Extracellular Formation of Body and Tunic Spicules in the New Zealand Solitary Ascidian Pyura pachydermatina (Urochordata, Ascidiacea)

The New Zealand ascidian Pyura pachydermatina has a 7–10 cm long body at the end of a stalk up to 1 m long and 1–2 cm in diameter. Two different spicule types are present: dumbbell-shaped spicules of calcite in the fibrous tunic that covers the body and stalk, and antler-shaped spicules of amorphous calcium carbonate in the soft body tissues. Both types form extracellularly within a closed compartment surrounded by an epithelium of sclerocytes. In adults the tunic spicules form in 2–3 weeks in the lumen of the tunic blood vessels, as determined by calcein uptake studies. They add mineral only while surrounded by the sclerocyte epithelium, which is anchored to the vessel wall. Ultimately the sclerocytes rupture at one or more leading points on the spicule. The blood vessel epithelium also becomes very thin at these points and either ruptures or the cells separate. allowing the spicules to migrate out into the tunic. The sclerocytes degenerate and the blood vessel closes behind the migrating spicule, thus maintaining the vessel's integrity. Tunic spicules accumulate in the subcuticular region of the stalk, but the outermost layer of tunic covering the body is periodically sloughed off along with some spicules. This gives the "neck" between body and stalk a flexibility that allows it to orient to currents, and prevents an accumulation of epizoic organisms on the body. The antler spicules form within blood sinuses of the body tissues. The mineral and organic material are arranged in concentric layers. In the branchial sac, oral tentacles, gut and endostyle, where antler spicules occur most densely, the branches interlock, providing support to the soft tissues. They are of many sizes and apparently remain where they form, increasing in number and size throughout the animal's lifespan.     

Ultrastructural Aspects of Spicule Formation in the Solitary Ascidian Herdmania momus (Urochordata,Ascidiacea)

The solitary stolidobranch ascidian Herdmania momus contains numerous calcium carbonate spicules in its tunic and body tissues. The slender body spicules form inside complex sheaths in the body wall and branchial basket, where they remain for the life of the animal. The much smaller tunic spicules form inside the tunic blood vessels and then migrate to the tunic surface, where they become anchored by their spiny base. This paper is an ultrastructural investigation of the formation of the body spicules; the tunic spicules, which apparently form quite differently, will be the focus of a future study. The body spicules are composed of rows of closely packed acicular spines which form completely extracellularly. The spine tips are covered by flattened, highly pseudopodial sclerocytes bound together by tightly interdigitating cell processes. The basal regions of contiguous spines are covered by very thin sclerocyte cell processes. An organic matrix is present within the spines; its exact nature is not clear. A very dense extracellular inter-spine matrix is located between the spine tips and the contiguous basal regions. Presclerocytes within the sheaths between the spicules are probably responsible for formation of the extracellular structures of the sheaths. The presclerocytes appear to aggregate and transform into sclerocytes at the apical end of the spicule. New spines are added at the apical end of the spicule as well as between larger spines. Comparisons are made between body spicule formation in H. momus and skeletogenesis in echinoids.     

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.     

In vivo study of microsclere formation in sponges of the genus <Emphasis Type="Italic">Mycale</Emphasis> (Demospongiae,Poecilosclerida)

The process of microsclere secretion was examined in vivo through glass coverslip implants in three species of the genus Mycale from São Sebastião channel, southeastern Brazil: Mycale (Aegogropila) angulosa, Mycale (Arenochalina) laxissima, and Mycale (Carmia) microsigmatosa. All three species adhered well to coverslips and developed normally through at least 2 weeks. Similar experiments with different species (Cinachyrella alloclada, Amphimedon viridis, Haliclona melana, and Aplysina caissara) were also successful with one exception (the cartilaginous Chondrilla nucula), indicating that the method can be applied to most demosponges. Microsclerocyte size varied according to the type of microsclere secreted, but all were elongated to fusiform and had small, anucleolated nuclei. Spicules were transported by microsclerocytes alone, without any other cell type ("helper cells") involved. Secretion of a microsclere was performed by a single sclerocyte. Although some axial filaments were found free in the mesohyl, all microsclere secretion in these animals was fully intracellular. Normal axial filaments were observed in most types of microscleres of the Mycale species (sigmas, toxas, and microxeas). Timed observations of sclerocytes suggest that immature spicules with the aspect of short straight rods with thick ends might be the precursors of the anisochelae. Observed differences in the size versus number of toxa secreted may indicate either the presence of two distinct subpopulations of toxa-producing microsclerocytes or that the initial number of axial filaments at the beginning of silica deposition may determine the final size of the spicules. Although other microscleres such as sigmas and chelae are secreted in a one cell-one spicule basis, several toxas and microxeas can be secreted simultaneously in a single cell.     

