 Gγ subtypes, respectively. In addition to displaying selectivity towards each other to form the Gßγ dimer, numerous studies have identified preferences of distinct Gßγ combinations for specific GPCRs, Gα subtypes and effector molecules. Importantly, Gß and Gγ subtype-dependent regulation of downstream effectors, representing a diverse range of signalling pathways and physiological functions have been found. Here, we review the literature on the repercussions of Gß and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes. Our discussion additionally provides perspective in understanding the intricacies underlying molecular regulation of subtype-specific roles of Gßγ signalling and associated diseases.

Blue light-triggered photochemistry and cytotoxicity of retinal.

The chemical- and photo- toxicity of chromophore retinal on cells have long been debated. Although we recently showed that retinal and blue light exposure interrupt cellular signaling, a comprehensive study examining molecular underpinnings of this perturbation and its consequences to cellular fate is lacking. Here, we report molecular evidence for blue light excited-retinal induced oxidative damage of polyunsaturated lipid anchors in membrane-interacting signaling molecules and DNA damage in cells using live-cell imaging and in vitro experimentation. The incurred molecular damage irreversibly disrupted subcellular localization of these molecules, a crucial criterion for their signaling. We further show retinal accumulation in lipid-bilayers of cell membranes could enhance the lifetime of retinal in cells. Comparative response-signatures suggest that retinal triggers reactions upon photoexcitation similar to photodynamic therapy agents and generate reactive oxygen species in cells. Additionally, data also shows that exposing retinal-containing cells to sunlight induces substantial cytotoxicity. Collectively, our results explain a likely in vivo mechanism and reaction conditions under which bio-available retinal in physiological light conditions damages cells.

Blue light excited retinal intercepts cellular signaling.

Photoreceptor chromophore, 11-cis retinal (11CR) and the photoproduct, all-trans retinal (ATR), are present in the retina at higher concentrations and interact with the visual cells. Non-visual cells in the body are also exposed to retinal that enters the circulation. Although the cornea and the lens of the eye are transparent to the blue light region where retinal can absorb and undergo excitation, the reported phototoxicity in the eye has been assigned to lipophilic non-degradable materials known as lipofuscins, which also includes retinal condensation products. The possibility of blue light excited retinal interacting with cells; intercepting signaling in the presence or absence of light has not been explored. Using live cell imaging and optogenetic signaling control, we uncovered that blue light-excited ATR and 11CR irreversibly change/distort plasma membrane (PM) bound phospholipid; phosphatidylinositol 4,5 bisphosphate (PIP2) and disrupt its function. This distortion in PIP2 was independent of visual or non-visual G-protein coupled receptor activation. The change in PIP2 was followed by an increase in the cytosolic calcium, excessive cell shape change, and cell death. Blue light alone or retinal alone did not perturb PIP2 or elicit cytosolic calcium increase. Our data also suggest that photoexcited retinal-induced PIP2 distortion and subsequent oxidative damage incur in the core of the PM. These findings suggest that retinal exerts light sensitivity to both photoreceptor and non-photoreceptor cells, and intercepts crucial signaling events, altering the cellular fate.

Regulation of G Protein ßγ Signaling.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) deliver external signals to the cell interior, upon activation by the external signal stimulated G protein-coupled receptors (GPCRs).While the activated GPCRs control several pathways independently, activated G proteins control the vast majority of cellular and physiological functions, ranging from vision to cardiovascular homeostasis. Activated GPCRs dissociate GαGDPßγ heterotrimer into GαGTP and free Gßγ. Earlier, GαGTP was recognized as the primary signal transducer of the pathway and Gßγ as a passive signaling modality that facilitates the activity of Gα. However, Gßγ later found to regulate more number of pathways than GαGTP does. Once liberated from the heterotrimer, free Gßγ interacts and activates a diverse range of signaling regulators including kinases, lipases, GTPases, and ion channels, and it does not require any posttranslation modifications. Gßγ family consists of 48 members, which show cell- and tissue-specific expressions, and recent reports show that cells employ the subtype diversity in Gßγ to achieve desired signaling outcomes. In addition to activated GPCRs, which induce free Gßγ generation and the rate of GTP hydrolysis in Gα, which sequester Gßγ in the heterotrimer, terminating Gßγ signaling, additional regulatory mechanisms exist to regulate Gßγ activity. In this chapter, we discuss structure and function, subtype diversity and its significance in signaling regulation, effector activation, regulatory mechanisms as well as the disease relevance of Gßγ in eukaryotes.

