Spicule Matrix Protein Research Articles

EchinoDB: an update to the web-based application for genomic and transcriptomic data on echinoderms

BackgroundHere we release a new version of EchinoDB, EchinoDB v2.0 (https://echinodb.uncc.edu). EchinoDB is a database of genomic and transcriptomic data on echinoderms. The initial database consisted of groups of 749,397 orthologous and paralogous transcripts arranged in orthoclusters by sequence similarity.ResultsThe updated version of EchinoDB includes two new major datasets: the RNA-Seq data of the brittle star Ophioderma brevispinum and the high-quality genomic assembly data of the green sea urchin Lytechinus variegatus. In addition, we enabled keyword searches for annotated data and installed an updated version of Sequenceserver to allow Basic Local Alignment Search Tool (BLAST) searches. The data are downloadable in FASTA format. The first version of EchinoDB appeared in 2016 and was implemented in GO on a local server. The new version has been updated using R Shiny to include new features and improvements in the application. Furthermore, EchinoDB now runs entirely in the cloud for increased reliability and scaling.ConclusionEchinoDB serves a user base drawn from the fields of phylogenetics, developmental biology, genomics, physiology, neurobiology, and regeneration. As use cases, we illustrate the function of EchinoDB in retrieving components of signaling pathways involved in the tissue regeneration process of different echinoderms, including the emerging model species Ophioderma brevispinum. Moreover, we use EchinoDB to shed light on the conservation of the molecular components involved in two echinoderm-specific phenomena: spicule matrix proteins involved in the formation of stereom endoskeleton and the tensilin protein that contributes to the capacity of the connective tissues to quickly change its mechanical properties. The genes involved in the former had been previously studied in echinoids, while gene sequences involved in the latter had been previously described in holothuroids. Specifically, we ask (a) if the biomineralization-related proteins previously reported only in sea urchins are also present in other, non-echinoid, echinoderms and (b) if tensilin, the protein responsible for the control of stiffness of the mutable collagenous tissue, previously described in sea cucumbers, is conserved across the phylum.

Skeletal development in the sea urchin relies upon protein families that contain intrinsic disorder, aggregation-prone, and conserved globular interactive domains.

The formation of the sea urchin spicule skeleton requires the participation of hydrogel-forming protein families that regulate mineral nucleation and nanoparticle assembly processes that give rise to the spicule. However, the structure and molecular behavior of these proteins is not well established, and thus our ability to understand this process is hampered. We embarked on a study of sea urchin spicule proteins using a combination of biophysical and bioinformatics techniques. Our biophysical findings indicate that recombinant variants of the two most studied spicule matrix proteins, SpSM50 and SpSM30B/C (S. purpuratus) have a conformational landscape that include a C-terminal random coil/intrinsically disordered MAPQG sequence coupled to a conserved, folded N-terminal C-type lectin-like (CTLL) domain, with SpSM50 > SpSM30B/C with regard to intrinsic disorder. Both proteins possess solvent-accessible unfolded MAQPG sequence regions where Asn, Gln, and Arg residues may be accessible for protein hydrogel interactions with water molecules. Our bioinformatics study included seven other spicule matrix proteins where we note similarities between these proteins and rare, unusual proteins that possess folded and unfolded traits. Moreover, spicule matrix proteins possess three types of sequences: intrinsically disordered, amyloid-like, and folded protein-protein interactive. Collectively these reactive domains would be capable of driving protein assembly and hydrogel formation. Interestingly, three types of global conformations are predicted for the nine member protein set, wherein we note variations in the arrangement of intrinsically disordered and interactive globular domains. These variations may reflect species-specific requirements for spiculogenesis. We conclude that the molecular landscape of spicule matrix protein families enables them to function as hydrogelators, nucleators, and assemblers of mineral nanoparticles.

Glycosylation Fosters Interactions between Model Sea Urchin Spicule Matrix Proteins. Implications for Embryonic Spiculogenesis and Biomineralization.

The formation of embryonic mineralized skeletal elements (spicules) in the sea urchin requires the participation of proteins, many of which may interact with one another and assist in the creation of an extracellular matrix wherein mineral formation takes place. To probe this, we created a sea urchin spicule recombinant model protein pair system wherein we tested the interactions between two major spicule proteins, SpSM50 and the glycoprotein, SpSM30B/C. Both proteins are strong hydrogelators that manipulate early and later events in mineral formation. We discovered that the anionic glycan moieties of SpSM30B/C are required for interaction with the SpSM50 protein and that these interactions are Ca(II)-independent. In addition, when these proteins form a complex, they create hybrid hydrogel particles that are physically distinct from their individual counterparts. Thus, glycan-mediated interactions play an important role in in vitro spicule protein assembly and most likely within the spicule itself.

