Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.     

Axial growth of hexactinellid spicules: formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS–PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30–60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules.     

Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets

Incubation of human platelets with unilamellar vesicles composed of dilauroylphosphatidylcholine (DLPC) induces shedding of small vesicular structures from the platelet plasma membrane. No significant cell lysis is observed during the process of shedding. Isolated spicules contain the major membrane glycoproteins, Ib, IIb, and IIIa, which are used to define the sidedness of the spicule membrane. These glycoproteins are completely susceptible to chymotrypsin treatment, whereas cytoskeletal proteins are inaccessible towards this enzyme. This demonstrates that the spicule membranes have a right-side-out orientation in as far as membrane proteins are concerned. Isolated spicules were 30-fold more active than platelets in stimulating prothrombin conversion to thrombin by the prothrombinase complex (factors Xa, Va and Ca2+). The increased prothrombinase activity reflects an increased amount of phosphatidylserine in the outer leaflet of the spicule membrane. Protein analysis of platelet spicules and native platelets reveals a number of differences, the most conspicuous of which is the virtual absence of myosin in the spicule preparations. It is proposed that a lack of myosin produces a different cytoskeletal organization in the spicules. This enables phosphatidylserine to become exposed at the outer surface of the spicule membrane.     

Magnetic resonance imaging of the siliceous skeleton of the demosponge Lubomirskia baicalensis

The skeletal elements (spicules) of the demosponge Lubomirskia baicalensis were analyzed; they are composed of amorphous, non-crystalline silica, and contain in a central axial canal the axial filament which consists of the enzyme silicatein. The axial filament, that orients the spicule in its longitudinal axis exists also in the center of the spines which decorate the spicule. During growth of the sponge, new serially arranged modules which are formed from longitudinally arranged spicule bundles are added at the tip of the branches. X-ray analysis revealed that these serial modules are separated from each other by septate zones (annuli). We describe that the longitudinal bundles of spicules of a new module originate from the apex of the earlier module from where they protrude. A cross section through the oscular/apical-basal axis shows that the bundle rays are organized in a concentric and radiate pattern. High resolution magnetic resonance microimaging studies showed that the silica spheres of the spicules in the cone region contain high amounts of 'mobile' water. We conclude that the radiate accretive growth pattern of sponges is initiated in the apical region (cones) by newly growing spicules which are characterized by high amounts of 'mobile' water; subsequently spicule bundles are formed laterally around the cones.     

Plasmodesmata: intercellular tunnels facilitating transport of macromolecules in plants

The major structural and enzymatically active protein in spicules from siliceous sponges, e.g., for Suberites domuncula studied here, is silicatein. Silicatein has been established to be the key enzyme that catalyzes the formation of biosilica, a polymer that represents the inorganic scaffold for the spicule. In the present study, it is shown, by application of high-resolution transmission and scanning transmission electron microscopy that, during the initial phase of spicule synthesis, nanofibrils with a diameter of around 10 nm are formed that comprise bundles of between 10 and 20 nanofibrils. In intracellular vacuoles, silicasomes, the nanofibrils form polar structures with a pointed tip and a blunt end. In a time-dependent manner, these nanofibrillar bundles become embedded into a Si-rich matrix, indicative for the formation of biosilica via silicatein molecules that form the nanofibrils. These biosilicified nanofibrillar bundles become extruded from the intracellular space, where they are located in the silicasomes, to the extracellular environment by an evagination process, during which a cellular protrusion forms the axial canal in the growing spicule. The nanofibrillar bundles condense and progressively form the axial filament that becomes localized in the extracellular space. It is concluded that the silicatein-composing nanofibrils act not only as enzymatic silica bio-condensing platforms but also as a structure-giving guidance for the growing spicule.     

