Histone demethylase Lsd1 is required for the differentiation of neural cells in Nematostella vectensis.

Chromatin regulation is a key process in development but its contribution to the evolution of animals is largely unexplored. Chromatin is regulated by a diverse set of proteins, which themselves are tightly regulated in a cell/tissue-specific manner. Using the cnidarian Nematostella vectensis as a basal metazoan model, we explore the function of one such chromatin regulator, Lysine specific demethylase 1 (Lsd1). We generated an endogenously tagged allele and show that NvLsd1 expression is developmentally regulated and higher in differentiated neural cells than their progenitors. We further show, using a CRISPR/Cas9 generated mutant that loss of NvLsd1 leads to developmental abnormalities. This includes the almost complete loss of differentiated cnidocytes, cnidarian-specific neural cells, as a result of a cell-autonomous requirement for NvLsd1. Together this suggests that the integration of chromatin modifying proteins into developmental regulation predates the split of the cnidarian and bilaterian lineages and constitutes an ancient feature of animal development.

Symbiont population control by host-symbiont metabolic interaction in Symbiodiniaceae-cnidarian associations.

In cnidarian-Symbiodiniaceae symbioses, algal endosymbiont population control within the host is needed to sustain a symbiotic relationship. However, the molecular mechanisms that underlie such population control are unclear. Here we show that a cnidarian host uses nitrogen limitation as a primary mechanism to control endosymbiont populations. Nitrogen acquisition and assimilation transcripts become elevated in symbiotic Breviolum minutum algae as they reach high-densities within the sea anemone host Exaiptasia pallida. These same transcripts increase in free-living algae deprived of nitrogen. Symbiotic algae also have an elevated carbon-to-nitrogen ratio and shift metabolism towards scavenging nitrogen from purines relative to free-living algae. Exaiptasia glutamine synthetase and glutamate synthase transcripts concomitantly increase with the algal endosymbiont population, suggesting an increased ability of the host to assimilate ammonium. These results suggest algal growth and replication in hospite is controlled by access to nitrogen, which becomes limiting for the algae as their population within the host increases.

The CYP443C1 (CYP74 clan) Cytochrome of Sea Anemone Nematostella vectensis-the First Metazoan Enzyme Possessing Hydroperoxide Lyase/Epoxyalcohol Synthase Activity.

A novel CYP74 clan gene CYP443С1 of the starlet sea anemone (Nematostella vectensis, Cnidaria) has been cloned, and the properties of the corresponding recombinant protein have been studied. Depending on the substrate, CYP443С1 exhibited double function hydroperoxide lyase/epoxyalcohol synthase activity.

Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation.

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the ~3 Å resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post-filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2.

Phylogenetic analysis of cnidarian peroxiredoxins and stress-responsive expression in the estuarine sea anemone Nematostella vectensis.

Peroxiredoxins (PRXs) are a family of antioxidant enzymes present in all domains of life. To date, the diversity and function of peroxiredoxins within animals have only been studied in a few model species. Thus, we sought to characterize peroxiredoxin diversity in cnidarians and to gain insight into their function in one cnidarian-the sea anemone Nematostella vectensis. Phylogenetic analysis using all six known PRX subfamilies (PRX1-4, PRX5, PRX6, PRXQ/AHPE1, TPX, BCP-PRXQ) revealed that like bilaterians, cnidarians contain representatives from three subfamilies (PRX1-4, PRX5, PRX6). Within the PRX1-4 subfamily, cnidarian sequences fall into two clades: PRX4, and a cnidarian-specific clade, which we term CNID-PRX. This phylogenetic analysis demonstrates that the three PRX subfamilies present in Bilateria were also present in the last common ancestor of the Cnidaria and Bilateria, and further that diversification of the PRX1-4 subfamily has occurred within the cnidarian lineage. We next examined the impact of decreased salinity, increased temperature, and peroxide exposure on the expression of four prx genes in N. vectensis (cnid-prx, prx4, prx5, and prx6). These genes exhibited unique expression patterns in response to these environmental stressors. Expression of prx4 decreased with initial exposure to elevated temperature, cnid-prx increased with exposure to elevated temperatures as well as with hydrogen peroxide exposure, and expression of all prxs transiently decreased with reduced salinity. Predicted subcellular localization patterns also varied among PRX proteins. Together these results provide evidence that peroxiredoxins in N. vectensis serve distinct physiological roles and lay a groundwork for understanding how peroxiredoxins mediate cnidarian developmental processes and environmental responses.

Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis.

The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano. Two versions of fused homing endonuclease gene (HEG) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA, a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function.

Identification of CYP443D1 (CYP74 clan) of Nematostella vectensis as a first cnidarian epoxyalcohol synthase and insights into its catalytic mechanism.