The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth

ABSTRACTBackground: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. Materials and Methods: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as “Predicted uncharacterized proteins” and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer “hypothetical” but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. Results: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. Conclusion: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both “high” and “very high” magnesium calcite, [Ca(1–X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.

Noninvasive Microcomputerized X-ray Tomography Visualization of Mineralization Directed by Sea Urchin- and Nacre-Specific Proteins

The biomineralization process offers novel principles for crystal engineering and solid-state chemistry, but to achieve this we must first understand how organisms such as the mollusk and sea urchin craft skeletal elements such as the shell and embryonic spicule, respectively. In vitro studies of mollusk- and sea urchin-associated proteins reveal that these proteins form hydrogel particles that control the nucleation process, assemble mineral nanoparticles, and modify the surfaces and interiors of existing crystals. However, visualization of these processes is hampered by destructive and invasive methods such as those used for conventional scanning electron microscopy/transmission electron microscopy. In this report we detail the novel use of microcomputerized X-ray tomography (μCT) imaging to nondestructively investigate the in vitro calcium carbonate mineralization process in the presence of a recombinant sea urchin spicule matrix protein, rSpSM50, and a recombinant mollusk shell nacre protein, rPif97. Relative to the protein-free control scenario, both proteins generate calcite crystals that are consistent with the results obtained from previous studies, but with μCT we discovered new features; each protein generates a different number of mineral deposits, exhibits unique domains or motifs, and creates a stratification of mineral phases into layers. These results coincide with the known function of these proteins in vitro and in situ, and provide new information regarding biomineralization protein hydrogels and how they influence nucleation and crystal growth. We foresee that μCT imaging could, for appropriately sized systems, offer a bridge between in vitro experiments and in situ investigations of time-resolved nucleation and crystal growth phenomena in organisms, tissues, and synthetic materials.

Endocytosis in primary mesenchyme cells during sea urchin larval skeletogenesis

The sea urchin larval embryo elaborates two calcitic endoskeletal elements called spicules. Spicules are synthesized by the primary mesenchyme cells (PMCs) and begin to form at early gastrula stage. It is known that the calcium comprising the spicules comes from the seawater and we wish to further consider the mode of calcium transport from the extracellular seawater to the PMCs and then onto the forming spicules. We used PMC in vitro cultures, calcein, fluorescently labeled dextran, and fluorescently labeled Wheat Germ Agglutinin (WGA) to track calcium transport from the seawater into PMCs and spicules and to determine how molecules from the surface of PMCs interact with the incoming calcium. Labeling of PMC endocytic vesicles and forming spicules by both calcein and fluorescently tagged dextran indicate that calcium is taken up from the seawater by endocytosis and directly incorporated into spicules. Calcein labeling studies also indicate that calcium from the extracellular seawater begins to be incorporated into spicules within 30min of uptake. In addition, we demonstrate that fluorescently labeled WGA and calcein are taken up by many of the same endocytic vesicles and are incorporated into growing spicules. These findings suggest that PMC specific surface molecules accompany calcium ions as they enter PMCs via endocytosis and are incorporated together in the growing spicule. Using anti-spicule matrix protein antibodies, we pinpoint a subset of spicule matrix proteins that may accompany calcium ions from the surface of the PMCs until they are incorporated into spicules. Msp130 is identified as one of these spicule matrix proteins.

The skeletal proteome of the sea star Patiria miniata and evolution of biomineralization in echinoderms

