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Strong pH-Dependent Near-Infrared Fluorescence in a Microbial Rhodopsin Reconstituted with a Red-Shifting Retinal Analogue
摘要： Near-infrared (NIR)-driven rhodopsins are of great interest in optogenetics and other optobiotechnological developments such as artificial photosynthesis and deep-tissue voltage imaging. Here we report the proton pump proteorhodopsin (PR) containing a NIR-active retinal analogue (PR:MMAR) exhibits intense NIR fluorescence at a quantum yield of 3.3%. This is 130 times higher than native PR (Lenz, M. O.; et al. Biophys J. 2006, 91, 255?262) and 3?8 times higher than the QuasAr and PROPS voltage sensors (Kralj, J.; et al. Science 2011, 333, 345?348; Hochbaum, D. R.; et al. Nat. Methods 2014, 11, 825?833). The NIR fluorescence strongly depends on the pH in the range of 6?8.5, suggesting potential application of MMAR-binding proteins as ultrasensitive NIR-driven pH and/or voltage sensors. Femtosecond transient absorption spectroscopy showed that upon near-IR excitation, PR:MMAR features an unusually long fluorescence lifetime of 310 ps and the absence of isomerized photoproducts, consistent with the high fluorescence quantum yield. Stimulated Raman analysis indicates that the NIR-absorbing species develops upon protonation of a conserved aspartate, which promotes charge delocalization and bond length leveling due to an additional methylamino group in MMAR, in essence providing a secondary protonated Schiff base. This results in much smaller bond length alteration along the conjugated backbone, thereby conferring significant single-bond character to the C13C14 bond and structural deformation of the chromophore, which interferes with photoinduced isomerization and extends the lifetime for fluorescence. Hence, our studies allow for a molecular understanding of the relation between absorption/emission wavelength, isomerization, and fluorescence in PR:MMAR. As acidification enhances the resonance state, this explains the strong pH dependence of the NIR emission.
关键词： stimulated Raman analysis,fluorescence,voltage sensor,rhodopsins,optogenetics,artificial photosynthesis,proteorhodopsin,femtosecond transient absorption spectroscopy,pH sensor,Near-infrared,voltage imaging
更新于2025-09-23 15:21:01
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LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution
摘要： Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.
关键词： protein translocation,eukaryotic cells,optogenetics,type III secretion system,LITESEC-T3SS
更新于2025-09-23 15:21:01
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Optogenetic Long-Term Depression Induction in the PVT-CeL Circuitry Mediates Decreased Fear Memory
摘要： The dysregulation of fear learning and abnormal activities of cerebral networks may contribute to the etiologies of anxiety disorders. Although it has been proposed that decreased activity in the paraventricular nucleus of the thalamus (PVT) to the lateral central nucleus of amygdala (CeL) pathway could induce an attenuation of learned fear, no study has shown the effect of the direct optogenetic activation of PVT projecting CeL neurons in vivo on unconditioned fear-related behaviors or learned fear expression. The mechanisms that control the neuronal activity of the PVT-CeL pathway involved in anxiety are rare. Here, we found that CeL neurons have varied responses to optogenetic excitation of PVT terminals in the CeL: neurons with relative high excitability(~ 30%), neurons with relative low excitability(~ 60%), and neurons with no excitability (~ 10%). We next explored the role of the PVT-CeL pathway in unconditioned and conditioned fear-related behaviors by using optogenetics and anxiety assays in freely moving mice. We observed that temporally precise optogenetic activation of the CeL-projecting PVT neurons had no effect on unconditioned fear-related behaviors on the elevated plus maze test and the open field test. But optogenetic activation of the CeL-projecting PVT neurons increased conditioned fear expression. We then found that optogenetic long-term depression (LTD) induction in the CeL receiving PVT afferents effectively exerted a persistent attenuation of learned fear. The percentage of neurons with relative high excitability was decreased by the LTD induction, and the percentage of neurons with relative low excitability was increased by the LTD induction. Taking these results together, we identify that increased activity of the PVT-CeL pathway could lead to as excessive learned fear. The CeL neurons with relative high responses to the photo-stimulation of PVT afferents in the CeL may be the key neurons that regulate the output of learned fear expression. Our optogenetic LTD protocol may inspire the development of novel treatments for anxiety disorders involving deep brain stimulation to induce plasticity at relevant brain areas.
关键词： Optogenetics,LTD,Fear memory,The PVT-CeL circuitry
更新于2025-09-23 15:21:01
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Guided by light: optogenetic control of microtubule gliding assays
摘要： Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays, in which surface-immobilized motor proteins drive microtubule propulsion, are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, bio-computation and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ~5-fold enrichment within 6 seconds upon illumination. Subsequently, proteins are released with a half-life of 13 seconds when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of non-uniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.