The CYP74 clan enzymes are responsible for the biosynthesis of numerous bioactive oxylipins in higher plants, some Proteobacteria, brown and green algae, and Metazoa. A novel putative CYP74 clan gene CYP443D1 of the starlet sea anemone (Nematostella vectensis, Cnidaria) has been cloned, and the properties of the corresponding recombinant protein have been studied in the present work. The recombinant CYP443D1 was incubated with the 9- and 13-hydroperoxides of linoleic and α-linolenic acids (9-HPOD, 13-HPOD, 9-HPOT, and 13-HPOT, respectively), as well as with the 9-hydroperoxide of γ-linolenic acid (γ-9-HPOT) and 15-hydroperoxide of eicosapentaenoic acid (15-HPEPE). The enzyme was active towards all C18-hydroperoxides with some preference to 9-HPOD. In contrast, 15-HPEPE was a poor substrate. The CYP443D1 specifically converted 9-HPOD into the oxiranyl carbinol 1, (9S,10R,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both 18O atoms from [18O2-hydroperoxy]9-HPOD were virtually quantitatively incorporated into product 1. Thus, the CYP443D1 exhibited epoxyalcohol synthase (EAS) activity. The 18O labelling data demonstrated that the reaction mechanism included three sequential steps: (1) hydroperoxyl homolysis, (2) oxy radical rearrangement into epoxyallylic radical, (3) hydroxyl rebound, resulting in oxiranyl carbinol formation. The 9-HPOT and γ-9-HPOT were also specifically converted into the oxiranyl carbinols, 15,16- and 6,7-dehydro analogues of compound 1, respectively. The 13-HPOD was converted into erythro- and threo-isomers of oxiranyl carbinol, as well as oxiranyl vinyl carbinols. The obtained results allow assignment of the name "N. vectensis EAS" (NvEAS) to CYP443D1. The NvEAS is a first EAS detected in Cnidaria.

Evolution of miRNA Tailing by 3' Terminal Uridylyl Transferases in Metazoa.

In bilaterian animals the 3' ends of microRNAs (miRNAs) are frequently modified by tailing and trimming. These modifications affect miRNA-mediated gene regulation by modulating miRNA stability. Here, we analyzed data from three nonbilaterian animals: two cnidarians (Nematostella vectensis and Hydra magnipapillata) and one poriferan (Amphimedon queenslandica). Our analysis revealed that nonbilaterian miRNAs frequently undergo modifications like the bilaterian counterparts: the majority are expressed as different length isoforms and frequent modifications of the 3' end by mono U or mono A tailing are observed. Moreover, as the factors regulating miRNA modifications are largely uncharacterized in nonbilaterian animal phyla, in present study, we investigated the evolution of 3' terminal uridylyl transferases (TUTases) that are known to involved in miRNA 3' nontemplated modifications in Bilateria. Phylogenetic analysis on TUTases showed that TUTase1 and TUTase6 are a result of duplication in bilaterians and that TUTase7 and TUTase4 are the result of a vertebrate-specific duplication. We also find an unexpected number of Drosophila-specific gene duplications and domain losses in most of the investigated gene families. Overall, our findings shed new light on the evolutionary history of TUTases in Metazoa, as they reveal that this core set of enzymes already existed in the last common ancestor of all animals and was probably involved in modifying small RNAs in a similar fashion to its present activity in bilaterians.

Regulation of AP-1 by MAPK Signaling in Metal-Stressed Sea Anemone.

BACKGROUND/AIMS: AP-1 transcription factor plays a conserved role in the immediate response to stress. Activation of AP-1 members jun and fos is mediated by complex signaling cascades to control cell proliferation and survival. To understand the evolution of this broadly-shared pathway, we studied AP-1 regulation by MAPK signaling in a basal metazoan. METHODS: Metal- stressed cnidarian Nematostella vectensis anemones were tested with kinase inhibitors and analyzed for gene expression levels and protein phosphorylation. RESULTS: We show that in cnidarian, AP-1 is regulated differently than in bilaterian models. ERK2 and ERK5, the main MAPK drivers of AP-1 activation in Bilateria, down-regulated fos1 and jun1 transcription in anemones exposed to metal stress, whereas p38 MAPK, triggered transcription of jun1 but not fos1. Furthermore, our results reveal that GSK3-ß is the main driver of the immediate stress response in Nematostella. GSK3-ß triggered transcription of AP-1 and two other stress-related genes, egr1 and hsp70. Finally, phylogenetic analysis and protein characterization show that while MAPKs and GSK3-ß are evolutionarily conserved, Fos and Jun proteins in Nematostella and other cnidarians lack important regulatory and phosphorylation sites found in Bilateria. CONCLUSION: These findings reveal alternative network interactions of conserved signaling kinases, providing insight into the evolutionary plasticity of immediate stress response mechanisms.

