Two-photon absorption is a nonlinear optical process in which a molecule simultaneously absorbs two photons. It finds use in two-photon fluorescence microscopy, providing high spatial resolution and deep imaging of biological tissues. Understanding the physical mechanisms of two-photon absorption will help optimize excitation conditions and design brighter probes for two-photon microscopy. The two-photon polarization ratio (Ω), defined as the ratio of the two-photon absorption strength of circularly and linearly polarized light─carries indispensable information on the symmetry of electronic, vibronic, and excitonic transitions. Here, we present a physical model based on few-state approximations to derive analytical expressions for Ω as a function of molecular dipole moment matrix elements. The model accounts for an unusual, Herzberg-Teller vibronic coupling of the permanent dipole moments to the bond-length alternating vibrational coordinate(s). Using this framework, we analyze both monomeric systems (Rhodamine 6G, TM-BODIPY, and the chromophore in fluorescent protein Venus) and a dimeric Venus protein. For the monomers, Ω measurements allow us to resolve the two-photon absorption spectra into components corresponding to the Franck-Condon vibronic progressions similar to those observed in one-photon absorption, as well as new vibronic progressions built upon Herzberg-Teller replicas of the bond-length alternating vibration(s), only pertinent to two-photon absorption. Our model explains previously reported broadening and blue shift of the two-photon absorption spectra of dyes relative to their one-photon absorption counterparts. For the Venus dimer protein, spectral dependence of Ω helps to resolve strongly overlapping excitonic J- and H-transitions and specify mutual geometrical arrangement of the two chromophores.

Objectives Obesity-induced steatotic liver disease (SLD) is driven by the uptake of adipocyte-derived fatty acids (FAs) into hepatocytes via the FA translocase CD36, which also prevents their consumption by inhibiting AMP kinase (AMPK)-mediated FA oxidation (FAO). We explored the role of hepatocyte CB1 receptors (hCB1R) in controlling hepatic triglyceride (TG) content by regulating CD36 and its downstream targets. Methods hCB1R knockout (hCB1Rko) mice and their control littermates kept on standard or high-fat diet were used to analyze hCB1R-mediated hepatic gene expression profile and lipid metabolism in intact mice and in cultured hepatocytes. Results Multi-omics data indicate that hCB1R target a distinct set of genes associated with SLD, including Cd36. In mice with diet-induced obesity, hCB1R signaling via CD36-AMPK-FAO pathway contributes to both the development of SLD and its reversal by CB1R blockade. But, in lean mice hCB1R signaling inhibits CD36 expression and activates AMPK-mediated FAO. These opposite effects were replicated in AML12 mouse hepatocytes incubated with or without oleic acid (OA). OA, an endogenous ligand of GPR3, induced a switch in hCB1R signaling from a Gi/oα-mediated reduction in cAMP to a Gsα-mediated increase in cAMP in a GPR3/Gsα -dependent manner, facilitated by increasing the ratio of Gsα:Gi/oα proteins in the steatotic compared to lean liver. Conclusions In the lean state, endocannabinoid activation of hCB1R increases FAO, which mitigates SLD, as reported for chronic marihuana smokers, whereas in obese mice hCB1R tonically inhibit FAO, which promotes SLD and underlies the anti-steatotic effect of peripheral CB1R blockade.

Fluorescent proteins (FPs) comprise hundreds of different genetically encoded biosensors. Anomalous FP photo-physics, consistent with excitonic coupling, i.e., delocalized excitation, has been previously reported. Since delocalized excitation can potentially alter fluorescence lifetimes, intensities, and spectra at long distances, its impact needs to be evaluated for the proper design and interpretation of biosensor experiments, as well as for the development of genetically encoded excitonic materials. In addition, it is unclear if excitonic coupling is a shared trait of all β-barrel FPs, nor are the distances requirements for FP excitonic coupling known. To address these questions, we engineered FP constructs having either one or two functional chromophores. We found red shifts in absorption, circular dichroism Davydov splitting, and shorter fluorescence lifetimes in evolutionarily divergent FP tandem dimers having two chromophores, supporting the existence of excitonic coupling. Photon antibunching statistics indicated that tandem dimers with two chromophores emit as a single quantum unit. Sub-Poissonian photon statistics was observed even with 20-nm Venus-Venus chromophore separation, twice the limit of Förster's resonance energy transfer, but not at 60-nm. Our findings support the hypothesis that the conserved β-barrel structure is the common structural attribute associated with allowing fluorescent protein exciton delocalization under physiological conditions.

