Curated Optogenetic Publication Database

Abstract: Dominant gain-of-function alleles of Arabidopsis phytochrome B were recently shown to confer light-independent, constitutive photomorphogenic (cop) phenotypes to transgenic plants (Su and Lagarias, 2007). In the present study, comparative transcription profiling experiments were performed to assess whether the pattern of gene expression regulated by these alleles accurately reflects the process of photomorphogenesis in wild-type Arabidopsis. Whole-genome transcription profiles of dark-grown phyAphyB seedlings expressing the Y276H mutant of phyB (YHB) revealed that YHB reprograms about 13% of the Arabidopsis transcriptome in a light-independent manner. The YHB-regulated transcriptome proved qualitatively similar to but quantitatively greater than those of wild-type seedlings grown under 15 or 50 micromol m(-2) m(-1) continuous red light (Rc). Among the 2977 genes statistically significant two-fold (SSTF) regulated by YHB in the absence of light include those encoding components of the photosynthetic apparatus, tetrapyrrole/pigment biosynthetic pathways, and early light-responsive signaling factors. Approximately 80% of genes SSTF regulated by Rc were also YHB-regulated. Expression of a notable subset of 346 YHB-regulated genes proved to be strongly attenuated by Rc, indicating compensating regulation by phyC-E and/or other Rc-dependent processes. Since the majority of these 346 genes are regulated by the circadian clock, these results suggest that phyA- and phyB-independent light signaling pathway(s) strongly influence clock output. Together with the unique plastid morphology of dark-grown YHB seedlings, these analyses indicate that the YHB mutant induces constitutive photomorphogenesis via faithful reconstruction of phyB signaling pathways in a light-independent fashion.

Abstract: An important contributing factor to the success of terrestrial flowering plants in colonizing the land was the evolution of a developmental strategy, termed skotomorphogenesis, whereby postgerminative seedlings emerging from buried seed grow vigorously upward in the subterranean darkness toward the soil surface.

Abstract: To control many physiological responses, phytochromes directly modulate gene expression. A key regulatory event in this signal transduction pathway is the light-controlled translocation of the photoreceptor from the cytoplasm into the nucleus. Recent publications are beginning to shed light on the molecular mechanisms underlying this central control point. Interestingly, there is a specific mechanism for phytochrome A (phyA) nuclear accumulation. The dedicated phyA nuclear import pathway might be important for the distinct photosensory specificity of this atypical phytochrome. Recent studies in the field also provide a starting point for investigating how the different subcellular pools of phytochrome can control distinct responses to light.

Abstract: General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs.

Abstract: Cyanobacteriochromes are a newly recognized group of photoreceptors that are distinct relatives of phytochromes but are found only in cyanobacteria. A putative cyanobacteriochrome, CcaS, is known to chromatically regulate the expression of the phycobilisome linker gene (cpcG2) in Synechocystis sp. PCC 6803. In this study, we isolated the chromophore-binding domain of CcaS from Synechocystis as well as from phycocyanobilin-producing Escherichia coli. Both preparations showed the same reversible photoconversion between a green-absorbing form (Pg, lambda(max) = 535 nm) and a red-absorbing form (Pr, lambda(max) = 672 nm). Mass spectrometry and denaturation analyses suggested that Pg and Pr bind phycocyanobilin in a double-bond configuration of C15-Z and C15-E, respectively. Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, was higher in Pr than in Pg. From these results, we conclude that CcaS phosphorylates CcaR under green light and induces expression of cpcG2, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. CcaS is the first recognized green light receptor in the expanded phytochrome superfamily, which includes phytochromes and cyanobacteriochromes.

Abstract: The spectroscopic identification of sensory rhodopsin I by Bogomolni and Spudich in 1982 provided a molecular link between the light environment and phototaxis in Halobacterium salinarum, and thus laid the foundation for the study of signal transducing photosensors in prokaryotes. In recent years, a number of new prokaryotic photosensory receptors have been discovered across a broad range of taxa, including dozens in chemotrophic species. Among these photoreceptors are new classes of rhodopsins, BLUF-domain proteins, bacteriophytochromes, cryptochromes, and LOV-family photosensors. Genetic and biochemical analyses of these receptors have demonstrated that they can regulate processes ranging from photosynthetic pigment biosynthesis to virulence.

Abstract: Spatiotemporal regulation of protein function is a key feature of living systems; experimental tools that provide such control are of great utility. Here we report a genetically encoded system for controlling a post-translational process, protein splicing, with light. Studies in Saccharomyces cerevisiae demonstrate that fusion of a photodimerization system from Arabidopsis thaliana to an artificially split intein permits rapid activation of protein splicing to yield a new protein product.