The sphingosine rheostat is involved in the cnidarian heat stress response but not necessarily in bleaching.

Sphingolipids play important roles in mitigating cellular heat and oxidative stress by altering membrane fluidity, receptor clustering and gene expression. Accumulation of signaling sphingolipids that comprise the sphingosine rheostat, pro-apoptotic sphingosine (Sph) and pro-survival sphingosine-1-phosphate (S1P) is key to determining cell fate. Reef-building corals and other symbiotic cnidarians living in shallow tropical waters can experience elevated seawater temperature and high UV irradiance, two stressors that are increasing in frequency and severity with climate change. In symbiotic cnidarians, these stressors disrupt the photosynthetic machinery of the endosymbiont and ultimately result in the collapse of the partnership (dysbiosis), known as cnidarian bleaching. In a previous study, exogenously applied sphingolipids altered heat-induced bleaching in the symbiotic anemone Aiptasia pallida, but endogenous regulation of these lipids is unknown. Here, we characterized the role of the rheostat in the cnidarian heat stress response (HSR) and in dysbiosis. Gene expression of rheostat enzymes sphingosine kinase (AP-SPHK) and S1P phosphatase (AP-SGPP), and concentrations of sphingolipids were quantified from anemones incubated at elevated temperatures. We observed a biphasic HSR in A. pallida. At early exposure, rheostat gene expression and lipid levels were suppressed while gene expression of a heat stress biomarker increased and 40% of symbionts were lost. After longer incubations at the highest temperature, AP-SGPP and then Sph levels both increased. These results indicate that the sphingosine rheostat in A. pallida does not participate in initiation of dysbiosis, but instead functions in the chronic response to prolonged heat stress that promotes host survival.

The discovery of novel LPMO families with a new Hidden Markov model.

BACKGROUND: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs. RESULTS: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of ß-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin. CONCLUSIONS: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.

Characterization of glutathione peroxidase diversity in the symbiotic sea anemone Anemonia viridis.

Cnidarians living in symbiosis with photosynthetic dinoflagellates (commonly named zooxanthellae) are exposed to high concentrations of reactive oxygen species (ROS) upon illumination. To quench ROS production, both the cnidarian host and zooxanthellae express a full suite of antioxidant enzymes. Studying antioxidative balance is therefore crucial to understanding how symbiotic cnidarians cope with ROS production. We characterized glutathione peroxidases (GPx) in the symbiotic cnidarian Anemonia viridis by analysis of their isoform diversity, their activity distribution in the three cellular compartments (ectoderm, endoderm and zooxanthellae) and their involvement in the response to thermal stress. We identified a GPx repertoire through a phylogenetic analysis showing 7 GPx transcripts belonging to the A. viridis host and 4 GPx transcripts strongly related to Symbiodinium sp. The biochemical approach, used for the first time with a cnidarian species, allowed the identification of GPx activity in the three cellular compartments and in the animal mitochondrial fraction, and revealed a high GPx electrophoretic diversity. The symbiotic lifestyle of zooxanthellae requires more GPx activity and diversity than that of free-living species. Heat stress induced no modification of GPx activities. We highlight a high GPx diversity in A. viridis tissues by genomic and biochemical approaches. GPx activities represent an overall constitutive enzymatic pattern inherent to symbiotic lifestyle adaptation. This work allows the characterization of the GPx family in a symbiotic cnidarian and establishes a foundation for future studies of GPx in symbiotic cnidarians.

Warm preconditioning protects against acute heat-induced respiratory dysfunction and delays bleaching in a symbiotic sea anemone.

Preconditioning to non-stressful warming can protect some symbiotic cnidarians against the high temperature-induced collapse of their mutualistic endosymbiosis with photosynthetic dinoflagellates (Symbiodinium spp.), a process known as bleaching. Here, we sought to determine whether such preconditioning is underpinned by differential regulation of aerobic respiration. We quantified in vivo metabolism and mitochondrial respiratory enzyme activity in the naturally symbiotic sea anemone Exaiptasia pallida preconditioned to 30°C for >7 weeks as well as anemones kept at 26°C. Preconditioning resulted in increased Symbiodinium photosynthetic activity and holobiont (host+symbiont) respiration rates. Biomass-normalised activities of host respiratory enzymes [citrate synthase and the mitochondrial electron transport chain (mETC) complexes I and IV] were higher in preconditioned animals, suggesting that increased holobiont respiration may have been due to host mitochondrial biogenesis and/or enlargement. Subsequent acute heating of preconditioned and 'thermally naive' animals to 33°C induced dramatic increases in host mETC complex I and Symbiodinium mETC complex II activities only in thermally naive E. pallida These changes were not reflected in the activities of other respiratory enzymes. Furthermore, bleaching in preconditioned E. pallida (defined as the significant loss of symbionts) was delayed by several days relative to the thermally naive group. These findings suggest that changes to mitochondrial biogenesis and/or function in symbiotic cnidarians during warm preconditioning might play a protective role during periods of exposure to stressful heating.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian.