The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP -driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin -binding CaMKII 6 isoform using single -molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface -immobilized Venus-CaMKII 6 were resolved with 0.5 s resolution from video records, batch -processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP -dependent increase of the CAM association requires dimer formation for both the a and 6 isoforms. The study of mutant 6 holoenzymes revealed that the ATP -dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306 -307A) holoenzyme resides only in the low -affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306 -307A. In the absence of ATP, the affinity of CAM for the T287.306 -307A mutant and the wild -type monomer are comparable. The affinity of the ATP -binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low -affinity state. The phosphomimetic mutant (T287D) resides only in a 1000 -fold higher -affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP -dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild -type population had a two -state distribution with both low- and high -affinity states represented. The low -affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action -potential frequency decoding in vivo.

Quantum mechanics has significantly advanced our understanding of fundamental properties. While biological studies have traditionally been assumed to be governed by classical physics, recent studies have shown that coherent excitonic coupling between two chromophores in a homodimer of the yellow fluorescent protein, Venus, is possible at room temperature [Y. Kim et al., Biophys. J., 2019, 116, 1918-1930]. In this study, we present a genetically encoded sensor, inspired by recent finding of the ultrafast photoinduced energy transfer between dimeric enhanced green fluorescent proteins (dEGFPs), to monitor hydrophobic environmental changes [A. Sanchez-Pedreno Jimenez, et al., Phys. Chem. Chem. Phys., in press]. This sensor can be used to investigate the spatial and temporal dynamics biocondensates in cells. This biosensor offers an innovative approach to unravel the dynamics of biocondensates, to elucidate their biological functions and potential implications in health and disease. Experiments are being conducted to test this sensor in living cells.

The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons.

Advances in ultra-fast photonics have enabled monitoring of biochemical interactions on a sub nano-second time scale. In addition, picosecond dynamics of intermolecular energy transfer in fluorescent proteins has been observed. Here, we present the development of a genetically encoded fluorescent sensor that can detect changes in hydrophobicity by monitoring ultrafast fluorescence depolarisation. Our sensor is composed of a pair of dimeric enhanced green fluorescent proteins (dEGFPs) linked by a flexible amino-acid linker. We show dimerisation is perturbed by the addition of glycerol which interferes with the hydrophobic interaction of the two proteins. Time-resolved fluorescence anisotropy revealed a systematic attenuation of ultrafast fluorescence depolarisation when the sensor was exposed to increasing glycerol concentrations. This suggests that as hydrophobicity increases, dEGFP pairing decreases within a tandem dimer. Un-pairing of the protein fluorophores dramatically alters the rate of energy transfer between the proteins, resulting in an increase in the limiting anisotropy of the sensor.

Two-photon polarization ratio ()Ω measures the angle between optical transition dipole moment and permanent dipole moments difference. Linking two VenusA206 copies together results in changes of Ω value, reflecting different dipoles arrangement in excitonically coupled chromophores.

Under physiological conditions fluorescent proteins (FP) in close proximity unexpectedly exhibit photophysical effects that are consistent with excitonic coupling: (1) ultrafast drop in anisotropy, (2) Davydov splitting in its CD spectra and (3) multiple FPs behaving as a single quantum emitter in photon antibunching experiments. We hypothesize that the FP β-barrel structure protects its internal chromophore from environmental decoherence, and thus slows its dephasing time relative to its energy transfer rate.

Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium-calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity.

Seven-transmembrane receptors signal via G-protein- and β-arrestin-dependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced β-arrestin-2 (βArr2) recruitment over G-protein activation. In obese wild-type and βArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB1R occupancy. By contrast, the unbiased global CB1R antagonist rimonabant elicits anxiety in both strains, indicating no βArr2 involvement. Interestingly, obesity-induced muscle insulin resistance is improved by MRI-1891 in wild-type but not in βArr2-KO mice. In C2C12 myoblasts, CB1R activation suppresses insulin-induced akt-2 phosphorylation, preventable by MRI-1891, βArr2 knockdown or overexpression of CB1R-interacting protein. MRI-1891, but not rimonabant, interacts with nonpolar residues on the N-terminal loop, including F108, and on transmembrane helix-1, including S123, a combination that facilitates βArr2 bias. Thus, CB1R promotes muscle insulin resistance via βArr2 signaling, selectively mitigated by a biased CB1R antagonist at reduced risk of central nervous system (CNS) side effects.