Intra-epithelial spicules in a homosclerophorid sponge

Attempts to understand the intricacies of biosilicification in sponges are hampered by difficulties in isolating and culturing their sclerocytes, which are specialized cells that wander at low density within the sponge body, and which are considered as being solely responsible for the secretion of siliceous skeletal structures (spicules). By investigating the homosclerophorid Corticium candelabrum, traditionally included in the class Demospongiae, we show that two abundant cell types of the epithelia (pinacocytes), in addition to sclerocytes, contain spicules intracellularly. The small size of these intracellular spicules, together with the ultrastructure of their silica layers, indicates that their silicification is unfinished and supports the idea that they are produced "in situ" by the epithelial cells rather than being incorporated from the intercellular mesohyl. The origin of small spicules that also occur (though rarely) within the nucleus of sclerocytes and the cytoplasm of choanocytes is more uncertain. Not only the location, but also the structure of spicules are unconventional in this sponge. Cross-sectioned spicules show a subcircular axial filament externally enveloped by a silica layer, followed by two concentric extra-axial organic layers, each being in turn surrounded by a silica ring. We interpret this structural pattern as the result of a distinctive three-step process, consisting of an initial (axial) silicification wave around the axial filament and two subsequent (extra-axial) silicification waves. These findings indicate that the cellular mechanisms of spicule production vary across sponges and reveal the need for a careful re-examination of the hitherto monophyletic state attributed to biosilicification within the phylum Porifera.     

Effects of germanium on the morphology of silica deposition in a freshwater sponge

The effect of germanium on the secretion of siliceous spicules by the freshwater sponge Spongilla lacustris was investigated by exposing germinating and hatching gemmules to varying concentrations of germanium (Ge) in the presence of silicon (Si). Results were analyzed quantitatively and qualitatively and demonstrate that a [Ge]/[Si] (= molar ratio) of 1.0 completely inhibits silicon deposition. Intermediate ratios (0.5, 0.1, 0.01) which are permissive to spicule appearance result in fewer, shorter, and thinner spicules, in proportionately fewer microscleres, and in short bulbous megascleres. The size of the bulb increases with increasing [Ge]/[Si], while the length of the bulbous megascleres decreases with increasing [Ge]/[Si]. Microscleres do not demonstrate these graded responses suggesting that they are secreted in an all or none manner. Swellings produced in pond water and bulbs produced in germanium appear to decrease in size with time indicating a spreading of the accumulated silica. The effect of germanium on spicule secretion can be partially explained by its ability to uncouple the growth in length of the axial filament from the growth of the surrounding silicalemma. Under these conditions excess silicalemma is produced in which silica accumulates as bulbs in short spicules. Continuous exposure to Ge is necessary to produce this altered morphology. It is conjectured that the bulbs may be retained due to an inhibition of spreading. which in turn may be caused by the incorporation of germanium into the silica.     

Spicule formation in the calcareous sponge Sycon ciliatum

Summary The spicule primordium is formed in an intercellular cavity within a group of sclerocytes. This cavity contains organic material which ensheaths the growing spicule but does not appear to determine the nature of the mineral morph (magnesian calcite) or the crystallographic orientation of the spicule. The tip of each growing spicule ray is seated in a dense cup in the cytoplasm of the sclerocyte concerned. Both ends of monaxons are initially inserted each into a dense cup. As rays elongate the sclerocyte membrane around the tip becomes invaginated and forms a system of converging spaces that possibly indicate high secretory activity in that region. Spicule growth involves the displacement and expansion of the organic sheath by the enlarging spicule. Fully formed spicules which are exposed to the mesohyl become surrounded by collagen fibrils. However, these fibrils are in no way concerned with the process of mineral deposition and are never found within the spicule calcite.     