BackgroundProteomic studies of skeletal proteins have revealed large, complex mixtures of proteins occluded within the mineral. Many skeletal proteomes contain rapidly evolving proteins with repetitive domains, further complicating our understanding. In echinoderms, proteomic analysis of the skeletal proteomes of mineralized tissues of the sea urchin Strongylocentrotus purpuratus prominently featured spicule matrix proteins with repetitive sequences linked to a C-type lectin domain. A comparative study of the brittle star Ophiocoma wendtii skeletal proteome revealed an order of magnitude fewer proteins containing C-type lectin domains. A number of other proteins conserved in the skeletons of the two groups were identified. Here we report the complete skeletal proteome of the sea star Patiria miniata and compare it to that of the other echinoderm groups.ResultsWe have identified eighty-five proteins in the P. miniata skeletal proteome. Forty-two percent of the proteins were determined to be homologous to proteins found in the S. purpuratus skeletal proteomes. An additional 34 % were from similar functional classes as proteins in the urchin proteomes. Thirteen percent of the P. miniata proteins had homologues in the O. wendtii skeletal proteome with an additional 29% showing similarity to brittle star skeletal proteins. The P. miniata skeletal proteome did not contain any proteins with C-lectin domains or with acidic repetitive regions similar to the sea urchin or brittle star spicule matrix proteins. MSP130 proteins were also not found. We did identify a number of proteins homologous between the three groups. Some of the highly conserved proteins found in echinoderm skeletons have also been identified in vertebrate skeletons.ConclusionsThe presence of proteins conserved in the skeleton in three different echinoderm groups indicates these proteins are important in skeleton formation. That a number of these proteins are involved in skeleton formation in vertebrates suggests a common origin for some of the fundamental processes co-opted for skeleton formation in deuterostomes. The proteins we identify suggest transport of proteins and calcium via endosomes was co-opted to this function in a convergent fashion. Our data also indicate that modifications to the process of skeleton formation can occur through independent co-option of proteins following species divergence as well as through domain shuffling.

A Model Sea Urchin Spicule Matrix Protein, rSpSM50, Is a Hydrogelator That Modifies and Organizes the Mineralization Process.

In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as SpSM50. However, because of its limited abundance and solubility issues, it has been difficult to pursue extensive in vitro biochemical studies of SpSM50 protein and deduce its role in spicule formation and mineralization. To circumvent these problems, we expressed a tag-free bacterial model recombinant spicule matrix protein, rSpSM50. Bioinformatics and biophysical experiments confirm that rSpSM50 is an intrinsically disordered, aggregation-prone C-type lectin-like domain-containing protein that forms dimensionally and internally heterogeneous protein hydrogels that control the in vitro mineralization process in three ways. The hydrogels (1) kinetically stabilize the aqueous calcium carbonate system against nucleation and thermodynamically destabilize the initially formed ACC in bulk solution, (2) promote and organize faceted single-crystal calcite and polycrystalline vaterite nanoparticles, and (3) promote surface texturing of calcite crystals and induce subsurface nanoporosities and channels within both calcite and vaterite crystals. Many of these features are also common to mollusk shell nacre proteins and the sea urchin spicule matrix glycoprotein, SpSM30B/C, and we conclude that rSpSM50 is a spiculogenesis hydrogelator protein that exhibits traits found in other calcium carbonate mineral-modification proteins.

A Model Sea Urchin Spicule Matrix Protein Self-Associates To Form Mineral-Modifying Protein Hydrogels.

In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as the SpSM30A-F isoforms. However, because of limited abundance, it has been difficult to pursue extensive biochemical studies of the SpSM30 proteins and deduce their role in spicule formation and mineralization. To circumvent these problems, we expressed a model recombinant spicule matrix protein, rSpSM30B/C, which possesses the key sequence attributes of isoforms "B" and "C". Our findings indicate that rSpSM30B/C is expressed in insect cells as a single polypeptide containing variations in glycosylation that create microheterogeneity in rSpSM30B/C molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic mono- and bisialylated and mono- and bisulfated monosaccharides on the protein molecules and enhance its aggregation propensity. Bioinformatics and biophysical experiments confirm that rSpSM30B/C is an intrinsically disordered, aggregation-prone protein that forms porous protein hydrogels that control the in vitro mineralization process in three ways: (1) increase the time interval for prenucleation cluster formation and transiently stabilize an ACC polymorph, (2) promote and organize single-crystal calcite nanoparticles, and (3) promote faceted growth and create surface texturing of calcite crystals. These features are also common to mollusk shell nacre proteins, and we conclude that rSpSM30B/C is a spiculogenesis protein that exhibits traits found in other calcium carbonate mineral modification proteins.

