Literatura científica selecionada sobre o tema "Fluorescent opsins"

Vertebrate color vision requires spectrally selective opsin-based pigments, expressed in distinct cone photoreceptor populations. In primates and in fish, spectrally divergent opsin genes may reside in head-to-tail tandem arrays. Mechanisms underlying differential expression from such arrays have not been fully elucidated. Regulation of human red (LWS) vs. green (MWS) opsins is considered a stochastic event, whereby upstream enhancers associate randomly with promoters of the proximal or distal gene, and one of these associations becomes permanent. We demonstrate that, distinct from this stochastic model, the endocrine signal thyroid hormone (TH) regulates differential expression of the orthologous zebrafish lws1/lws2 array, and of the tandemly quadruplicated rh2-1/rh2-2/rh2-3/rh2-4 array. TH treatment caused dramatic, dose-dependent increases in abundance of lws1, the proximal member of the lws array, and reduced lws2. Fluorescent lws reporters permitted direct visualization of individual cones switching expression from lws2 to lws1. Athyroidism increased lws2 and reduced lws1, except within a small ventral domain of lws1 that was likely sustained by retinoic acid signaling. Changes in lws abundance and distribution in athyroid zebrafish were rescued by TH, demonstrating plasticity of cone phenotype in response to this signal. TH manipulations also regulated the rh2 array, with athyroidism reducing abundance of distal members. Interestingly, the opsins encoded by the proximal lws gene and distal rh2 genes are sensitive to longer wavelengths than other members of their respective arrays; therefore, endogenous TH acts upon each opsin array to shift overall spectral sensitivity toward longer wavelengths, underlying coordinated changes in visual system function during development and growth.

The goal of this study was to determine whether the jellyfish green fluorescent protein (GFP) could be used in transgenic mice to label and purify cone photoreceptors from the living retina. We created a transgene containing the 5′ regulatory sequence of the human red pigment gene (pR6.5 lacZ clone; kindly provided by J. Nathans & Y. Wang), fused to the GFP coding sequence. This transgene was used to generate seven lines of PCR-positive founders. Three of the lines had bright green fluorescent cone photoreceptors. The GFP fills the entire cell. Two mouse lines had only a few (∼10–100) fluorescent cells per retina, and one line (R6.85933) had many thousands. In the latter, double labeling of the cones with RITC-conjugated peanut agglutinin reveals that in the ventral retina a small proportion of the cones express GFP, while in the dorsal retina the majority do. Cells dissociated from the retinae of line R6.85933 continue to fluoresce and can be readily detected and enriched with flow cytometry. The signal provides a log unit of separation between the fluorescent cone soma and the remaining retinal cells. Roughly 3% of the cells are this fluorescent, and it is possible to purify up to 30,000 cells from one mouse. RT-PCR analysis of the mRNA from these isolated cells detects both the middle and short wavelength opsins with little if any contamination from rhodopsin.

Abstract The progress in optogenetics largely depends on the development of light-activated proteins as new molecular tools. Using cultured hippocampal neurons, we compared the properties of two light-activated cation channels – classical channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) and recently described channelrhodopsin isolated from the alga Platymonas subcordiformis (PsChR2). PsChR2 ensured generation of action potentials by neurons when activated by the pulsed light stimulation with the frequencies up to 40-50 Hz, while the upper limit for CrChR2 was 20-30 Hz. An important advantage of PsChR2 compared to classical channelrhodopsin CrChR2 is the blue shift of its excitation spectrum, which opens the possibility for its application in all-optical electrophysiology experiments that require the separation of the maxima of the spectra of channelrhodopsins used for the stimulation of neurons and the maxima of the excitation spectra of various red fluorescent probes. We compared the response (generation of action potentials) of neurons expressing CrChR2 and PsChR2 to light stimuli at 530 and 550 nm commonly used for the excitation of red fluorescent probes. The 530-nm light was significantly (3.7 times) less efficient in the activation of neurons expressing PsChR2 vs. CrChR2-expressing neurons. The light at 550 nm, even at the maximal used intensity, failed to stimulate neurons expressing either of the studied opsins. This indicates that the PsChR2 channelrhodopsin from the alga P. subcordiformis is a promising optogenetic tool, both in terms of its frequency characteristics and possibility of its application for neuronal stimulation with a short-wavelength (blue, 470 nm) light accompanied by simultaneous recording of various physiological processes using fluorescent probes.

Mutations in rod opsin—the light-sensitive protein of rod cells—cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT–green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP–expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.

Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11–14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the locus control region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses.

We have examined the binding of anti-opsin antibodies to the plasma membrane of frog retinal rod outer segments (ROS) by fluorescence light microscopy and electron microscopy. Polyclonal and monoclonal antibodies specific for the N-terminal domain of opsin were observed to bind to the extracellular surface of ROS plasma membrane of aldehyde-fixed but not of unfixed retinas. This reaction was found regardless of whether purified ROS, rhodopsin, opsin, or an N-terminal peptide of opsin was used as the immunogen. The fixation-induced binding of these antibodies contrasts with the more frequently noted loss of antigenicity upon fixation. Concanavalin A, however, binds to unfixed ROS plasma membranes. Its binding sites in the plasma membrane may be oligosaccharides in the N-terminal region of opsin. These results suggest that the N-terminal domain of opsin is latent in the native membrane and that changes in conformation may account for its detectability in fixed membranes.