关键词： motor proteins,optogenetics,optical control,Microtubules
更新于2025-09-23 15:21:01
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Optogenetic control of perforant pathway kindled seizures by photoinhibition of CA3 after insertion of Jaws into CA3 neuronal cells
摘要： Medial temporal lobe epilepsy (MTLE) is among the most common and most drug-resistant types of epilepsies associated with remodeling of the trisynaptic circuit of the hippocampus. The cornu ammonis (CA)3 region, as the “pacemaker” of the circuit, and CA3 → CA1 synapse (Scha?er collaterals) are potential targets for suppression of MTLE. We examined optogenetic manipulation of CA3 neurons in controlling the perforant pathway kindled seizures. One week after implantation of stimulating electrodes in perforant pathway, a recording electrode in CA1, and an optic ?ber in CA3, rats underwent rapid kindling procedure. A lentivector with capability to move in retrograde monosynaptic direction and to insert the gene of red light sensitive opsin Jaws in neurons was injected into CA1 of the kindled rats. One week later, the kindled rats were stimulated at after-discharge (AD) threshold under red light illumination to CA3; and duration of AD (ADD), generalized seizures (S5D), and total seizure behavior (SD) were recorded. Encoding Jaws in CA1, CA3, and entorhinal neuronal cells of the vector injected rats was veri?ed by immunohistochemistry. More than 90% of CA1, CA3, and entorhinal neurons of the counted sections expressed Jaws. Red light (625 nm) illumination to CA3 of the kindled rats expressing Jaws entirely suppressed generalized seizures and signi?cantly diminished ADD and SD. Encoding the light-sensitive chloride pump Jaws in the CA3, is an e?cient optogenetic strategy to stop perforant pathway kindled seizures.
关键词： Optogenetics,Entorhinal cortex,Scha?er collaterals,Perforant pathway kindling,CA3,Jaws
更新于2025-09-23 15:19:57
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Optogenetic control of iPS cell‐derived neurons in 2D and 3D culture systems using channelrhodopsin‐2 expressed by the SYN1 and CaMKII promoters
摘要： Development of optogenetically controllable human neural network model in three-dimensional (3D) can provide an investigative system that is physiologically relevant or mimic to the human brain. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cells (hiPSCs) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells was obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of sub-populations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for hPSCs and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.
关键词： channelrhodopsin-2 (ChR2),Optogenetics,induced pluripotent stem cell (iPSC),3D culture,calcium-calmodulin kinase II (CaMKII),synapsin-1 (SYN1),alginate hydrogel,neural tissue engineering
更新于2025-09-23 15:19:57
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Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing
摘要： From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue–green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.
关键词： near-infrared fluorescent protein,spectral multiplexing,cyanobacteriochrome,FRET biosensors,biliverdin,optogenetics
更新于2025-09-19 17:15:36
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In vivo neurovascular response to focused photoactivation of Channelrhodopsin-2
摘要： The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics’ potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed ~40% larger LFP responses (p=0.05) and twice as large cerebrovascular responses (p=0.002) under focused vs. diffuse photostimulation (focused: 1.64±0.84mV LFP amplitude and 75±48% increase in vRBC; diffuse: 1.14±0.75mV LFP amplitude and 35±23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a ~65% increase in the yield of cerebrovascular responses (73±10% for focused and 42±29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9±14.7 for focused and 10.4±1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.
关键词： Thy1-ChR2,neurovascular coupling,optogenetics,two-photon fluorescence microscopy
更新于2025-09-19 17:15:36
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Near-infrared light remotely up-regulate autophagy with spatiotemporal precision via upconversion optogenetic nanosystem
摘要： In vivo noninvasively manipulating biological functions by the mediation of biosafe near infrared (NIR) light is becoming increasingly popular. For these applications, upconversion rare-earth nanomaterial holds great promise as a novel photonic element, and has been widely adopted in optogenetics. In this article, an upconversion optogenetic nanosystem that was promised to achieve autophagy up-regulation with spatiotemporal precision was designed. The biocompatible system worked via two separated parts: blue light-receptor optogenetics-autophagy upregulation plasmids for import; and upconversion rods-encapsulated flexible capsule for converting tissue-penetrative NIR light into local visible blue light. Results validated that this system could achieve up-regulation of autophagy in vitro (in both HeLa and 293T cell lines) and remotely penetrate tissue (~3.5mm) in vivo. Since autophagy serves at a central position in intracellular signalling pathways, which is correlative with diverse pathologies, we expect that this method could establish an upconversion material-based autophagy up-regulation strategy for fundamental and clinical applications.
关键词： optogenetics,autophagy,upconversion materials,protein-protein interaction (PPI),near-infrared (NIR) light
更新于2025-09-19 17:15:36
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[Methods in Molecular Biology] Vertebrate Embryogenesis Volume 1920 (Embryological, Cellular, and Genetic Methods) || Optogenetic Control of Subcellular Protein Location and Signaling in Vertebrate Embryos
摘要： This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an “anchor” protein and a “bait” protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117–126, 2016) and describe the imaging parameters used to achieve subcellular light patterning.
关键词： Polarity,Heterodimerization,Signaling,Phytochrome,Optogenetics,Zebrafish
更新于2025-09-19 17:15:36