From hemoglobin to urchin spicules.

A retrospective of an academic career. The article poses the question about the values involved in a life in the academy, starting with the role of hemoglobin in the chick embryo and ending with the role of calcium in the sea urchin spine.

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.

Investigating protein function in biomineralized tissues using molecular biology techniques.

We describe modern molecular biology methods currently used in the study of biomineralization. We focus our descriptions on two areas of biomineralization research in which these methods have been particularly powerful. The first area is the use of modern molecular methods to identify and characterize the so-called occluded matrix proteins present in mineralized tissues. More specifically, we describe the use of RNA-seq and the next generation of DNA sequencers and the use of direct protein sequencing and mass spectrometers as ways of identifying proteins present in mineralized tissues. The second area is the use of molecular methods to examine the function of proteins in biomineralization. RNA interference (RNAi), morpholino antisense, and other methods are described and discussed as ways of elucidating protein function.

SM30 protein function during sea urchin larval spicule formation.

A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO's) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins.

Phase transitions in biogenic amorphous calcium carbonate.

Crystalline biominerals do not resemble faceted crystals. Current explanations for this property involve formation via amorphous phases. Using X-ray absorption near-edge structure (XANES) spectroscopy and photoelectron emission microscopy (PEEM), here we examine forming spicules in embryos of Strongylocentrotus purpuratus sea urchins, and observe a sequence of three mineral phases: hydrated amorphous calcium carbonate (ACC · H(2)O) → dehydrated amorphous calcium carbonate (ACC) → calcite. Unexpectedly, we find ACC · H(2)O-rich nanoparticles that persist after the surrounding mineral has dehydrated and crystallized. Protein matrix components occluded within the mineral must inhibit ACC · H(2)O dehydration. We devised an in vitro, also using XANES-PEEM, assay to identify spicule proteins that may play a role in stabilizing various mineral phases, and found that the most abundant occluded matrix protein in the sea urchin spicules, SM50, stabilizes ACC · H(2)O in vitro.

Molecular aspects of biomineralization of the echinoderm endoskeleton.

Echinoderms possess a rigid endoskeleton composed of calcite and small amounts of occluded organic matrix proteins. The test (i.e., the shell-like structure of adults), spines, pedicellariae, tube feet, and teeth of adults, as well as delicate endoskeletal spicules found in larvae of some classes, are the main skeletal structures. They have been intensively studied for insight into the mechanisms of biomineralization. Recent work on characterization of the mineral phase and occluded proteins in embryonic skeletal spicules shows that these simple-looking structures contain scores of different proteins, and that the mineral phase is composed of amorphous calcium carbonate (ACC), which then transforms to an anhydrous ACC and eventually to calcite. Likewise, the adult tooth shows a similar transition from hydrated ACC to anhydrous ACC to calcite during its formation, and a similar transition is likely occurring during adult spine regeneration. We speculate that: (1) the ACC precursor is a general strategy employed in biomineralization in echinoderms, (2) the numerous occluded proteins play a role in post-secretion formation of the mature biomineralized structure, and (3) proteins with "multi-valent" intrinsically disordered domains are important for formation of occluded matrix structures, and regulation of crucial matrix-mineral interactions, such as ACC to calcite transitions and polymorph selection.

Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix.

BACKGROUND: The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously. RESULTS: Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components. CONCLUSIONS: The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins.

SpSM30 gene family expression patterns in embryonic and adult biomineralized tissues of the sea urchin, Strongylocentrotus purpuratus.

