Mineo Iseki

Photoactivated Adenylyl Cyclase Controls Phototaxis in the Flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena

Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.

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.

Origins of a cyanobacterial 6-phosphogluconate dehydrogenase in plastid-lacking eukaryotes

BackgroundPlastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. Although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive.ResultsHere we show an extended phylogenetic analysis including novel gnd gene sequences from Excavata and Glaucophyta. Our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. The Diplonema gene was related to cytosol-type genes in red algae and Opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. Statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. In addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in Euglenida were robustly monophyletic with Stramenopiles, and this monophyletic clade was moderately separated from those of red algae. These data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses.ConclusionWe propose an evolutionary scenario in which plastid-lacking Excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene.

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.

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

Significance Optogenetics is a rapidly growing field in which light is used to control biological systems. We show that Oscillatoria acuminata photoactivated adenylate cyclase (OaPAC) protein produces the fundamental second messenger cyclic-AMP (cAMP) in response to blue light, is stable and functional in different mammalian cell types, and can be used to trigger events by raising cAMP level. OaPAC consists of a catalytic domain controlled by a photosensitive blue light using flavin (BLUF) domain. We have solved the crystal structure to show how activity is triggered by light, and guide mutagenesis experiments. Although the catalytic domain resembles known cyclases, the BLUF domains form an unusual intertwined structure. The protein activity is the same in solution as in the crystal, showing that the activation mechanism involves only small molecular movements. 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.

The origin of photoactivated adenylyl cyclase (PAC), the Euglena blue-light receptor: phylogenetic analysis of orthologues of PAC subunits from several euglenoids and trypanosome-type adenylyl cyclases from Euglena gracilisElectronic supplementary information (ESI) available: Multiple alignment of deduced amino acid sequences. See http://www.rsc.org/suppdata/pp/b3/b316075k/

Photoactivated adenylyl cyclase (PAC) is the blue-light receptor flavoprotein recently identified as a photoreceptor for photoavoidance of the unicellular flagellate, Euglena gracilis. To gain an insight into the evolution of this unique protein, similar sequences were searched for in several euglenoids by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers. Two similar transcripts were detected in each of the four phototrophic euglenoids, Euglena stellata, Colacium sideropus, Eutreptia viridis, Eutreptiella gymnastica, and in an osmotrophic (i.e., obtaining nutrients by absorption) one, Khawkinea quartana, but not in a phagotrophic euglenoid, Petalomonas cantuscygni. Each of them seemed to be orthologous to PACalpha and PACbeta, respectively, and had the same domain structure as PAC subunits each of which is composed of two flavin binding domains, F1 and F2, each followed by an adenylyl cyclase catalytic domain, C1 and C2, respectively. This fact implies that they constitute a functional photoactivated adenylyl cyclase like PAC. Phylogenetic analysis of the adenylyl cyclase catalytic domains revealed that they belong to a bacterial cluster, not to a trypanosomal one. In addition, two trypanosome-type adenylyl cyclases were discovered in E. gracilis. In contrast to PAC, deduced amino acid sequences of the trypanosome-type adenylyl cyclases indicated that they are integral membrane proteins with a membrane spanning region at the midpoint of them, followed by an adenylyl cyclase catalytic domain which seems cytoplasmic. Overall, we propose that PAC might have been transferred to euglenoids on the occasion of secondary endosymbiosis.

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 Ca2+ 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.

Possible Involvement of a Tetrahydrobiopterin in Photoreception for UV-B-induced Anthocyanin Synthesis in Carrot

Our previous studies of action spectra for UV‐B‐induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV‐B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV‐B light sensing. UV‐B‐induced phenylalanine ammonia‐lyase (PAL) activity was considerably suppressed by N‐acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N‐acetylserotonin‐treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV‐B‐induced PAL activity. These results suggest the occurrence of an unidentified UV‐B photoreceptor (other than UVR8, the tryptophan‐based UV‐B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV‐B sensors.

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

Significance We have previously shown that photoactivated adenylate cyclase from Oscillatoria acuminata (OaPAC) is stable and functional in human cells and can be used to raise intracellular cAMP levels by exposure to blue light. Two prior crystal structures of OaPAC in the dark state, and mutagenesis experiments, indicate that the activation mechanism involves only very small movements, but we have now succeeded in refining the structure of the light-activated protein to high resolution, showing in molecular detail the changes at the chromophore on light exposure and allowing precise comparison of the structure in the light-exposed and dark states. The differences between these structures indicate the very small but concerted shifts that trigger enzyme activity tens of ångstroms from the chromophore. 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.