Secondary Male Sex Characteristics of Hoplolaimus galeatus

The secondary male sex characteristics of Hoplolaimus galeatus consisted of caudal alae, two independently retractable spicules and a gubernaculum with two bilobed titillae. The spicules were dimorphic, with the outer one possessing a velum. When both spicules were completely extruded, the only open orifice on the ventral surface of the posterior region was formed by the close association of these two appendages. In specimens where the inner spicule was slightly retracted, the velum almost completely surrounded the inner spicule. When the inner spicule was retracted further, the velum appeared to convolute, closing the orifice described above.     

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.     

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.     

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.     

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE*

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl₂ and NaHCO₃. The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.     

Spicule Formation in the Invertebrates with Special Reference to the Gorgonian Leptogorgia virgulata

Many of the invertebrates possess calcium carbonate spicules.This paper is a review of the formation of these structuresin the Porifera, Coelenterata, Platyhelminthes, Mollusca, Echinodermataand Ascidiacea. Mature spicules appear to be extracellular structures.Sponge spicules initiate intercellularly then become extracellular.Alcyonarian, turbellarian, echinoid and ascidian spicule depositionbegins intracellularly and then becomes extracellular. The continuationof growth in the extracellular environment has not been documentedexcept for the echinoids. Placophoran spicules initiate andremain as extracellular structures. Early spicule growth seemsto occur from or within a single cell. However, cell aggregationand/or neighboring cells appear to be important to the processof spicule formation. The spicule forming cells, in general,are found in a collagenous medium which may be associated withspicule growth. The organic matrix from the spicules of the gorgonian Leptogorgiavirgulata is a glycoprotein. Autoradiography reveals that thismatrix is apparently synthesized in the rough endoplasmic reticulumand Golgi complexes and then transported to the spicule formingvacuole via Golgi vesicles. To gain information about the entryand transport of calcium ions, the effects of ouabain and vanadateon calcium uptake were examined. Ouabain had no effect on calciumuptake. Vanadate treatment increased the uptake of calcium inscleroblasts and epithelial tissue and decreased its uptakein spicules. This may suggest that vanadate sensitive ATPasesare involved in the pumping of calcium out of scleroblasts,out of epithelial cells into the mesoglea, and into scleroblastorganelles. Autoradiography using ⁴⁵Ca indicates that the majorityof these ions initially accumulate in the branch axis. The labelmoves through the axial epithelium to the mesoglea and reachesthe spiculeforming vacuoles in the scleroblasts via dense bodies     

Dynamics of spicule production in the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis> during in vitro cell culture and seasonal development in the field

To characterize the formation of silica spicules, the dynamics of spiculogenesis of an intertidal marine sponge Hymeniacidon perlevis (Montagu 1818) (Porifera: Demospongiae) were investigated by measuring the gene expression of silicatein (the enzyme responsible for spicule silicification) and the dimensional changes of spicules during the developmental process of individual sponges and in cell cultures of primmorphs of archaeocyte-dominant cell populations. The different developmental stages of spicules were documented by time-lapse microscopy and observed by transmission electron microscopy during a 1-month culture period. During its annual life cycle, H. perlevis has four different developmental stages: dormancy, resuscitation, bloom, and decline. Field-grown individual sponge samples at different stages were collected over 7 months (March to September 2005). The dimensions of the silica spicules from these samples were microscopically measured and statistically analyzed. This analysis and the material properties of the spicules allowed them to be classified into four groups representing the different developmental stages of spiculogenesis. Silicatein expression in the bloom stage was more than 100 times higher than that in the other stages and was correlated with the spicule developmental stage. The trend of spicule formation in field-grown sponges was consistent with the trend in cell culture. A new parameter, the maturation degree (MD) of spicules (defined as the ratio of actual to theoretical silica deposition of mature spicules), was introduced to quantify spicule development. Silica spiculogenesis during H. perlevis development was delineated by comparing MD and silicatein expression.     