Responses of the sea anemone, Exaiptasia pallida, to ocean acidification conditions and copper exposure.

Ocean acidification (OA) is a growing concern due to its deleterious effects on aquatic organisms. Additionally, the combined effects of OA and other local stressors like metal pollution are largely unknown. In this study, we examined physiological effects in the sea anemone, Exaiptasia pallida after exposure to the global stressor carbon dioxide (CO2), as well as the local stressor copper (Cu) over 7 days. Cu accumulated in the tissues of E. pallida in a concentration-dependent manner. At some time points, sea anemones exposed to 1000 ppm CO2 had higher tissue Cu concentrations than those exposed to 400 ppm CO2 at the same Cu exposure concentrations. In general, the activities of all anti-oxidant enzymes measured (catalase, CAT; glutathione peroxidase, GPx, glutathione reductase, GR) increased with exposure to increasing Cu concentrations. Significant differences in GR, CAT and to some degree GPx activity, were observed due to increasing CO2 exposure in control treatments. Sea anemones exposed to Cu in combination with higher CO2 generally had higher anti-oxidant enzyme activities than those exposed to the same concentration of Cu and lower CO2. Activity of the enzyme, carbonic anhydrase (CA), involved in acid-base balance, was significantly decreased with increasing Cu exposure. At the two lowest Cu concentrations, the extent of CA inhibition was lessened with increasing CO2 concentration. These results provide insight into toxic mechanisms of both Cu and CO2 exposure to the sensitive cnidarian E. pallida and have implications for environmental exposure of multiple contaminants.

Cooperativity and evolution of Tetrahymena two-domain arginine kinase.

Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura.

Structure of a double-domain phosphagen kinase reveals an asymmetric arrangement of the tandem domains.

Tandem duplications and fusions of single genes have led to magnificent expansions in the divergence of protein structures and functions over evolutionary timescales. One of the possible results is polydomain enzymes with interdomain cooperativities, few examples of which have been structurally characterized at the full-length level to explore their innate synergistic mechanisms. This work reports the crystal structures of a double-domain phosphagen kinase in both apo and ligand-bound states, revealing a novel asymmetric L-shaped arrangement of the two domains. Unexpectedly, the interdomain connections are not based on a flexible hinge linker but on a rigid secondary-structure element: a long α-helix that tethers the tandem domains in relatively fixed positions. Besides the connective helix, the two domains also contact each other directly and form an interdomain interface in which hydrogen bonds and hydrophobic interactions further stabilize the L-shaped domain arrangement. Molecular-dynamics simulations show that the interface is generally stable, suggesting that the asymmetric domain arrangement crystallographically observed in the present study is not a conformational state simply restrained by crystal-packing forces. It is possible that the asymmetrically arranged tandem domains could provide a structural basis for further studies of the interdomain synergy.

Improved purification and enzymatic properties of a mixture of Sticholysin I and II: isotoxins with hemolytic and phospholipase A(2) activities from the sea anemone Stichodactyla helianthus.

Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10(-3)U/mg of protein). In this work, a mixture StI-StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI-StII was relatively high (1.85U/mg) and dependent of Ca(2+). The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40mM Ca(2+) at pH 8.0. This Ca(2+) concentration did not exert any effect on binding of StI-StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.

Stress and death of cnidarian host cells play a role in cnidarian bleaching.

Coral bleaching occurs when there is a breakdown of the symbiosis between cnidarian hosts and resident Symbiodinium spp. Multiple mechanisms for the bleaching process have been identified, including apoptosis and autophagy, and most previous work has focused on the Symbiodinium cell as the initiator of the bleaching cascade. In this work we show that it is possible for host cells to initiate apoptosis that can contribute to death of the Symbiodinium cell. First we found that colchicine, which results in apoptosis in other animals, causes cell death in the model anemone Aiptasia sp. but not in cultured Symbiodinium CCMP-830 cells or in cells freshly isolated from host Aiptasia (at least within the time frame of our study). In contrast, when symbiotic Aiptasia were incubated in colchicine, cell death in the resident Symbiodinium cells was observed, suggesting a host effect on symbiont mortality. Using live-cell confocal imaging of macerated symbiotic host cell isolates, we identified a pattern where the initiation of host cell death was followed by mortality of the resident Symbiodinium cells. This same pattern was observed in symbiotic host cells that were subjected to temperature stress. This research suggests that mortality of symbionts during temperature-induced bleaching can be initiated in part by host cell apoptosis.