Characterization and Expression of Conserved Skeletal Proteins in Echinoderms

Specification of the cell fate of skeleton‐forming cells in developing sea urchin embryos has been well characterized and the skeletal proteome of both adult and larval skeleton has been determined. Little is known, however, of the mechanism and proteins involved in the mineralization process. A number of spicule matrix proteins have been identified in sea urchins and are present in the organic matrix during spicule formation. However, loss of function studies have indicated they may not be required for mineralization. We have taken a comparative, evolutionary approach to try and elucidate what proteins may be required for the process of mineralization. Here we report the characterization of the skeletal proteome of the sea star Patiria miniata. Our hypothesis is that there are proteins utilized in skeleton formation that are conserved between all echinoderm groups, and possibly among all deuterostomes. First we have isolated skeletal proteins from P. miniata, and then used LC/MS/MS to obtain peptide sequences of those proteins. Characterization of skeletal proteins was accomplished utilizing the NCBI non‐redundant protein database. By comparing the proteins present in all mineralized tissues of the sea urchin with those found in other echinoderm groups we have identified a set of conserved proteins containing similar functional domains. The skeletal proteome from the two echinoderms, P. miniata and Strongylocentrotus purpuratus, groups also shared novel proteins with similar functional domains. We have analyzed the sequences of the proteins we identified as potentially important to determine the evolutionary relationship and to identify the sequences most highly conserved. Additionally, some of these proteins are also found in vertebrate bone. We have also shown that some skeletal proteins prevalent in one group are not found in other echinoderms. This suggests independent co‐option of proteins utilized in mineralization in some groups. The conserved proteins were then analyzed for temporal expression at eight different stages of the developing embryo of S. purpuratus. Some of these stages, such as mesenchyme blastula, were predicted to show higher level of expression for some of the conserved skeletal proteins. Utilizing gel electrophoresis and quantitative polymerase chain reaction (qPCR), we analyzed the temporal expression of conserved skeletal proteins and the relative expression of those proteins during the eight stages of the developing embryo. We found that expression of the conserved skeletal proteins varied, but all were present when skeleton formation occurred in the embryo.Support or Funding InformationNIH/NIGMS T34GM008074, NSF grant 09097979

Expression of SM30(A–F) genes encoding spicule matrix proteins in intact and damaged sea urchin Strongylocentrotus intermedius at the six-armed pluteus

In this study we investigated expression of the SM30(A–F) gene family encoding Strongylocentrotus intermedius spicule matrix proteins during the normal and regenerative pluteus II stage (three pairs of arms). We found that SiSM30A and SiSM30B genes are expressed at high levels in the normal pluteus II sea urchin. SiSM30A is expression was also significantly upregulated in the reparative pluteus II stage 3 hours after damage. Conversely, SiSM30B was downregulated during the reparative pluteus II stage. Our findings reveal a substantial similarity between the activity of SiSM30A and SiSM30B activity in the processes of regenerative growth during the pluteus II stage and during normal development at the prism stage in Strongylocentrotus purpuratus. On the basis of our findings, we propose that normal developmental mechanisms corresponding to the preceding developmental stage are reactivated during pluteus regeneration.

The skeletal proteome of the brittle star <em>Ophiothrix spiculata</em> identifies C-type lectins and other proteins conserved in echinoderm skeleton formation

Determining the identity and functional role of proteins involved in biomineralization and the formation of skeletons is critical to our understanding of the process. Proteomics has allowed rapid characterization of the proteins occluded within mineralized tissue, but the large numbers of proteins detected makes it difficult to assign the relative importance of each protein. We have taken a comparative approach, examining the skeletal proteome of different species of echinoderms in order to identify the proteins that are conserved and likely to be important. Our previous study comparing the skeletal proteome of the brittle star Ophiocoma wendtii to the published proteomes of the sea urchin Strongylocentrotus purpuratus revealed some conservation of proteins, but indicated that the C-type lectin domain-containing spicule matrix proteins abundant in the sea urchin skeletal proteome were not conserved in the brittle star. Here we examine the skeletal proteome of a different species of brittle star, Ophiothrix spiculata. We have isolated the proteins from the skeleton of O. spiculata and performed LC/MS/MS to identify peptides present. Comparison to transcriptome and genome databases revealed the proteins present in the O. spiculata proteome. Despite being diverged for several million years, the two brittle stars have very similar proteins in their skeletons. Included is a fibrinogen C-like lectin and several C-type lectins proteins, which we describe in detail. The unusual number of C-type lectins found in the S. purpuatus skeleton and the repetitive regions seen in those spicule matrix proteins are not present in O. spiculata.

