Temperature-responsive optogenetic probes of cell signaling.
Ontology highlight
ABSTRACT:
SUBMITTER: Benman W
PROVIDER: S-EPMC9252025 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The application of NIR to optogenetic systems is in great demand due to its superior properties enabling in vivo deep tissue penetration. Irradiation of NIR to tissue samples or cells rapidly generates heat locally. The resultant elevation in temperature affects cells at the molecular level because of the activation of the heat shock pathway and ROS generation. Nevertheless, few reports have presented detailed comparisons of the effects of the temperature change rate on signaling pathway biomolecules, especially those of rapid heat changes. Aiming at broadening the understanding of temperature sensitivity, we investigated seven insulin signaling pathway biomolecules (INSR, IRS1, Akt, GSK3β, p70S6K, FoxO1, and ERK1/2) in three cell lines (C2C12, HepG2, and Fao) at temperatures between 25 and 45 °C. The results show that, except for INSR, pAkt(T308), and FoxO1, biomolecules are sensitive to rapid temperature changes at temperatures higher than 42 °C, at which they are significantly phosphorylated. At 25 °C, around a 50% reduction in phosphorylation occurred. Moreover, p70S6K is sensitive over time. It dephosphorylates quickly (5 min) and then phosphorylates over time. Our findings extend the temperature range to 45 °C, while providing additional time course information about the signaling pathway biomolecule response necessary to advance NIR optogenetic research.
| S-EPMC9564263 | biostudies-literature
Project description:OBJECTIVE:Optogenetic modulation of neural activity is a ubiquitous tool for basic investigation of brain circuits. While the majority of optogenetic paradigms rely on short light pulses to evoke synchronized activity of optically sensitized cells, many neurobiological processes are associated with slow local field potential (LFP) oscillations. Therefore, we developed a hybrid fiber probe capable of simultaneous electrophysiological recording and optical stimulation and used it to investigate the utility of sinusoidal light stimulation for evoking oscillatory neural activity in vivo across a broad frequency range. APPROACH:We fabricated hybrid fiber probes comprising a hollow cylindrical array of 9 electrodes and a flexible optical waveguide integrated within the core. We implanted these probes in the hippocampus of transgenic Thy1-ChR2-YFP mice that broadly express the blue-light sensitive cation channel channelrhodopsin 2 (ChR2) in excitatory neurons across the brain. The effects of the sinusoidal light stimulation were characterized and contrasted with those corresponding to pulsed stimulation in the frequency range of physiological LFP rhythms (3-128 Hz). MAIN RESULTS:Within hybrid probes, metal electrode surfaces were vertically aligned with the waveguide tip, which minimized optical stimulation artifacts in neurophysiological recordings. Sinusoidal stimulation resulted in reliable and coherent entrainment of LFP oscillations up to 70 Hz, the cutoff frequency of ChR2, with response amplitudes inversely scaling with the stimulation frequencies. Effectiveness of the stimulation was maintained for two months following implantation. SIGNIFICANCE:Alternative stimulation patterns complementing existing pulsed protocols, in particular sinusoidal light stimulation, are a prerequisite for investigating the physiological mechanisms underlying brain rhythms. So far, studies applying sinusoidal stimulation in vivo were limited to single stimulation frequencies. We show the feasibility of sinusoidal stimulation in vivo to induce coherent LFP oscillations across the entire frequency spectrum supported by the gating dynamics of ChR2 and introduce a hybrid fiber probe tailored to continuous light stimulation.
| S-EPMC6125198 | biostudies-other
Project description:The purpose of this study was to characterize a novel optogenetic kinase system, a Light-Regulated (LightR) switch domain, designed to improve spatiotemporal control of signaling. Using LC-MS/MS, tyrosine phosphorylation was measured after 10 seconds, 30 seconds, 1 minute, 5 minutes and 60 minutes of LightR Src activation. TMT labeling was utilized to allow multiplexed relative quantification across time points. Results provide insight into the timing of Src and Src substrate activation.
