Natural Photoreceptors Research Articles

Perovskite Quantum Dot-Based Photovoltaic Biointerface for Photostimulation of Neurons.

A promising avenue for vision restoration against retinal degeneration is the use of semiconductor-based photovoltaic biointerfaces to substitute natural photoreceptors. Instead of silicon, perovskite has emerged as an exciting material for solar energy harvesting, and its nanocrystalline forms generally offer better stability than their bulk counterparts in addition to the distinct synthesis and fabrication steps. Herein, we synthesize tetramethylammonium lead iodide (TMAPbI3) perovskite quantum dots (QDs) as a novel photoactive material for photovoltaic biointerfaces. While the TMAPbI3 quantum dots and electrolyte interface induces Faradaic photocurrent under light illumination, the heterojunction with P3HT converts the charge-transfer process to a safe capacitive photocurrent with an improved ionic responsivity of 17.4 mA/W. The integration of the 18-nm quantum dot thickness shows good biocompatibility with primary cultures of hippocampal neurons and the photoresponse of the biointerface triggered photostimulation of the neurons. The rise of perovskite materials can stimulate novel forms of photovoltaic retina implants.

Self‐powered transparent photodetector for subretinal visual functions of wide‐field‐of‐view and broadband perception

AbstractNatural photoreceptors enable color vision in humans, wherein the eyes detect colors and their corresponding intensities via cone and rod photoreceptors, respectively. Herein, we developed an artificial broadband photoreceptor with light‐color intensity detection similar to that of natural photoreceptors. The developed photoreceptor operates in the self‐powered mode and is capable of broadband perception (365–940 nm). The designed metal‐oxide heterojunction (n‐ZnO/p‐NiO) photoreceptor with a thin tin sulfide layer embedded in between is capable of perceiving various colors. It exhibits good transparency in the visible range and displays excellent integration with flexible substrates, highlighting its potential for use in flexible electronics. The fabricated structure has an exceptional response time (≈1 ms) and a wide‐field‐of‐view (150°) compared to the human eye's sensing range (50–100 ms and 108°). The transparent photoreceptor mimics cones and rods to detect a various wavelength‐dependent signals and explicitly differentiate between the intensities of the detected signals, respectively. This is further illustrated by employing the developed photoreceptor to detect colors in real time by generating unique signals corresponding to each color. The demonstration provides the proof of concept for self‐biased flexible bioelectronics emulating high‐performing visual functions of artificial eyes.image

Light controlled signaling initiated by subretinal semiconducting-polymer layer in developing-blind-retina mimics the response of the neonatal retina

Optoelectronic semiconducting polymer material interfaced with a blind-developing chick-retina (E13–E18) in subretinal configuration reveals a response to full-field flash stimulus that resembles an elicited response from natural photoreceptors in a neonatal chick retina. The response manifests as evoked-firing of action potentials and was recorded using a multi-electrode array in contact with the retinal ganglion layer. Characteristics of increasing features in the signal unfold during different retina-development stages and highlight the emerging network mediated pathways typically present in the vision process of the artificial photoreceptor interfaced retina.

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance.

Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

A guide to the optogenetic regulation of endogenous molecules.

Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.

Enlightening Allostery: Designing Switchable Proteins by Photoreceptor Fusion.

Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-component optogenetic systems with fast on- and off-switching kinetics. However, designing protein-photoreceptor chimeras, in which structural changes of the photoreceptor are effectively propagated to the fused effector protein, is a challenging engineering problem and often relies on trial and error. Here, recent advances in the design and application of optogenetic allosteric switches are reviewed. First, an overview of existing optogenetic tools based on inducible allostery is provided and their utility for cell biology applications is highlighted. Focusing on light-oxygen-voltage domains, a widely applied class of small blue light sensors, the available strategies for engineering light-dependent allostery are presented and their individual advantages and limitations are highlighted. Finally, high-throughput screening technologies based on comprehensive insertion libraries, which could accelerate the creation of stimulus-responsive receptor-protein chimeras for use in optogenetics and beyond, are discussed.