Monohydroxylated polycyclic aromatic hydrocarbons influence spicule formation in the early development of sea urchins (Hemicentrotus pulcherrimus)

We previously demonstrated that monohydroxylated polycyclic aromatic hydrocarbons (OHPAHs), which are metabolites of polycyclic aromatic hydrocarbons (PAHs), act on calcified tissue and suppress osteoblastic and osteoclastic activity in the scales of teleost fish. The compounds may possibly influence other calcified tissues. Thus, the present study noted the calcified spicules in sea urchins and examined the effect of both PAHs and OHPAHs on spicule formation during the embryogenesis of sea urchins. After fertilization, benz[a]anthracene (BaA) and 4-hydroxybenz[a]anthracene (4-OHBaA) were added to seawater at concentrations of 10−8 and 10−7M and kept at 18°C. The influence of the compound was given at the time of the pluteus larva. At this stage, the length of the spicule was significantly suppressed by 4-OHBaA (10−8 and 10−7M). BaA (10−7M) decreased the length of the spicule significantly, while the length did not change with BaA (10−8M). The expression of mRNAs (spicule matrix protein and transcription factors) in the 4-OHBaA (10−7M)-treated embryos was more strongly inhibited than were those in the BaA (10−7M)-treated embryos. This is the first study to demonstrate that OHPAHs suppress spicule formation in sea urchins.

Examination of the skeletal proteome of the brittle star Ophiocoma wendtii reveals overall conservation of proteins but variation in spicule matrix proteins.

BackgroundWhile formation of mineralized tissue is characteristic of many animal taxa, the proteins that interact with mineral are diverse and appear in many cases to be of independent origin. Extracellular matrix proteins involved in mineralization do share some common features. They tend to be disordered, secreted proteins with repetitive, low complexity. The genes encoding these proteins are often duplicated and undergo concerted evolution, further diversifying the repetitive domains. This makes it difficult to identify mineralization genes and the proteins they encode using bioinformatics techniques. Here we describe the use of proteomics to identify mineralization genes in an ophiuroid echinoderm, Ophiocoma wendtii (O. wendtii).ResultsWe have isolated the occluded proteins within the mineralized tissue of the brittle star Ophiocoma wendtii. The proteins were analyzed both unfractionated and separated on SDS-PAGE gels. Each slice was analyzed using mass spectroscopy and the amino acid sequence of the most prevalent peptides was obtained. This was compared to both an embryonic transcriptome from the gastrula stage when skeleton is being formed and a tube foot (an adult mineralized tissue) transcriptome. Thirty eight proteins were identified which matched known proteins or protein domains in the NCBI databases. These include C-type lectins, ECM proteins, Kazal-type protease inhibitors, matrix metalloproteases as well as more common cellular proteins. Many of these are similar to those found in the sea urchin Strongylocentrotus purpuratus (S. purpuratus) skeleton. We did not, however, identify clear homologs to the sea urchin spicule matrix proteins, and the number of C-type lectin containing genes was much reduced compared to sea urchins. Also notably absent was MSP-130.ConclusionsOur results show an overall conservation of the types of proteins found in the mineralized tissues of two divergent groups of echinoderms, as well as in mineralized tissues in general. However, the extensive gene duplication and concerted evolution seen in the spicule matrix proteins found in the sea urchin skeleton was not observed in the brittle star.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0064-7) contains supplementary material, which is available to authorized users.

Effects of seawater acidification on gene expression: resolving broader-scale trends in sea urchins.

Sea urchins are ecologically and economically important calcifying organisms threatened by acidification of the global ocean caused by anthropogenic CO2 emissions. Propelled by the sequencing of the purple sea urchin (Strongylocentrotus purpuratus) genome, profiling changes in gene expression during exposure to high pCO2 seawater has emerged as a powerful and increasingly common method to infer the response of urchins to ocean change. However, analyses of gene expression are sensitive to experimental methodology, and comparisons between studies of genes regulated by ocean acidification are most often made in the context of major caveats. Here we perform meta-analyses as a means of minimizing experimental discrepancies and resolving broader-scale trends regarding the effects of ocean acidification on gene expression in urchins. Analyses across eight studies and four urchin species largely support prevailing hypotheses about the impact of ocean acidification on marine calcifiers. The predominant expression pattern involved the down-regulation of genes within energy-producing pathways, a clear indication of metabolic depression. Genes with functions in ion transport were significantly over-represented and are most plausibly contributing to intracellular pH regulation. Expression profiles provided extensive evidence for an impact on biomineralization, epitomized by the down-regulation of seven spicule matrix proteins. In contrast, expression profiles provided limited evidence for CO2-mediated developmental delay or induction of a cellular stress response. Congruence between studies of gene expression and the ocean acidification literature in general validates the accuracy of gene expression in predicting the consequences of ocean change and justifies its continued use in future studies.