Abstract Channelrhodopsin-2 is a light-gated cation channel from the green alga Chlamydomonas reinhardtii. It is functional in animal cells and therefore widely used for light-activated depolarization, especially in neurons. To achieve a fully functional protein, the chromophore all-trans-retinal is needed. It has not been investigated whether or not the apoprotein is stable without its cofactor until now. Here we show that channelopsin-2 (Chop2, protein without bound retinal) is much more prone to degradation than channelrhodopsin-2 (protein with retinal). Constructs of Chop2 fused to yellow fluorescent protein (Chop2::YFP) in the absence and presence of retinal confirm this observation by exhibiting strongly differing fluorescence. We present mutants of Chop2 with highly increased stability in the absence of retinal. Substitution of threonine 159 with aromatic amino acids causes enhanced resistance to degradation in the absence of retinal, which is confirmed by fluorescence intensity, the increase in photocurrents on the addition of retinal to previously expressed protein, and Western blot analysis. Exchanging threonine 159 with cysteine, however, increases photocurrents due to better binding of retinal, without obvious stabilization against degradation of the retinal-free opsin. We also show that the light-activated hyperpolarizing chloride pump halorhodopsin from Natronomonas pharaonis (NpHR) is not prone to retinal-dependent degradation.

Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45–50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

In recent years, world economy and technological advancement have been transformed by Genomics, which allows us to study, design and build biologically relevant molecules. Genomics is already deeply embedded in industries as diverse as pharmaceutical, food and agricultural, environmental and bio-tech in general. Fast and cheap tools for gene sequencing, protein expression and analysis are commonly used for high-throughput genomic-related studies. However, due to experimental difficulties and long time scales (e.g., protein crystallization), protein structure determination, and thus the fundamental structure function rationalization, cannot presently be performed at the same fast pace: a fact that is slowing down the discovery of proteins with new features, as well as ex novo design. These difficulties are particularly felt in the field of photobiology, where the crystal structure of Bovine rhodopsin (Rh, retina dim-light visual photo-receptor), still remains the only structure of a vertebrate photo-receptor sensor available for photobiological studies since the year 2000. Rhodopsins constitute a class of light-triggered proteins that can be found throughout the whole spectrum of living organisms, and represent the perfect blue-print for building light-activated bio-molecular machines. In principle, the problem of not having a sufficient number of rhodopsins molecular structures could be circumvented and overcome with the construction of accurate atomistic computer models of the set of studied photoreceptors, which would allow: (i) in silico fundamental structure-function characterization, (ii) thorough and detailed screening of mutant series, and even (iii) ex novo design. Nevertheless, such models should also be constructed using a fast, relatively cheap, reliable and standardized protocol, of known accuracy. In this thesis, we refine and test the Automatic Rhodopsin Modeling (ARM) computational protocol, which we demonstrate as being capable of helping to address the above issues. Such protocol has the primary target of generating congruous quantum mechanical/molecular mechanical (QM/MM) models of rhodopsins, with the aim of facilitating systematic rhodopsin-mutants studies. The cornerstone of this thesis is the validation of the ARM protocol as a successful attempt to provide a basis for the standardization and reproducibility of rhodopsin QM/MM models, aimed to study the behaviour of photoactive molecules. First, we validate the ARM protocol, which employs a CASPT2//CASSCF/AMBER scheme, for a benchmark set of rhodopsins from different biological kingdoms. We show that ARM is able to reproduce and predict absorption trends in rhodopsin protein sets, with blue-shifted values not much displaced (a few kcal/mol) from the observed data. Secondly, we present how to use this protocol towards a better design of novel mutations as applications for Optogenetics, an innovative biological tool aimed to visualize and control neuron signals through light. Two different microbial rhodopsins are studied: Krokinobacter eikastus rhodopsin 2 (KR2), a light-driven outward sodium pump, and Anabaena sensory rhodopsin (ASR), a light sensor. In both cases, the qualitative and quantitative information acquired from the ARM-obtained QM/MM models reveal nature (electrostatic or steric) and extent of the mutation-induced changes on the retinal configuration, which, in turn, are the cause of the shift in the absorption wavelength of the relative mutants. Finally, we explore the fluorescence of ASR mutants, particularly useful for the visualization of neuronal activity. The target of this work is to use QM/MM simulations to understand the opposite behaviour observed in two blue-shifted ASR mutants, where one presents a negligible fluorescence, while the other displays one order of magnitude enhanced fluorescence, with respect to the wild type protein. Our QM/MM models show that specific electrostatic and steric interactions control the character mixing of different electronic states, opening a path to the rational engineering of highly fluorescent rhodopsins. In conclusion, within the limits of its automation, the ARM protocol allows the study of ground and excited states of specific photoactive proteins: rhodopsins. This opens the way to an improved molecular-level understanding of rhodopsin photochemistry and photobiology. The results obtained highlight the importance of having a standardized, effective and automatic protocol, which renders this kind of studies more efficient and accessible, by drastically shortening the time required to produce accurate and congruous QM/MM models. For the above reasons the author of the present thesis believes that ARM stands as an important cogwheel in the virtuous cycle between experimental and theoretical work, aimed to prepare the photobiological tools for tomorrow’s needs.

AbstractAll-optical physiology of neuronal microcircuits requires the integration of optogenetic perturbation and optical imaging, efficient opsin and indicator co-expression, and tailored illumination schemes. It furthermore demands concepts for system integration and a dedicated analysis pipeline for calcium transients in an event-related manner. Here, firstly, we put forward a framework for the specific requirements for technical system integration particularly focusing on temporal precision. Secondly, we devise a step-by-step guide for the image analysis in the context of an all-optical physiology experiment. Starting with the raw image, we present concepts for artifact avoidance, the extraction of fluorescence intensity traces on single-neuron basis, the identification and binarization of putatively action-potential-related calcium transients, and finally ensemble activity analysis.