Lichtsignale für die Hefe

Natural photoreceptors from plants and microorganisms are used for synthetic approaches to control cell behaviour. Light perception by the photoreceptor, often by a cofactor, induces a conformational change, which is transduced to the effector and regulates its activity. Synthetic combinations of photoreceptors and effectors resulted in a wealth of cellular events that are controlled by optogenetic tools. A general approach is to regulate protein abundance controlling either protein stability, protein biosynthesis or both with optogenetic tools.

Generic and reversible opto-trapping of biomolecules

Molecular traps can control activity and abundance of many biological factors. Here, we report the development of a generic opto-trap to reversibly bind and release biomolecules with high spatiotemporal control by illumination with non-invasive and cell-compatible red and far-red light. We use the Arapidopsis thaliana photoreceptor phytochrome B to regulate the release of diverse proteins from a variety of material scaffolds. Fusion of a short 100 amino acids “PIF-tag”, derived from the phytochrome interacting factor 6, renders arbitrary molecules opto-trap-compatible. Reversible opto-trapping of target molecules enables novel possibilities for future developments in diagnostics, therapeutics, and basic research. Statement of SignificanceThe investigation of cellular signaling events or the development of complex therapeutics and integrative diagnostic devices requires the deliberate control of biomolecule abundance and activity. During recent years, the use of natural photoreceptors within cells leveraged the control of diverse cellular events, benefiting from the superior spatial and temporal control characteristics of light as compared to conventional chemical stimuli. Concurrently, biological switches entailing intrinsic compatibility toward biological environments increasingly found application in biohybrid materials. We employ the plant red/far-red photoreceptor phytochrome B, which reversibly interacts with its phytochrome interacting factors (PIFs), for developing a generic opto-trap. This platform allows the use of red and far-red light to spatiotemporally control binding and release of arbitrary PIF-fused biomolecules from various material scaffolds.

Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Blue-Light Receptors for Optogenetics.

Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Controlling Biological Pathways with Photopharmacology

Light can be used to control biological events with unmatched temporal and spatial precision. A case in point is optogenetics, which relies on natural photoreceptors that employ retinal, flavins or tetrapyrrols as chromophores. Recently, the incorporation of synthetic photoswitches, such as azobenzenes, into naturally “blind” native receptors has been explored as well. These molecules can bind covalently or non-covalently to a wide variety of proteins, including ion channels, GPCRs, enzymes, molecular motors, and components of the cytoskeleton, effectively turning them into photoreceptors. As such, photoswitchable molecules add another dimension to pharmacology. I will discuss the advantages and disadvantages of photopharmacology and its potential in biology and medicine, in particular with respect to restoring vision and fighting cancer.

Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence.

The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics.

Guidelines for Photoreceptor Engineering.

Sensory photoreceptors underpin optogenetics by mediating the noninvasive and reversible perturbation of living cells by light with unprecedented temporal and spatial resolution. Spurred by seminal optogenetic applications of natural photoreceptors, the engineering of photoreceptors has recently garnered wide interest and has led to the construction of a broad palette of novel light-regulated actuators. Photoreceptors are modularly built of photosensors that receive light signals, and of effectors that carry out specific cellular functions. These modules have to be precisely connected to allow efficient communication, such that light stimuli are relayed from photosensor to effector. The engineering of photoreceptors benefits from a thorough understanding of the underlying signaling mechanisms. This chapter gives a brief overview of key characteristics and signal-transduction mechanisms of sensory photoreceptors. Adaptation of these concepts in photoreceptor engineering has enabled the generation of novel optogenetic tools that greatly transcend the repertoire of natural photoreceptors.

Orthogonal Optical Control of a G Protein-Coupled Receptor with a SNAP-Tethered Photochromic Ligand.

The covalent attachment of synthetic photoswitches is a general approach to impart light sensitivity onto native receptors. It mimics the logic of natural photoreceptors and significantly expands the reach of optogenetics. Here we describe a novel photoswitch design—the photoswitchable orthogonal remotely tethered ligand (PORTL)—that combines the genetically encoded SNAP-tag with photochromic ligands connected to a benzylguanine via a long flexible linker. We use the method to convert the G protein-coupled receptor mGluR2, a metabotropic glutamate receptor, into a photoreceptor (SNAG-mGluR2) that provides efficient optical control over the neuronal functions of mGluR2: presynaptic inhibition and control of excitability. The PORTL approach enables multiplexed optical control of different native receptors using distinct bioconjugation methods. It should be broadly applicable since SNAP-tags have proven to be reliable, many SNAP-tagged receptors are already available, and photochromic ligands on a long leash are readily designed and synthesized.

Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools.

Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.

Shedding New Light on Retinal Protein Photochemistry

The ultrafast spectroscopic investigation of novel retinal proteins challenges existing notions concerning the course of primary events in these natural photoreceptors. We review two illustrations here. The first demonstrates that changes in the initial retinal configuration can alter the duration of photochemistry by nearly an order of magnitude in Anabaena sensory rhodopsin, making it as rapid as the ballistic photoisomerization in visual pigments. This prompted a reinvestigation of the much studied bacteriorhodopsin, leading to a similar trend as well, contrary to earlier reports. The second involves the study of xanthorhodopsin, an archaeal proton pump that includes an attached light-harvesting carotenoid. Pump-probe experiments demonstrate the efficient transfer of energy from carotenoid to retinal, providing a first glimpse at a cooperative multichromophore function, which is probably characteristic of many other proteins as well. Finally, we discuss measures required to advance our knowledge from kinetics to mode-specific dynamics concerning this expanding family of biological photoreceptors.

Optogenetic control of cell function using engineered photoreceptors

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.

Biophysical Studies of Natural Photoreceptors: Application to Optogenetic Tool Optimization

Many biological processes are regulated by environmental control of protein conformation. This is commonly achieved by sensory protein domains that bind both protein targets and environmentally-sensitive cofactors, using these compounds to trigger allosteric changes in the sensory domain. These general principles are exemplified by photosensory proteins, several of which have been amenable to biophysical study and subsequent use in applied settings.

Single-Pixel, Single-Layer Polymer Device as a Tricolor Sensor with Signals Mimicking Natural Photoreceptors

Color sensing procedures typically involve multiple active detectors or a photodetector coupled to a filter array. We demonstrate the possibility of using a single polymer layer based device structure for multicolor sensing. The device structure does not require any color filters or any subpixelation, and it distinguishes colors without any external bias. The color sensing relies on an appropriate thickness of the active polymer layer that results in a characteristic polarity and temporal profile of the photocurrent signal in response to various incident colors. The device characteristics reveal interesting similarities to the features observed in natural photosensitive systems including retinal cone cells.

Does calcium diffusional global feedback leads to slow light adaptation in Drosophila photoreceptors? - A 3D biophysical modelling approach

Drosophila photoreceptors transduce light signals to voltage responses. In natural environment, light intensity can change over 10 log range, whereas the coding range of photoreceptors is only about 50 mV. Thus, to reliably represent natural light changes photoreceptors rely upon powerful adaptation. How do different molecular mechanisms of phototransduction enable light adaptation? Insect photoreceptors are highly polarized, containing light-sensitive and light-insensitive parts of separate functions. The light-sensitive rhabdomere contains a matrix of 30,000 independent phototransduction units (microvilli), which capture and covert light energy into bursts of currents, fluxing through TRP/TRPL channels inside the microvilli. The light insensitive cell-body, which contains voltage-gated channels, then shapes up the resulting voltage responses. Recently, we produced a two-compartmental biophysical model of a Drosophila photoreceptor [1]. Using this model we showed that the dynamic ratio of the used and unused microvilli (ultrastucture), their stochastic reactions and calcium feedbacks cause fast adaptation of the responses, while a global feedback though the membrane voltage compresses the responses to their limited size. However, this model fails to capture slow adaptive changes in the data, which could be important for adjusting the photoreceptor dynamics to changing image statistics in a more Bayesian way. As a first step, we wish to test whether calcium could diffuse from the responding microvillus to its neighbours, affecting the speed and sensitivity of their separate reaction cascades. We call this hypothetical cross-talk between microvilli global-diffusional-calcium feedback, and ask if it could induce the slow adaptation. To investigate this question, we extend our biophysical model of Drosophila photoreceptor to a 3D-reaction-diffusion model; taking into account the 3D diffusion of calcium. Here, the signalling molecules are loaded in a reconstructed macro-cell structure, described by a 3D tetrahedral mesh. The reaction pathways are simulated using STEPS (STochastic Engine for Pathway Simulation), based on Gillespie’s Stochastic Simulation Algorithm, extended for diffusion.