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to generate eCBs do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRABeCB2.0, was expressed presynaptically in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients that were generated predominantly by 2-arachidonoylglycerol (2-AG) mobilization. Efficient 2-AG mobilization required coactivation of AMPA and NMDA ionotropic glutamate receptors and muscarinic M1 receptors. Dopamine D2 receptors expressed on cholinergic interneurons inhibited 2-AG mobilization by inhibiting acetylcholine release. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

Endocannabinoids (eCBs) are retrograde neuromodulators with important functions in a wide range of physiological processes, but their in vivo dynamics remain largely uncharacterized. Here we developed a genetically encoded eCB sensor called GRAB(eCB2.0). GRAB(eCB2.0) consists of a circular-permutated EGFP and the human CB1 cannabinoid receptor, providing cell membrane trafficking, second-resolution kinetics with high specificity for eCBs, and shows a robust fluorescence response at physiological eCB concentrations. Using GRAB(eCB2.0), we monitored evoked and spontaneous changes in eCB dynamics in cultured neurons and acute brain slices. We observed spontaneous compartmentalized eCB transients in cultured neurons and eCB transients from single axonal boutons in acute brain slices, suggesting constrained, localized eCB signaling. When GRAB(eCB2.0) was expressed in the mouse brain, we observed foot shock-elicited and running-triggered eCB signaling in the basolateral amygdala and hippocampus, respectively. In a mouse model of epilepsy, we observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. GRAB(eCB2.0) is a robust probe for eCB release in vivo. A genetically encoded sensor reveals the dynamics of endocannabinoid signaling.

Interoceptive and exteroceptive signals, and the corresponding coordinated control of internal organs and sensory functions, including pain, are received and orchestrated by multiple neurons within the peripheral, central and autonomic nervous systems. A central aim of the present report is to obtain a molecularly informed basis for analgesic drug development aimed at peripheral rather than central targets. We compare three key peripheral ganglia: nodose, sympathetic (superior cervical), and dorsal root ganglia in the rat, and focus on their molecular composition using next-gen RNA-Seq, as well as their neuroanatomy using immunocytochemistry and in situ hybridization. We obtained quantitative and anatomical assessments of transmitters, receptors, enzymes and signaling pathways mediating ganglion-specific functions. Distinct ganglionic patterns of expression were observed spanning ion channels, neurotransmitters, neuropeptides, G-protein coupled receptors (GPCRs), transporters, and biosynthetic enzymes. The relationship between ganglionic transcript levels and the corresponding protein was examined using immunohistochemistry for select, highly expressed, ganglion-specific genes. Transcriptomic analyses of spinal dorsal horn and intermediolateral cell column (IML), which form the termination of primary afferent neurons and the origin of preganglionic innervation to the SCG, respectively, disclosed pre- and post-ganglionic molecular-level circuits. These multimodal investigations provide insight into autonomic regulation, nodose transcripts related to pain and satiety, and DRG-spinal cord and IML-SCG communication. Multiple neurobiological and pharmacological contexts can be addressed, such as discriminating drug targets and predicting potential side effects, in analgesic drug development efforts directed at the peripheral nervous system.

Multimodal microscopy can detect multiple changes in molecular conformation and has become a powerful tool for studying protein-protein interactions within and between complexes. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homo-FRET, hetero-FRET and FCS was developed for this purpose. FPFA was recently extended by developing auto-FPFA, a robotic version of FPFA that can measure up to 96 biological samples automatically and/or repeatedly. Here we use auto-FPFA to investigate time-dependent changes of calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzyme in response to activation and subsequent interaction with the T-site ligand motif of NR2B in vitro. Homo-FRET analysis showed that upon binding Ca2+/CaM, catalytic-domain pairing, a fundamental structural motif of the autoinhibited holoenzyme, rapidly disjoined, and then slowly reformed. The rate of this subsequent re-dimerization was influenced by ATP: the higher the ATP concentration, the slower the re-pairing rate. CaMKII binding to NR2B maintained the opening of catalytic-domain pairs, therefore suppressed the reformation of catalytic-domain pairing. Interestingly, hetero-FRET efficiency, an indicator of binding between mVenus-CaMKII and mCherry-NR2B, was proportional to the fraction of unpaired CaMKII subunits. These results suggest the existence of an ATP regulated time window for gating CaMKII interactions with T-site ligands.

