Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Physiological basis of phototaxis to near-infrared light in Nephotettix cincticeps.

In a previous study of the phototaxis of green rice leafhoppers, Nephotettix cincticeps (Hemiptera, Cicadellidae), we found positive responses to 735 nm light. Here, we investigated the mechanism underlying this sensitivity to near-infrared light. We first measured the action spectrum using a Y-maze with monochromatic lights from 480 to 740 nm. We thus found that the action spectrum peaks at 520 nm in the tested wavelength range, but that a significant effect is still observed at 740 nm, albeit with a sensitivity 5 log units lower than the peak. Second, we measured the spectral sensitivity of the eye, and found that the sensitivity in the long-wavelength region parallels the behaviorally determined action spectrum. We further identified mRNAs encoding opsins of ultraviolet, blue, and green-absorbing visual pigments, and localized the mRNAs in the ommatidia by in situ hybridization. The electrophysiology, molecular biology and the anatomy of the eye together indicate that the eyes of N. cincticeps do not contain true "red" receptors, but rather that the behavioral response to near-infrared light is mediated by the tail sensitivity of the green receptors in the long-wavelength region of the spectrum.

Identification of 4-hydroxy-2-nonenal-histidine adducts that serve as ligands for human lectin-like oxidized LDL receptor-1.

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.

How 5000 independent rowers coordinate their strokes in order to row into the sunlight: phototaxis in the multicellular green alga Volvox.

BACKGROUND: The evolution of multicellular motile organisms from unicellular ancestors required the utilization of previously evolved tactic behavior in a multicellular context. Volvocine green algae are uniquely suited for studying tactic responses during the transition to multicellularity because they range in complexity from unicellular to multicellular genera. Phototactic responses are essential for these flagellates because they need to orientate themselves to receive sufficient light for photosynthesis, but how does a multicellular organism accomplish phototaxis without any known direct communication among cells? Several aspects of the photoresponse have previously been analyzed in volvocine algae, particularly in the unicellular alga Chlamydomonas. RESULTS: In this study, the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta) was analyzed. In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. Moreover, positive phototaxis is facilitated by gravitational forces. Under our conditions, V. rousseletii spheroids showed no negative phototaxis. CONCLUSIONS: On the basis of our results, we developed a mechanistic model that predicts the phototactic behavior in V. rousseletii. The model involves photoresponses, periodically changing light conditions, morphological polarity, rotation of the spheroid, two modes of flagellar beating, and the impact of gravity. Our results also indicate how recently evolved multicellular organisms adapted the phototactic capabilities of their unicellular ancestors to multicellular life.

Silicone-urethane adhesive for improved coverslip mounting and leak-free preparation of living cell observation chambers.

Using a combination of silicone and urethane resin, we established a rapid technique for preparing living specimens for microscopy. One major advantage of this technique is that the coverslip is rigidly attached and does not detach during handling. As a result, it is possible to continuously observe living cells at high magnification and resolution using an oil immersion objective. Another advantage is that living cells are quickly confined to the space between the glass slide and coverslip, protecting them from environmental changes, which can cause serious effects on cell function and morphology. Moreover, high-resolution observations of real-time responses of cells are possible, using the combination of the mounting technique and a simple flow chamber.

The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata.

Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

Ca2+ efflux via plasma membrane Ca2+-ATPase mediates chemotaxis in ascidian sperm.

When a spermatozoon shows chemotactic behavior, transient [Ca2+]i increases in the spermatozoon are induced by an attractant gradient. The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa.

Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta.

We report here the whole-genome sequence of Nostoc cycadae strain WK-1, which was isolated from cyanobacterial colonies growing in the coralloid roots of the gymnosperm Cycas revoluta It can provide valuable resources to study the mutualistic relationships and the syntrophic metabolisms between the cyanobacterial symbiont and the host plant, C. revoluta.

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL.

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

Light spectrum regulates cell accumulation during daytime in the raphidophyte Chattonella antiqua causing noxious red tides.

Most marine raphidophyte species cause noxious red tides in temperate coastal areas around the world. It is known that swimming abilities enable raphidophytes to accumulation of cells and to actively acquire light at surface layers and nutrients over a wide depth range. However, it remains unclear how the swimming behavior is affected by environmental conditions, especially light condition. In the present study, we observed the accumulation of the harmful red-tide raphidophyte Chattonella antiqua under various light conditions during the daytime in the laboratory. When exposed to ultraviolet-A/blue light (320-480nm) or red light (640-680nm) from above, cells moved downward. In the case of blue light (455nm), cells started to swim downward after 5-15min of irradiation at a photon flux density≥10µmolm(-2)s(-1). When exposed to monochromatic lights (400-680nm) from the side, cells moved away from the blue light source and then descended, but just moved downward under red light. However, mixing of green/orange light (520-630nm) diminished the effects of blue light. When exposed to a mixture of 30µmolm(-2)s(-1) of blue light (440nm) and ≥6µmolm(-2)s(-1) of yellow light (560nm) from above, cells did not move downward. These results indicate that blue light induces negative phototaxis and ultraviolet-A/blue and red lights induce descending, and green/orange light cancels out their effects in C. antiqua.

Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis.

Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a unique scavenger receptor that plays important roles in atherogenesis and has been thought to function as a monomer. Using coimmunoprecipitation studies, we demonstrate that human LOX-1 (hLOX-1) forms constitutive homo-interactions in vivo. Western blot analysis of cell lysates under nonreducing or reducing conditions revealed one clear immunoreactive species corresponding to the size of a putative receptor dimer or a monomer, respectively, consistent with the presence of disulfide-linked hLOX-1 complexes. Site-directed mutagenesis studies indicated that cysteine 140 has a key role in the formation of these disulfide-linked hLOX-1 dimers. Eliminating this intermolecular disulfide bond markedly impairs the recognition of Escherichia coli by hLOX-1. Furthermore, these dimers can act as a "structural unit" to form noncovalently associated oligomers, as demonstrated by a membrane-impermeant crosslinker, which resulted in immunoreactive species corresponding to the sizes of putative tetramers and hexamers. These results provide the first evidence for the existence of hLOX-1 dimers/oligomers.

Screening effect diverts the swimming directions from diaphototactic to positive phototactic in a disk-shaped green flagellate Mesostigma viride.

We found diaphototactic behavior (i.e. the cells swim perpendicularly with respect to the incident light) in a strain with colorless eyespot of a unicellular disk-shaped green flagellate Mesostigma viride. Lacking pigments completely in the eyespot, the screening effect in this strain was due only to the central part of the chloroplast whose cross section was thin. The diaphototaxis was most obvious when unilateral green stimulus light (520-580 nm) was given, whereas positive phototaxis appeared when given blue light (430-490 nm). The choice between diaphototaxis and (ordinary) phototaxis depended entirely on the transmission (%T) of the cell body against each wavelength of the stimulus: the green light penetrated well (%T > 90%), whereas the blue light was considerably shaded by the chloroplast (50% < %T < 70%). The fraction of positive phototactically behaving cells against each wavelength was in proportion to the front-to-back contrast value obtained at each individual wavelength. The fraction of diaphototaxis was inversely proportional to it. In addition, bilateral stimulus irradiations to wild-type cell with colored eyespot provided useful information about the principle of the diaphototactic steering.

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus.

Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca(2+) influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation.

Flagellar motions in phototactic steering in a brown algal swarmer.

Using infrared high-speed video microscopy, we observed light-triggered transitory flagellar motions in flagellate reproductive cells (swarmers) of a brown alga, Scytosiphon lomentaria, under primary helical swimming conditions before and during negative phototactic orientation to unilateral actinic light. The posterior flagellum, which is autofluorescent and thought to be light-sensing, was passively dragged in the dark and exhibited one to several rapid lateral beats during orientation changes for phototactic steering. Notably, a brief cessation of anterior flagellar beating was occasionally observed concomitantly with rapid beats of the posterior flagellum. This behavior caused a pause in helical body rotation, which may contribute to the accuracy of phototactic steering. Thus, coordinated regulation of the movement of the two flagella plays a crucial role in phototactic steering.

Complex array of endobionts in Petalomonas sphagnophila, a large heterotrophic euglenid protist from Sphagnum-dominated peatlands.

Petalomonas sphagnophila is a poorly studied plastid-lacking euglenid flagellate living in Sphagnum-dominated peatlands. Here we present a broad-ranging microscopic, molecular and microspectrophotometric analysis of uncultured P. sphagnophila collected from four field locations in Nova Scotia, Canada. Consistent with its morphological characteristics, 18S ribosomal DNA (rDNA) phylogenies indicate that P. sphagnophila is specifically related to Petalomonas cantuscygni, the only other Petalomonas species sequenced to date. One of the peculiar characteristics of P. sphagnophila is the presence of several green-pigmented particles approximately 5 mum in diameter in its cytoplasm, which a previously published study suggested to be cyanobacterial endosymbionts. New data presented here, however, suggest that the green intracellular body may not be a cyanobacterium but rather an uncharacterized prokaryote yet to be identified by molecular sequencing. 16S rDNA library sequencing and fluorescence in situ hybridizations show that P. sphagnophila also harbors several other endobionts, including bacteria that represent five novel genus-level groups (one firmicute and four different proteobacteria). 16S rDNA phylogenies suggest that three of these endobionts are related to obligate intracellular bacteria such as Rickettsiales and Coxiella, while the others are related to the Daphnia pathogen Spirobacillus cienkowskii or belong to the Thermoactinomycetaceae. TEM, 16S rDNA library sequencing and a battery of PCR experiments show that the presence of the five P. sphagnophila endobionts varies markedly among the four geographic collections and even among individuals collected from the same location but at different time points. Our study adds significantly to the growing evidence for complex and dynamic protist-bacterial associations in nature.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density.

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS(3), we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.

In vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes.

Antibacterial peptides have been isolated from a wide range of species. Some of these peptides act on microbial membranes, disrupting their barrier function. With the increasing development of antibiotic resistance by bacteria, these antibacterial peptides, which have a new mode of action, have attracted interest as antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides that act selectively on microbial membranes. In vitro display techniques are powerful tools to select biologically functional peptides from peptide libraries. Here, we used the ribosome display system to form peptide-ribosome-mRNA complexes in vitro from nucleotides encoding a peptide library, as well as immobilized model membranes, to select specific sequences that recognize bacterial membranes. This combination of ribosome display and immobilized model membranes was effective as an in vitro high-throughput screening system and enabled us to identify motif sequences (ALR, KVL) that selectively recognized the bacterial membrane. Owing to host toxicity, it was not possible to enrich any sequence expected to show antimicrobial activity using another in vitro system, e.g. phage display. The synthetic peptides designed from these enriched motifs acted selectively on the bacterial model membrane and showed antibacterial activity. Moreover, the motif sequence conferred selectivity onto native peptides lacking selectivity, and decreased mammalian cell toxicity of native peptides without decreasing their antibacterial activity.