Abstract: In the purple photosynthetic bacterium Rhodopseudomonas palustris, far-red illumination induces photosystem synthesis via the action of the bacteriophytochrome RpBphP1. This bacteriophytochrome antagonizes the repressive effect of the transcriptional regulator PpsR2 under aerobic condition. We show here that, in addition to photosystem synthesis, far-red light induces a significant growth rate limitation, compared to cells grown in the dark, linked to a decrease in the respiratory activity. The phenotypes of mutants inactivated in RpBphP1 and PpsR2 show their involvement in this regulation. Based on enzymatic and transcriptional studies, a 30% decrease in the expression of the alpha-ketoglutarate dehydrogenase complex, a central enzyme of the Krebs cycle, is observed under far-red light. We propose that this decrease is responsible for the down-regulation of respiration in this condition. This regulation mechanism at the Krebs cycle level still allows the formation of the photosynthetic apparatus via the synthesis of key biosynthesis precursors but lowers the production of NADH, i.e. the respiratory activity. Overall, the dual action of RpBphP1 on the regulation of both the photosynthesis genes and the Krebs cycle allows a fine adaptation of bacteria to environmental conditions by enhancement of the most favorable bioenergetic process in the light, photosynthesis versus respiration.

Abstract: Bacteriophytochromes are bacterial photoreceptors that sense red/far red light using the biliverdin chromophore. Most bacteriophytochromes work as photoactivated protein kinases. The Rhodobacter sphaeroides bacteriophytochrome BphG1 is unconventional in that it has GGDEF and EAL output domains, which are involved, respectively, in synthesis (diguanylate cyclase) and degradation (phosphodiesterase) of the bacterial second messenger c-di-GMP. The GGDEF-EAL proteins studied to date displayed either diguanylate cyclase or phosphodiesterase activity but not both. To elucidate the function of BphG1, the holoprotein was purified from an Escherichia coli overexpression system designed to produce biliverdin. The holoprotein contained covalently bound biliverdin and interconverted between the red (dark) and far red (light-activated) forms. BphG1 had c-di-GMP-specific phosphodiesterase activity. Unexpectedly for a photochromic protein, this activity was essentially light-independent. BphG1 expressed in E. coli was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain of BphG1. It possessed c-di-GMP phosphodiesterase activity. Surprisingly, the larger species lacking EAL possessed diguanylate cyclase activity, which was dependent on biliverdin and strongly activated by light. BphG1 therefore is the first phytochrome with a non-kinase photoactivated enzymatic activity. This shows that the photosensory modules of phytochromes can transmit light signals to various outputs. BphG1 is potentially the first "bifunctional" enzyme capable of both c-di-GMP synthesis and hydrolysis. A model for the regulation of the "opposite" activities of BphG1 is presented.

Abstract: We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.

Abstract: A novel FAD-binding domain, BLUF, exemplified by the N-terminus of the AppA protein from Rhodobacter sphaeroides, is present in various proteins, primarily from Bacteria. The BLUF domain is involved in sensing blue-light (and possibly redox) using FAD and is similar to the flavin-binding PAS domains and cryptochromes. The predicted secondary structure reveals that the BLUF domain is a novel FAD-binding fold.

Abstract: Regulatable transgene systems providing easily controlled, conditional induction or repression of expression are indispensable tools in biomedical and agricultural research and biotechnology. Several such systems have been developed for eukaryotes. Most of these rely on the administration of either exogenous chemicals or heat shock. Despite the general success of many of these systems, the potential for problems, such as toxic, unintended, or pleiotropic effects of the inducing chemical or treatment, can impose limitations on their use. We have developed a promoter system that can be induced, rapidly and reversibly, by short pulses of light. This system is based on the known red light-induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light. We show here that yeast cells expressing two chimeric proteins, a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion, are induced by red light to express selectable or "scorable" marker genes containing promoters with a GAL4 DNA-binding site, and that this induction is rapidly abrogated by subsequent far-red light. We further show that the extent of induction can be controlled precisely by titration of the number of photons delivered to the cells by the light pulse. Thus, this system has the potential to provide rapid, noninvasive, switchable control of the expression of a desired gene to a preselected level in any suitable cell by simple exposure to a light signal.

Abstract: The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix-loop-helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.

Abstract: The phytochrome photoreceptor family directs plant gene expression by switching between biologically inactive and active conformers in response to the sequential absorption of red and farred photons. Several intermediates that act late in the phytochrome signalling pathway have been identified, but fewer have been identified that act early in the pathway. We have cloned a nuclear basic helix-loop-helix protein, PIF3, which can bind to non-photoactive carboxy-terminal fragments of phytochromes A and B and functions in phytochrome signalling in vivo. Here we show that full-length photoactive phytochrome B binds PIF3 in vitro only upon light-induced conversion to its active form, and that photoconversion back to its inactive form causes dissociation from PIF3. We conclude that photosensory signalling by phytochrome B involves light-induced, conformer-specific recognition of the putative transcriptional regulator PIF3, providing a potential mechanism for direct photoregulation of gene expression.