Lectin uptake and incorporation into the calcitic spicule of sea urchin embryos.

Primary mesenchyme cells (PMCs) are skeletogenenic cells that produce a calcareous endoskeleton in developing sea urchin larvae. The PMCs fuse to form a cavity in which spicule matrix proteins and calcium are secreted forming the mineralized spicule. In this study, living sea urchin embryos were stained with fluorescently conjugated wheat germ agglutinin, a lectin that preferentially binds to PMCs, and the redistribution of this fluorescent tag was examined during sea urchin development. Initially, fluorescence was associated primarily with the surface of PMCs. Subsequently, the fluorescent label redistributed to intracellular vesicles in the PMCs. As the larval skeleton developed, intracellular granular staining diminished and fluorescence appeared in the spicules. Spicules that were cleaned to remove membranous material associated with the surface exhibited bright fluorescence, which indicated that fluorescently labelled lectin had been incorporated into the spicule matrix. The results provide evidence for a cellular pathway in which material is taken up at the cell surface, sequestered in intracellular vesicles and then incorporated into the developing spicule.

Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule matrix protein SM50 from Strongylocentrotus purpuratus

Sea urchin spicules have a calcitic mesocrystalline architecture that is closely associated with a matrix of proteins and amorphous minerals. The mechanism underlying spicule formation involves complex processes encompassing spatio-temporally regulated organic-inorganic interactions. C-type lectin domains are present in several spicule matrix proteins in Strongylocentrotus purpuratus, implying their role in spiculogenesis. In this study, the C-type lectin domain of SM50 was overexpressed, purified and crystallized using a vapour-diffusion method. The crystal diffracted to a resolution of 2.85 Å and belonged to space group P212121, with unit-cell parameters a = 100.6, b = 115.4, c = 130.6 Å, α = β = γ = 90°. Assuming 50% solvent content, six chains are expected to be present in the asymmetric unit.

Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization

The larval spicule matrix protein SM50 is the most abundant occluded matrix protein present in the mineralized larval sea urchin spicule. Recent evidence implicates SM50 in the stabilization of amorphous calcium carbonate (ACC). Here, we investigate the molecular interactions of SM50 and CaCO3 by investigating the function of three major domains of SM50 as small ubiquitin-like modifier (SUMO) fusion proteins - a C-type lectin domain (CTL), a glycine rich region (GRR) and a proline rich region (PRR). Under various mineralization conditions, we find that SUMO-CTL is monomeric and influences CaCO3 mineralization, SUMO-GRR aggregates into large protein superstructures and SUMO-PRR modifies the early CaCO3 mineralization stages as well as growth. The combination of these mineralization and self-assembly properties of the major domains synergistically enable the full-length SM50 to fulfill functions of constructing the organic spicule matrix as well as performing necessary mineralization activities such as Ca(2+) ion recruitment and organization to allow for proper growth and development of the mineralized larval sea urchin spicule.

Temperature and CO2additively regulate physiology, morphology and genomic responses of larval sea urchins,Strongylocentrotus purpuratus

Ocean warming and ocean acidification, both consequences of anthropogenic production of CO2, will combine to influence the physiological performance of many species in the marine environment. In this study, we used an integrative approach to forecast the impact of future ocean conditions on larval purple sea urchins (Strongylocentrotus purpuratus) from the northeast Pacific Ocean. In laboratory experiments that simulated ocean warming and ocean acidification, we examined larval development, skeletal growth, metabolism and patterns of gene expression using an orthogonal comparison of two temperature (13°C and 18°C) and pCO2 (400 and 1100 μatm) conditions. Simultaneous exposure to increased temperature and pCO2 significantly reduced larval metabolism and triggered a widespread downregulation of histone encoding genes. pCO2 but not temperature impaired skeletal growth and reduced the expression of a major spicule matrix protein, suggesting that skeletal growth will not be further inhibited by ocean warming. Importantly, shifts in skeletal growth were not associated with developmental delay. Collectively, our results indicate that global change variables will have additive effects that exceed thresholds for optimized physiological performance in this keystone marine species.

Implication ofHpEtsin Gene Regulatory Networks Responsible for Specification of Sea Urchin Skeletogenic Primary Mesenchyme Cells

The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.