Silica deposition in Demosponges: spiculogenesis in Crambe crambe

Transmission electron-microscopy images coupled with dispersive X-ray analysis of the species Crambe crambe have provided information on the process of silica deposition in Demosponges. Sclerocytes (megasclerocytes) lie close to spicules or surround them at different stages of growth by means of long thin enveloping pseudopodia. Axial filaments occur free in the mesohyl, in close contact with sclerocytes, and are triangular in cross section, with an internal silicified core. The unit-type membrane surrounding the growing spicule coalesces with the plasmalemma. The axial filament of a growing spicule and that of a mature spicule contain 50%-70% Si and 30%-40% Si relative to that contained in the spicule wall, respectively. The extracellular space between the sclerocyte and the growing spicule contains 50%-65%. Mitochondria, vesicles and dense inclusions of sclerocytes exhibit less than 10%. The cytoplasm close to the growing spicule and that far from the growing spicule contain up to 50% and less than 10%, respectively. No Si has been detected in other parts of the sponge. The megascleres are formed extracellularly. Once the axial filament is extruded to the mesohyl, silicification is accomplished in an extracellular space formed by the enveloping pseudopodia of the sclerocyte. Si deposition starts at regularly distributed sites along the axial filament; this may be related to the highly hydroxylated zones of the silicatein-alpha protein. Si is concentrated in the cytoplasm of the sclerocyte close to the plasmalemma that surrounds the growing spicules. Orthosilicic acid seems to be pumped, both from the mesohyl to the sclerocyte and from the sclerocyte to the extracellular pocket containing the growing spicule, via the plasmalemma.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

Socket cells mediate spicule morphogenesis in Caenorhabditis elegans males.

Caenorhabditis elegans male spicule morphogenesis requires the coordinated cellular behaviors of several types of cells. We found that the spicule neurons and sheath cells, although important for spicule function, are dispensable for spicule morphology. In contrast, the spicule socket cells are essential for both spicule elongation and formation of spicule cuticle. The socket cells are not only necessary but also sufficient to produce spicule cuticle. This functional aspect of socket cells is genetically separable from their function in mediating spicule elongation: elongated spicules with defective spicule cuticle can be formed. During spicule morphogenesis, the expression of an egl-17::GFP reporter gene is found in the spicule socket cells and its expression appears to be regulated in the socket cells. Mutants defective in TGF-beta signaling display a crumpled spicules phenotype as a result of failure of socket cell movement during spicule morphogenesis. These observations suggest that both the FGF and the TGF-beta signaling pathways might be involved in spicule elongation.     

A new species of Dipetalonema (Filarioidea: Onchocercidae) from Ateles chamek from the Beni of Bolivia

We describe a new species of Dipetalonema occurring in the body cavity of Ateles chamek (Humboldt, 1812) from north-central Bolivia. Morphologic characters serving to separate Dipetalonema yatesi n. sp. from known forms include a vagina vera with a simple tube and thin walls and a left spicule, which possesses a handle shorter than the lamina (ratio 2.7); the latter displays an anterior membranous alae similar in length to the terminal flagellum, a distal extremity of the left spicule within a simple hook and a membrane, phasmids at the basis of the lappets, and heterogeneous muscles occupying the whole cavity. Dipetalonema yatesi n. sp. can be separated from Dipetalonema robini, Dipetalonema gracile, and Dipetalonema graciliformis, between other characters, in having a simple vagina vera instead of a sinuous one, and from Dipetalonema caudispina and Dipetalonema freitasi in having the lamina of the left spicule divided in a membranous alae and a terminal flagellum.     

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+-ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere-Derived Cells.

Embryos kept with omeprazole, a specific H⁺, K⁺-ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro-mere-derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole-sensitive H⁺, K⁺-ATPase, an H⁺pump, seems to be indispensable for CaCO₃ deposition (formation of spicule rod) in these spicule forming cells. H⁺, produced in overall reaction for CaCO₃ formation: Ca²⁺+ CO₂+H₂O°CaCO₃+2H⁺, is probably released from the cells by this H⁺pump and hence, this reaction tends to go to CaCO₃ production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H⁺, K⁺-ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H⁺ejection in H⁺, K⁺-ATPase reaction.     

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.     