We present an optical technique, called AB/FCS-fingerprinting, capable of characterizing fluorophore independence in aqueous solution using two-photon excitation microscopy and time-correlated single photon counting. Fluorescence correlation spectroscopy (FCS) is used to monitor fluctuations in the intensity of emissions. Auto- or cross-correlation analysis of these fluctuations can measure the average number of fluorescent molecules in an aqueous sample. Photon antibunching (AB) is observed from single quantum entities, which can emit only one photon at a time. By recording the number of coincident photons detected as a function of time between photon detections, AB analysis is used to determine the number of independent emitters in a sample. In AB/FCS-fingerprinting, the number of independent emitters in a sample is compared with the average number of fluorescent molecules in the same sample using a microscope that can be rapidly reconfigured to measure either AB or FCS from serial dilutions of a florescent sample. Since the number of fluorescent molecules is not necessarily equal to the number of independent emitters, a comparison of these values can provide insight into the independence of fluorophores in molecular assemblies. We validated this technique by measuring AB/FCS-fingerprinting of serial dilutions of mVenus, mNeonGreen, and an organic fluorophore, Alexa-Fluor-488. Experimental results were in good agreement with Monte-Carlo antibunching simulations for a single quantum emitter and with the predictions of a zero-truncated Poisson distribution model for photon antibunching from monomeric fluorophores in solution.

Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Förster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP β-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP VenusA206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a VenusA206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between VenusA206 fluorophores is possible at physiological temperatures.

NaV1.8 channels play a crucial role in regulating the action potential in nociceptive neurons. A single nucleotide polymorphism in the human NaV1.8 gene SCN10A, A1073V (rs6795970, G>A), has been linked to the diminution of mechanical pain sensation as well as cardiac conduction abnormalities. Furthermore, studies have suggested that this polymorphism may result in a “loss-of-function” phenotype. In the present study, we performed genomic analysis of A1073V polymorphism presence in a cohort of patients undergoing sigmoid colectomy who provided information regarding perioperative pain and analgesic use. Homozygous carriers reported significantly reduced severity in postoperative abdominal pain compared with heterozygous and wild-type carriers. Homozygotes also trended toward using less analgesic/opiates during the postoperative period. We also heterologously expressed the wild-type and A1073V variant in rat superior cervical ganglion neurons. Electrophysiological testing demonstrated that the mutant NaV1.8 channels activated at more depolarized potentials compared with wild-type channels. Our study revealed that postoperative abdominal pain is diminished in homozygous carriers of A1073V and that this is likely due to reduced transmission of action potentials in nociceptive neurons. Our findings reinforce the importance of NaV1.8 and the A1073V polymorphism to pain perception. This information could be used to develop new predictive tools to optimize patient pain experience and analgesic use in the perioperative setting. NEW & NOTEWORTHY We present evidence that in a cohort of patients undergoing sigmoid colectomy, those homozygous for the NaV1.8 polymorphism (rs6795970) reported significantly lower abdominal pain scores than individuals with the homozygous wild-type or heterozygous genotype. In vitro electrophysiological recordings also suggest that the mutant NaV1.8 channel activates at more depolarizing potentials than the wild-type Na+ channel, characteristic of hypoactivity. This is the first report linking the rs6795970 mutation with postoperative abdominal pain in humans.

Activation of short-chain free fatty acid receptors 3 (FFAR3) has been suggested to promote sympathetic outflow in postganglionic sympathetic neurons or hamper it by a negative coupling to N-type calcium (CaV2.2) channels. Heterogeneity of FFAR3 expression in sympathetic neurons, however, renders single neurons studies extremely time-consuming in wild-type mice. Previous studies demonstrated large variability of the degree of CaV2.2 channel inhibition by FFAR3 in a global population of rat sympathetic neurons. Therefore, we focused on a small subpopulation of mouse sympathetic neurons using an FFAR3 antibody and an Ffar3 reporter mouse to perform immunofluorescent and electrophysiological studies. Whole-cell patch-clamp recordings of identified FFAR3-expressing neurons from reporter mice revealed a 2.5-fold decrease in the CaV2.2-FFAR3 inhibitory coupling variability and 1.5-fold increase in the mean ICa2+ inhibition, when compared with unlabeled neurons from wild-type mice. Further, we found that the ablation of Ffar3 gene expression in two knockout mouse models led to a complete loss-of-function. Subpopulations of sympathetic neurons are associated with discrete functional pathways. However, little is known about the neural pathways of the FFAR3-expressing subpopulation. Our data indicate that FFAR3 is expressed primarily in neurons with a vasoconstrictor phenotype. Thus, fine-tuning of chemically-coded neurotransmitters may accomplish an adequate outcome.