2020-10-20 | PXD018162 | Pride
Project description:Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein-coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions.
| S-EPMC4116304 | biostudies-literature
Project description:We introduce a non-invasive approach for optogenetic regulation in biological cells through highly scattering skull tissue using wavefront shaping. The wavefront of the incident light was systematically controlled using a spatial light modulator in order to overcome multiple light-scattering in a mouse skull layer and to focus light on the target cells. We demonstrate that illumination with shaped waves enables spatiotemporal regulation of intracellular Ca(2+) level at the individual-cell level.
| S-EPMC4543936 | biostudies-literature
Project description:Genetically encoded photoswitches have enabled spatial and temporal control of cellular events to achieve tailored functions in living cells, but their applications to probe the structure-function relations of signaling proteins are still underexplored. We illustrate herein the incorporation of various blue light-responsive photoreceptors into modular domains of the stromal interaction molecule 1 (STIM1) to manipulate protein activity and faithfully recapitulate STIM1-mediated signaling events. Capitalizing on these optogenetic tools, we identify the molecular determinants required to mediate protein oligomerization, intramolecular conformational switch, and protein-target interactions. In parallel, we have applied these synthetic devices to enable light-inducible gating of calcium channels, conformational switch, dynamic protein-microtubule interactions and assembly of membrane contact sites in a reversible manner. Our optogenetic engineering approach can be broadly applied to aid the mechanistic dissection of cell signaling, as well as non-invasive interrogation of physiological processes with high precision.
| S-EPMC7042325 | biostudies-literature
Project description:The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.
| S-EPMC7220906 | biostudies-literature
Project description:Optogenetics is an advanced neuroscience technique that enables the dissection of neural circuitry with high spatiotemporal precision. Recent advances in materials and microfabrication techniques have enabled minimally invasive and biocompatible optical neural probes, thereby facilitating in vivo optogenetic research. However, conventional fabrication techniques rely on cleanroom facilities, which are not easily accessible and are expensive to use, making the overall manufacturing process inconvenient and costly. Moreover, the inherent time-consuming nature of current fabrication procedures impede the rapid customization of neural probes in between in vivo studies. Here, we introduce a new technique stemming from 3D printing technology for the low-cost, mass production of rapidly customizable optogenetic neural probes. We detail the 3D printing production process, on-the-fly design versatility, and biocompatibility of 3D printed optogenetic probes as well as their functional capabilities for wireless in vivo optogenetics. Successful in vivo studies with 3D printed devices highlight the reliability of this easily accessible and flexible manufacturing approach that, with advances in printing technology, can foreshadow its widespread applications in low-cost bioelectronics in the future.
| S-EPMC7942018 | biostudies-literature
Project description:Coacervates are a type of liquid-liquid phase separated (LLPS) droplets that can serve as models of membraneless organelles (MLOs) in living cells. Peptide-nucleotide coacervates have been widely used to mimic properties of ribonucleoprotein (RNP) granules, but the thermal stability and the role of base stacking is still poorly understood. Here, we report a systematic investigation of coacervates formed by five different nucleoside triphosphates (NTPs) with poly-l-lysine and poly-l-arginine as a function of temperature. All studied combinations exhibit an upper critical solution temperature (UCST), and a temperature-dependent critical salt concentration, originating from a significant nonelectrostatic contribution to the mixing free energy. Both the enthalpic and entropic parts of this nonelectrostatic interaction decrease in the order G/A/U/C/T, in accordance with nucleobase stacking free energies. Partitioning of two dyes proves that the local hydrophobicity inside the peptide-nucleotide coacervates is different for every nucleoside triphosphate. We derive a simple relation between the temperature and salt concentration at the critical point based on a mean-field model of phase separation. Finally, when different NTPs are mixed with one common oppositely charged peptide, hybrid coacervates were formed, characterized by a single intermediate UCST and critical salt concentration. NTPs with lower critical salt concentrations can remain condensed in mixed coacervates far beyond their original critical salt concentration. Our results show that NTP-based coacervates have a strong temperature sensitivity due to base stacking interactions and that mixing NTPs can significantly influence the stability of condensates and, by extension, their bioavailability.
| S-EPMC8020381 | biostudies-literature
Project description:The nucleus accumbens is a key mediator of cocaine reward, but the distinct roles of the two subpopulations of nucleus accumbens projection neurons, those expressing dopamine D1 versus D2 receptors, are poorly understood. We show that deletion of TrkB, the brain-derived neurotrophic factor (BDNF) receptor, selectively from D1+ or D2+ neurons oppositely affects cocaine reward. Because loss of TrkB in D2+ neurons increases their neuronal excitability, we next used optogenetic tools to control selectively the firing rate of D1+ and D2+ nucleus accumbens neurons and studied consequent effects on cocaine reward. Activation of D2+ neurons, mimicking the loss of TrkB, suppresses cocaine reward, with opposite effects induced by activation of D1+ neurons. These results provide insight into the molecular control of D1+ and D2+ neuronal activity as well as the circuit-level contribution of these cell types to cocaine reward.
| S-EPMC3011229 | biostudies-literature