Confocal Imaging and k-Means Clustering of GABAB and mGluR Mediated Modulation of Ca2+ Spiking in Hippocampal Neurons.

Imaging cytosolic calcium in neurons is emerging as a new tool in neurological disease diagnosis, drug screening, and toxicity testing. Ca2+ oscillation signatures show a significant variation depending on GPCR targeting agonists. Quantification of Ca2+ spike trains in ligand induced Ca2+ oscillations remains challenging due to their inherent heterogeneity in primary culture. Moreover, there is no framework available for identification of optimal number of clusters and distance metric to cluster Ca2+ spike trains. Using quantitative confocal imaging and clustering analysis, we show the characterization of Ca2+ spiking in GPCR targeting drug-treated primary culture of hippocampal neurons. A systematic framework for selection of the clustering method instead of an intuition-based method was used to optimize the cluster number and distance metric. The results discern neurons with diverse Ca2+ response patterns, including higher amplitude fast spiking and lower spiking responses, and their relative percentage in a neuron population in absence and presence of GPCR-targeted drugs. The proposed framework was employed to show that the  clustering pattern of Ca2+ spiking can be controlled using GABAB and mGluR targeting drugs. This approach can be used for unbiased measurement of neural activity and identification of spiking population with varying amplitude and frequencies, providing a platform for high-content drug screening.

Measurement of GPCR-G protein activity in living cells.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic genomes. They control a variety of cellular and physiological processes such as hormone secretion and heart rate, and therefore are associated with a majority of pathological conditions including cancer and heart diseases. Currently established assays to measure ligand-induced activation of GPCRs and G proteins possess limitations such as being time consuming, indirect, and expensive. Thus, an efficient method to measure GPCR-G protein activation is required to identify novel pharmacological modulators to control them and gain insights about molecular underpinnings of the associated pathways. Activation of GPCRs induces dissociation of G protein heterotrimers to form GαGTP and free Gßγ. Free Gßγ subunits have been shown to translocate reversibly from the plasma membrane to internal membranes. Gßγ translocation therefore represents the GPCR-G protein activation, and thus, imaging of this process can be used to quantify the kinetics and magnitude of the pathway activation-deactivation in real time in living cells. The objective of this chapter is to elaborate the protocols of (i) generation and optimization of the required sensor constructs; (ii) development of cell culture, transient transfection, imaging, and optogenetic procedures; (iii) imaging and data analysis methods; and (iv) stable cell line generation, pertaining to this assay to measure GPCR-G protein activation.

Facilitation of TRPV4 by TRPV1 is required for itch transmission in some sensory neuron populations.

The transient receptor potential channels (TRPs) respond to chemical irritants and temperature. TRPV1 responds to the itch-inducing endogenous signal histamine, and TRPA1 responds to the itch-inducing chemical chloroquine. We showed that, in sensory neurons, TRPV4 is important for both chloroquine- and histamine-induced itch and that TRPV1 has a role in chloroquine-induced itch. Chloroquine-induced scratching was reduced in mice in which TRPV1 was knocked down or pharmacologically inhibited. Both TRPV4 and TRPV1 were present in some sensory neurons. Pharmacological blockade of either TRPV4 or TRPV1 significantly attenuated the Ca(2+) response of sensory neurons exposed to histamine or chloroquine. Knockout of Trpv1 impaired Ca(2+) responses and reduced scratching behavior evoked by a TRPV4 agonist, whereas knockout of Trpv4 did not alter TRPV1-mediated capsaicin responses. Electrophysiological analysis of human embryonic kidney (HEK) 293 cells coexpressing TRPV4 and TRPV1 revealed that the presence of both channels enhanced the activation kinetics of TRPV4 but not of TRPV1. Biochemical and biophysical studies suggested a close proximity between TRPV4 and TRPV1 in dorsal root ganglion neurons and in cultured cells. Thus, our studies identified TRPV4 as a channel that contributes to both histamine- and chloroquine-induced itch and indicated that the function of TRPV4 in itch signaling involves TRPV1-mediated facilitation. TRP facilitation through the formation of heteromeric complexes could be a prevalent mechanism by which the vast array of somatosensory information is encoded in sensory neurons.