Fibronectin from the ovary of the sea urchin,Pseudocentrotus depressus

Summary Fibronectin, with a subunit molecular weight of 220,000 daltons, was isolated from the ovary of the sea urchin,Pseudocentrotus depressus, using affinity chromatography on heat-denatured mammalian collagen coupled to Sepharose 4B. The distribution of fibronectin in the sea urchin ovary was examined by indirect immunofluorescence using antifibronectin serum. The basement membrane and the connective tissues exhibited strong fluorescence. The fibronectin was localized closely together with collagen bundles in the sea urchin ovary. Biochemical and immunological examinations indicate that sea urchin fibronectin has similar properties as those of mammalian fibronectin.     

The compass depressors ofParacentrotus lividus (Echinodermata,Echinoida): ultrastructural and mechanical aspects of their variable tensility and contractility

Summary The compass depressors are bands of soft tissue which connect the compass ossicles of the echinoid lantern to the inner edge of the test. They are essentially ligaments with on one side a thin layer of muscle cells. The ligamentous component consists mainly of a parallel array of collagen fibrils with interspersed 12 nm microfibrils. The most notable cellular constituents are granule-containing cell bodies and their processes which resemble the juxtaligamental cells that have been found in all echinoderm mutable collagenous tissues and which may control the tensility of these tissues. The muscle cells occupy about 8% of the total cross-sectional area of the compass depressor and are located in a richly innervated pseudostratified myoepithelium. When subjected to constant low loads in creep tests the compass depressor stretches to a fixed length beyond which there is no further extension. The length at this creep limit coincides with the maximum length to which the compass depressor is stretched by natural movements of the intact lantern. Stress-strain tests show that treatment with 1 mM acetylcholine or 100 mM K⁺ ions can increase reversibly the stiffness of the compass depressor to an extent that cannot be due to contraction of the myoepithelium, suggesting that the mechanical properties of the ligament are under physiological control. Tension-length data on the myoepithelium suggest that it generates a maximum active tension when the compass depressor is stretched to the creep limit. The implications of these results for the function of the compass depressors are discussed.     

Biochemical and immunological characterization of collagen molecules from echinothurioid sea urchin Asthenosoma ijimai

Collagens collected from the test (the external hard covering of invertebrates) of the sea urchin, Asthenosoma ijimai, were characterized biochemically and immunologically. The amino-acid composition was typical of that of mammalian collagens. Crystals of segment-long-spacing showed that the molecules of sea urchin collagen were 300 nm long. Selective salt precipitation revealed that the collagen has the same solubility characteristics as type I collagen. The collagen was denatured at 23.1 degrees C. Anti-sea urchin collagen antisera were immunologically cross-reacted with collagens of the same species and the starfish Asterina pectinifera. However, the antisera showed no or slight responses to collagens of bovine type I, II, III, IV and V. The collagen molecules contained four alpha-chains, named alpha 1(SU), alpha 2(SU), alpha 3(SU) and alpha 4(SU), respectively. All of the four alpha-chains were eluted in the same fraction on gel filtration chromatography. Chains of alpha 1(SU) and alpha 2(SU) were extracted earlier than alpha 3(SU) and alpha 4(SU) during pepsin digestion. Other biochemical and immunological analyses clearly demonstrated that test of sea urchins contains two genetically different, but biochemically similar, species of collagens, one of which is composed of alpha 1(SU) and alpha 2(SU) chains, and the other of alpha 3(SU) and alpha 4(SU).     

Direct measurement of the rupture force of single pair of decorin interactions

Decorin is one important member of the family of small leucine-rich proteoglycans, which are widely distributed in connective tissues in the body such as tendon and ligament. Decorin may be responsible for collagen fibril connection in those tissues. A recent hypothesis suggests that decorin may bind to collagen with its core protein while binding to another decorin through the interaction with their glycosaminoglycan (GAG) chains. However, there is no direct evidence supporting this hypothesis to date. In this study, the interaction of decorin GAG chains was directly determined for the first time. The rupture force of single bonds between decorins (GAG chains interaction) was determined directly as 16.5+/-5.1 pN using a laser tweezers/interferometer single molecular nanomechanical testing system. This information can improve our understanding of the mechanical properties of connective tissues at the molecular level.     