Molecular shape of Lumbricus terrestris erythrocruorin studied by electron microscopy and image analysis

The molecular structure of erythrocruorin (hemoglobin) from Lumbricus terrestris has been studied by electron microscopy of negatively stained particles. Over 1000 molecular projections were selected from a number of electron micrographs and were then classified by multivariate statistical image-processing techniques. The two main groups of top and side views were each subdivided into smaller classes with significantly different features. About half of the top-view projections exhibit perfect hexagonal symmetry at the current resolution of about 2.0 nm, while the other top views lack this symmetry, probably as a result of tilting of the molecules relative to the carbon support film. The side views were separated into two 'families', each associated with the two different stable side-view positions the molecules can take. From these narrow stable side-views, the two families of projections are, again, generated by tilting. The symmetry properties of the three non-tilted projections show that Lumbricus erythrocruorin has a pointgroup D6 (622) symmetry rather than D3 (32).     

Effects of Germanium (Ge) on the silica spicules of the marine sponge Suberites domuncula: Transformation of spicule type

Germanium (Ge), in the form of germanic acid, at a Ge/Si molar ratio of 1.0 inhibits gemmule development and silica deposition in the marine demosponge Suberites domuncula. Lower Ge/Si ratios inhibit the growth in length of the silica spicules (tylostyles) producing short structures, but with relatively normal morphology and close to normal width; spherical protuberances occasionally occur on these spicules. A few of the short spicules possess completely round rather than pointed tips. Many of the latter develop when Ge is added (pulsed) to growing animals, thus inducing a change in spicule type. These results indicate that the growth in length of the axial filament is more sensitive to Ge inhibition than is silica deposition and that pointed spicule tips normally develop because the growth of the axial filament at the spicule tip is more rapid than silica deposition. Newly formed spicules initiate silica deposition at the spicule head but the absence of Ge-induced bulbs as in freshwater spicules (oxeas) leaves open the question of whether there is a silicification center(s) present in Suberites tylostyles. The morphogenesis of freshwater oxeas and of marine tyolstyles appears fundamentally different-bidirectional growth in the former and unidirectional growth in the latter. X-ray analysis demonstrate relatively uniform Ge incorporation into the silica spicules with considerable variation from spicule to spicule in the incorporated level. Increased silicic acid concentration induces the formation of siliceous spheres, suggesting that the axial filament becomes prematurely encased in silica.     

Spicule Formation-Inducing Substance in Sea Urchin Embryo

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro , their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation-inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat-labile and was inactivated by trypsin.     

Does Protein Phosphorylation by Protein Kinase C Support Pseudopodial Cable Growth in Cultured Micromere-Derived Cells of the Sea Urchin,Hemicentrotus pulcherrimus?

In cultured cells derived from micromeres, H-7 strongly inhibited the outgrowth of pseudopodial cables and the formation of spicule rods at concentrations around the Ki values for protein kinases. HA1004 did not inhibit the cable growth and spicule rod formation in these cells at higher concentrations than the Ki values for cyclic nucleotide-dependent protein kinases. Pseudopodial cable growth was also inhibited by H-7 in furosemide-treated cells which were able to undergo normal growth of the cables without the formation of spicule rods. Protein phosphorylation, measured by ³²P incorporation into proteins in the cells exposed to ³²Pi, was inhibited by H-7 at the concentrations for the blockage of the cable growth but was hardly blocked by HA1004. The cable growth and protein phosphorylation were activated by phorbol 12-myristate 13-acetate. The activity of Ca²⁺, phospholipid-dependent protein kinase (protein kinase C), which was inhibited by H-7, became appreciably high in micromere-derived cells at 16 hr of culture at 20°C, at which the outgrowth of pseudopodial cables was going to be initiated and gradually increased keeping pace with the cable growth. These suggest that the outgrowth of the cables is supported by protein phosphorylation catalyzed by protein kinase C.     

Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A--gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.     

Inhibitors of procollagen C-terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo

In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell-free extracts of gastrula-stage embryos of Strongylocentrotus purpuratus contain a procollagen C-terminal proteinase (PCP) activity. A rationally designed non-peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG-HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG-HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG-HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen-cleavage assay. However, FG-HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG-HL1 and FG-HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.