The SpSM30 gene family of the sea urchin, Strongylocentrotus purpuratus, is comprised of six members, designated SpSM30A through SpSM30F (Livingston et al., 2006). The SpSM30 proteins are found uniquely in embryonic and adult mineralized tissues of the sea urchin. Previous studies have revealed that SpSM30 proteins are occluded within the embryonic endoskeleton and adult mineralized tissues (Killian and Wilt, 1996; Mann et al., 2008a,b; Urry et al., 2000). Furthermore, some of the SpSM30 proteins are among the most abundant of the approximately four-dozen integral matrix proteins of the larval spicule (Killian and Wilt, 1996). The amino acid sequence, protein domain architecture, and contiguity within the genome strongly support the supposition that the six genes constitute a gene family. Reverse transcription-polymerase chain reaction (RT-PCR) is used in the present study to describe the time course of expression of the family members during embryonic development, and their expression in adult tissues. SpSM30A, B, C and E are expressed, albeit at different levels, during overt spicule deposition in the embryo with some differences in the precise timing of expression. SpSM30D is not expressed in the embryo, and SpSM30F is expressed transiently and at low levels just prior to overt spicule formation. Whole mount in situ hybridization studies show that SpSM30A, B, C, and E are expressed exclusively in primary mesenchyme (PMC) cells and their descendants. In addition, tissue fractionation studies indicate that SpSM30F expression is highly enriched in PMCs. Each adult tissue examined expresses a different cohort of the SpSM30 family members at varying levels: SpSM30A mRNA is not expressed in adult tissues. Its expression is limited to the embryo. Conversely, SpSM30D mRNA is not expressed in the embryo, but is expressed in adult spines and teeth. SpSM30B and SpSM30C are expressed at modest levels in all mineralized adult tissues; SpSM30E is expressed highly in tooth and test; and SpSM30F is expressed in spine and at low levels in the other adult tissues except the test. Relative levels of expression of the several family members in these different tissues vary widely. It is likely SpSM30 proteins play a vital, but still unknown, role in biomineralization of these tissues during development.

Mechanism of calcite co-orientation in the sea urchin tooth.

Sea urchin teeth are remarkable and complex calcite structures, continuously growing at the forming end and self-sharpening at the mature grinding tip. The calcite (CaCO(3)) crystals of tooth components, plates, fibers, and a high-Mg polycrystalline matrix, have highly co-oriented crystallographic axes. This ability to co-orient calcite in a mineralized structure is shared by all echinoderms. However, the physico-chemical mechanism by which calcite crystals become co-oriented in echinoderms remains enigmatic. Here, we show differences in calcite c-axis orientations in the tooth of the purple sea urchin ( Strongylocentrotus purpuratus ), using high-resolution X-ray photoelectron emission spectromicroscopy (X-PEEM) and microbeam X-ray diffraction (muXRD). All plates share one crystal orientation, propagated through pillar bridges, while fibers and polycrystalline matrix share another orientation. Furthermore, in the forming end of the tooth, we observe that CaCO(3) is present as amorphous calcium carbonate (ACC). We demonstrate that co-orientation of the nanoparticles in the polycrystalline matrix occurs via solid-state secondary nucleation, propagating out from the previously formed fibers and plates, into the amorphous precursor nanoparticles. Because amorphous precursors were observed in diverse biominerals, solid-state secondary nucleation is likely to be a general mechanism for the co-orientation of biomineral components in organisms from different phyla.

Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule.

Sea urchin larval spicules transform amorphous calcium carbonate (ACC) into calcite single crystals. The mechanism of transformation is enigmatic: the transforming spicule displays both amorphous and crystalline properties, with no defined crystallization front. Here, we use X-ray photoelectron emission spectromicroscopy with probing size of 40-200 nm. We resolve 3 distinct mineral phases: An initial short-lived, presumably hydrated ACC phase, followed by an intermediate transient form of ACC, and finally the biogenic crystalline calcite phase. The amorphous and crystalline phases are juxtaposed, often appearing in adjacent sites at a scale of tens of nanometers. We propose that the amorphous-crystal transformation propagates in a tortuous path through preexisting 40- to 100-nm amorphous units, via a secondary nucleation mechanism.

The dynamics of secretion during sea urchin embryonic skeleton formation.

Skeleton formation involves secretion of massive amounts of mineral precursor, usually a calcium salt, and matrix proteins, many of which are deposited on, or even occluded within, the mineral. The cell biological underpinnings of this secretion and subsequent assembly of the biomineralized skeletal element is not well understood. We ask here what is the relationship of the trafficking and secretion of the mineral and matrix within the primary mesenchyme cells of the sea urchin embryo, cells that deposit the endoskeletal spicule. Fluorescent labeling of intracellular calcium deposits show mineral precursors are present in granules visible by light microscopy, from whence they are deposited in the endoskeletal spicule, especially at its tip. In contrast, two different matrix proteins tagged with GFP are present in smaller post-Golgi vesicles only seen by electron microscopy, and the secreted protein are only incorporated into the spicule in the vicinity of the cell of origin. The matrix protein, SpSM30B, is post-translationally modified during secretion, and this processing continues after its incorporation into the spicule. Our findings also indicate that the mineral precursor and two well characterized matrix proteins are trafficked by different cellular routes.