Calcareous sponge biomineralization: Ultrastructural and compositional heterogeneity of spicules in Leuconia johnstoni

In contrast to siliceous sponge spicules, the biomineralization in calcareous sponges is poorly understood. In particular, the existence of a differentiated central core in calcareous spicules is still controversial. Here we combine high-spatial resolution analyses, including NanoSIMS, Raman, SXM, AFM, SEM and TEM to investigate the composition, mineralogy and ultrastructure of the giant tetractines of Leuconia johnstoni Carter, 1871 (Baeriidae, Calcaronea) and the organization of surrounding cells. A compositionally distinct core is present in these spicule types. The core measures 3.5–10 μm in diameter and is significantly depleted in Mg and lightly enriched in S compared with the adjacent outer layer in the spicule. Measured Mg/Ca ratios in the core range from 70 to 90 mmol/mol compared to 125–130 mmol/mol in the adjacent calcite envelope. However, this heterogeneous distribution of Mg and S is not reflected in the mineralogy and the microstructure. Raman spectroscopy demonstrates a purely calcitic mineralogy. SEM examination of slightly etched spicules indicates an ultrastructure organized hierarchically in a concentric pattern, with layers less than 250 nm in width inside layers averaging 535 ± 260 nm. No change in structural pattern corresponds to the Mg/Ca variation observed. AFM and TEM observations show a nanogranular organization of the spicules with a network of intraspicular organic material intercalated between nanograins 60–130 nm in diameter. Observations of sclerocyte cells in the process of spiculogenesis suggest that the compositionally distinct core is produced by a sub-apical sclerocyte “founder cell” that controls axial growth, while the envelope is secreted by lateral sclerocytes “thickener cells”, which control radial growth.     

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.     

Regulation of sclerocyte differentiation in the hydroxyurea treated sponge Ephydatia fluviatilis

Abstract. Freshwater sponges ( Ephydatia fluviatilis ) were raised in mineral medium containing hydroxyurea (HU) at a final concentration of 100 μg/ml. The spicules present in these sponges were counted daily. In HU-treated sponges, the regulation mechanisms of skeletogenesis remained functional despite the absence of an aquiferous system. Indeed, as in controls, the differentiation of sclerocytes from stem cells ceased when a critical number of spicules had been secreted. Stem cells again started to differentiate into sclerocytes when isolated from sponges that had completed their skeletogenesis. The number of spicules secreted was found to be an inverse function of silicate concentration. These results demonstrate that the regulation of skeletogenesis is not dependent on the differentiation of the aquiferous system.     

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.     

Differentiation of Sea Urchin Micromeres: Correlation between Specific Protein Synthesis and Spicule Formation

Sea urchin micromeres were isolated from the 16-cell stage embryos and cultured until they differentiated into spicule-forming cells. Electrophoretic analysis of proteins labeled with [³⁵S]-methionine showed that the differentiation accompanied the synthesis of five cell-specific proteins. These proteins appeared prior to spicule formation and were synthesized continuously or maintained stably while the cultured micromeres formed spicules. In contrast, these proteins were hardly detectable during development of the meso- and macromeres. Correlation between synthesis of the specific proteins and spicule formation was further examined in culture conditions which inhibit spicule formation. In Zn²⁺ -containing or serum-free medium, the micromere descendants failed to form spicules and exhibited markedly reduced synthesis of one of the specific proteins (32 K daltons). After removal of Zn²⁺, or addition of serum, however, spicules were formed with delay but concomitantly with an increase in the synthesis of this protein. This clear correlation suggests the participation of the 32 K protein in the process of spicule formation.     

Molecular tilting in dried elastoidin and its implications for the structures of other collagen fibrils

Wet elastoidin spicules (fish fin rays) yield low-angle meridional X-ray diffraction patterns which resemble those from tendons. However, when the spicule dries the meridian splits into the arms of a diagonal cross (sometimes only one arm appears). Of the possible explanations we reject shearing of the axial arrangement of molecules but confirm tilting. We suggest that, in three dimensions, the molecules are tilted at angles which vary from 0 degrees at the centre to some maximum value at the surface of the spicule, resembling torsion of the array of molecules. Molecular tilting probably occurs in other collagen fibrils.