Abstract: Divalent cations induce the aggregation of chromaffin granule ghosts (CG membranes) at millimolar concentrations. Monovalent cations produce the same effect at 100-fold higher concentrations. The kinetics of the dimerization phase were followed by light-scattering changes observed in stopped-flow rapid mixing experiments. The rate constant for Ca2+-induced dimerization (kapp) is 0.86-1.0 x 10(9) M-1sec-1, based on the "molar" vesicle concentration. This value is close to the values predicted by theory for the case of diffusion-controlled reaction (7.02 x 10(9) M-1sec-1), indicating that there is no energy barrier to dimerization. Arrhenius plots between 10 degrees and 42 degrees C support this; the activation energy observed, +4.4 Kcal, is close to the value (4.6-4.8 Kcal) predicted for diffusion control according to theory. Artificial vesicles prepared from CG lipids were also found to have cation-induced aggregation, but the rates (values of kapp) were less than 1/100 as large as those with native CG membranes. Also, significant differences were found with respect to cation specificity. It is concluded that the slow rates are due to the low probability that the segments of membrane which approach will be matched in polar head group composition and disposition. Thus large numbers of approaches are necessary before matched segments come into aposition. The salient features of the chromaffin granule membrane aggregation mechanism are as follows: (a) In the absence of cations capable of shielding and binding, the membranes are held apart by electrostatic repulsion of their negatively charged surfaces. (b) The divalent and monovalent cation effects on aggregation are due to their ability to shield these charges, allowing a closer approach of the membrane surfaces. (c) The major determinants of the aggregation rates of CG membranes are proteins which protrude from the (phospholipid) surface of the membrane and serve as points of primary contact. Transmembrane contact between these proteins does not require full neutralization of the surface charge and surface potential arising from the negatively charged phospholipids. (d) After contact between proteins is established, the interaction between membranes can be strengthened through transmembrane hydrogen bonding of phosphatidyl ethanolamine polar head groups, divalent cation-mediated salt bridging, and segregation of phosphatidylcholine out of the region of contact.

Abstract: Freeze-fracture electron microscopy demonstrates that in photosynthetic membranes of the blue-green alga Anacystis nidulans quenched from a temperature below growth temperature, areas devoid of membrane particles occur. We suggest that this phenomenon is related to phase transitions in the photosynthetic membrane.

Abstract: To evaluate responses to medical therapy in ulcerative colitis, rectal biopsies of patients with active untreated disease, individuals with positive and negative sigmoidoscopic findings treated with salicylazosulfapyridine, prednisone and 6-mercaptopurine, alone and in combinations and noncolitis controls were compared histologically. Predominant histological observations were analyzed statistically. There were fewer crypt abscesses but more mucosal edema after all forms of therapy. Quantitative histopathological analysis failed to demonstrate that the response to one drug was significantly different from another.

Abstract: The distribution of blood flow to the subendocardial, medium and subepicardial layers of the left ventricular free wall was studied in anaesthetized dogs under normoxic (A), hypoxic (B) conditions and under pharmacologically induced (etafenone) coronary vasodilation (C). Regional myocardial blood flow was determined by means of the particle distribution method. In normoxia a transmural gradient of flow was observed, with the subendocardial layers receiving a significantly higher flow rate compared with the subepicardial layers. In hypoxia induced vasodilation this transmural gradient of flow was persistent. In contrast a marked redistribution of regional flow was observed under pharmacologically induced vasodilation. The transmural gradient decreased. In contrast to some findings these experiments demonstrate that a considerable vasodilatory capacity exists in all layers of the myocardium and can be utilized by drugs. The differences observed for the intramural distribution pattern of flow under hypoxia and drug induced vasodilation support the hypothesis that this pattern reflects corresponding gradients of regional myocardial metabolism.

Abstract: Abstract not available.

Abstract: Fifteen normal volunteers without symptoms of gastroesophageal reflux and sixteen patients with symptoms of gastroesophageal reflux unresponsive to medical management and having endoscopic esophagitis had esophageal manometry and twenty-four hour pH monitoring of the distal esophagus. The symptomatic patients underwent a Nissen antireflux procedure and were restudied at four months. After surgery, patients had less reflux, a higher sphincteric pressure, and an equal amount of sphincter within the abdomen as did asymptomatic control subjects.