Probing the organic-mineral interface at the molecular level in model biominerals.

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.

Developmental biology meets materials science: Morphogenesis of biomineralized structures.

Biomineralization is the process by which metazoa form hard minerals for support, defense, and feeding. The minerals so formed, e.g., teeth, bones, shells, carapaces, and spicules, are of considerable interest to chemists and materials scientists. The cell biology underlying biomineralization is not well understood. The study of the formation of mineralized structures in developing organisms offers opportunities for understanding some intriguing aspects of cell and developmental biology. Five examples of biomineralization are presented: (1) the formation of siliceous spicules and frustules in sponges and diatoms, respectively; (2) the structure of skeletal spicules composed of amorphous calcium carbonate in some tunicates; (3) the secretion of the prism and nacre of some molluscan shells; (4) the development of skeletal spicules of sea urchin embryos; and (5) the formation of enamel of vertebrate teeth. Some speculations on the cellular and molecular mechanisms that support biomineralization, and their evolutionary origins, are discussed.

The localization of occluded matrix proteins in calcareous spicules of sea urchin larvae.

The sea urchin embryo forms calcareous endoskeletal spicules composed of calcite and an occluded protein matrix. Though the latter is approximately 0.1% of of the mass, the composite has substantially altered material properties, e.g., conchoidal fracture planes and increased hardness. Experiments were conducted to examine the localization of matrix proteins occluded in the mineral by use of immunocytochemistry coupled with scanning electron microscopy (SEM). The isolated, unfixed spicules were etched under relatively gentle conditions and exposed to affinity purified antibodies made against two different matrix proteins, as well as an antibody to the entire constellation of matrix proteins. Immunogold tagged secondary antibody was used to observe antibody localization in the back scatter mode of SEM. All proteins examined were very widely distributed throughout the calcite, supporting a model of the structure in which a multiprotein assemblage is woven with fine texture around microcrystalline domains of calcite. Gentle etching revealed a laminar arrangement of calcite solubility, consistent with a stepwise deposition of matrix and mineral to increase girth of the spicule.

Ultrastructural localization of spicule matrix proteins in normal and metalloproteinase inhibitor-treated sea urchin primary mesenchyme cells.

Studies of the sea urchin larval skeleton have contributed greatly to our understanding of the process of biomineralization. In this study we have undertaken an investigation of the morphology of skeleton formation and the localization of proteins involved in the process of spicule formation at the electron microscope level. Sea urchin primary mesenchyme cells undergo a number of morphological changes as they synthesize the larval skeleton. They form a large spicule compartment that surrounds the growing spicule and, as spicule formation comes to an end, the density of the cytoplasm decreases. Inhibition of spicule formation by specific matrix metalloproteinase inhibitors or serum deprivation has some subtle effects on the morphology of cells and causes the accumulation of specific classes of vesicles. We have localized proteins of the organic matrix of the spicule and found that one protein, SM30, is localized to the Golgi apparatus and transport vesicles in the cytoplasm as well as throughout the occluded protein matrix of the spicule itself. This localization suggests that SM30 is an important structural protein in the spicule. Another spicule matrix protein, SM50, has a similar cytoplasmic localization, but in the spicule much of it is localized at the periphery of the spicule compartment, and consequently it may play a role in the assembly of new material onto the growing spicule or in the maintenance of the integrity of the matrix surrounding the spicule.

Development of calcareous skeletal elements in invertebrates.

Most metazoans require skeletal support systems. While the formation of bones and teeth in vertebrates has been well studied, endo- and exoskeleton development of non-vertebrates, especially calcification during terminal differentiation, has been neglected. Biomineralization of skeletons in invertebrates presents interesting research opportunities. We undertake here to survey some of the better understood examples of skeletal development in selected invertebrates. The differentiation of the skeletal spicules of euechinoid larvae and other non-vertebrate deuterostomes, the shells of molluscs, and the calcification of crustacean carapaces are surveyed. The diversity of these different kinds of animals and our present limited understanding make it difficult to identify unifying themes, but there certainly are unifying questions: How is the mineral precursor secreted? What is the nature of the interaction of mineral with the matrix proteins of the skeleton? Is there any conservation of protein domains in matrix proteins found in skeletal elements from different phyla? Are there common strategies in the development of organs that form mineralized structures?

Biomineralization of the spicules of sea urchin embryos.

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.