Distinct maturations of N-propeptide domains in fibrillar procollagen molecules involved in the formation of heterotypic fibrils in adult sea urchin collagenous tissues

We have characterized the primary structure of a new sea urchin fibrillar collagen, the 5alpha chain, including nine repeats of the sea urchin fibrillar module in its N-propeptide. By Western blot and immunofluorescence analyses, we have shown that 5alpha is co-localized in adult collagenous ligaments with the 2alpha fibrillar collagen chain and fibrosurfin, two other extracellular matrix proteins possessing sea urchin fibrillar modules. At the ultrastructural level, the 5alpha N-propeptide is detected at the surface of fibrils, suggesting the retention of this domain in mature collagen molecules. Biochemical characterization of pepsinized collagen molecules extracted from the test tissue (the endoskeleton) together with a matrix-assisted laser desorption ionization time-of-flight analysis allowed us to determine that 5alpha is a quantitatively minor fibrillar collagen chain in comparison with the 1alpha and 2alpha chains. Moreover, 5alpha forms heterotrimeric molecules with two 1alpha chains. Hence, as in vertebrates, sea urchin collagen fibrils are made up of quantitatively major and minor fibrillar molecules undergoing distinct maturation of their N-propeptide regions and participating in the formation of heterotypic fibrils.     

Sea urchin fibrillar collagen 2alpha chain participates in heterotrimeric molecules of (1alpha)(2)2alpha stoichiometry.

In sea urchin, two fibrillar collagen chains (alpha1 and alpha2) have been characterized by molecular biology while two biochemically detected chains (alpha1 and alpha2) have been reported. Here, to determine the relationship between these results, Western-blotting and Edman degradation sequencing of the amino-termini of pepsinized sea urchin fibrillar collagen chains were performed. The data demonstrate that the 2alpha chain corresponds to the alpha2 chain and is involved in the formation of heterotrimeric molecules [(1alpha)(2)2alpha].     

Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

Characterization of fibrosurfin, an interfibrillar component of sea urchin catch connective tissues

The Sea URchin Fibrillar (SURF) domain is a four-cysteine module present in the amino-propeptide of the sea urchin 2alpha fibrillar collagen chain. Despite numerous international genome and expressed sequence tag projects, computer searches have so far failed to identify similar domains in other species. Here, we have characterized a new sea urchin protein of 2656 amino acids made up of a series of epidermal growth factor-like and SURF modules. From its striking similarity to the modular organization of fibropellins, we called this new protein fibrosurfin. This protein is acidic with a calculated pI of 4.12. Eleven of the 17 epidermal growth factor-like domains correspond to the consensus sequence of calcium-binding type. By Western blot and immunofluorescence analyses, this protein is not detectable during embryogenesis. In adult tissues, fibrosurfin is co-localized with the amino-propeptide of the 2alpha fibrillar collagen chain in several collagenous ligaments, i.e., test sutures, spine ligaments, peristomial membrane, and to a lesser extent, tube feet. Finally, immunogold labeling indicates that fibrosurfin is an interfibrillar component of collagenous tissues. Taken together, the data suggest that proteins possessing SURF modules are localized in the vicinity of mineralized tissues and could be responsible for the unique properties of sea urchin mutable collagenous tissues.     

New insights into mutable collagenous tissue: correlations between the microstructure and mechanical state of a sea-urchin ligament

The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs.     

Glycerinated catch apparatus of sea urchin spines: the effects of cations on its mechanical properties and ultrastructure

Sea urchin spinal ligaments (the catch apparatus) were extracted with glycerin, and electron microscopic observations comfirmed that no cell membranes remained intact after glycerination. We studied the effects of cations (Na(+), K(+), Ca(2+), Mg(2+)) on the mechanical properties of the glycerinated ligaments. Monovalent cations decreased whereas divalent cations increased the viscosity of the ligaments. The ion dependencies were similar to previous results with detergent-extracted holothurian dermis, which suggests that the echinoid ligament shares a similar mechanism for changes in mechanical properties with other catch connective tissues. This provides evidence against the hypothesis of that muscles in the catch apparatus are responsible for the changes in mechanical properties of the ligament. Fine projections cross-bridging collagen fibrils were observed in the glycerin-extracted ligaments as well as in the intact ligaments. They were found in all the ionic conditions studied.     

Innate immune response in the sea urchin Echinometra lucunter (Echinodermata)

Echinometra lucunter, (Pindá) is a sea urchin encountered in the Brazilian coast and exposed to high and low temperatures related to low and high tides. Despite their great distribution and importance, few studies have been done on the biological function of their coelomocytes. Thus, Echinometra lucunter perivisceral coelomocytes were characterized under optical and transmission electron microscopy. Phagocytic amoebocytes in the perivisceral coelom were labelled by injecting ferritin, and ferritin labelled phagocytic amoebocytes were found in the peristomial connective tissue after injecting India ink into the tissue, indicating the amoebocytes ability to respond to an inflammatory stimulus. Results showed that the phagocytic amoebocytes were the main inflammatory cells found in the innate immune response of E. lucunter. While other works have recorded these phenomena in sea urchins found in moderate and constant temperature, this study reports on these same phenomena in a tropical sea urchin under great variation of temperature, thus providing new data to inflammatory studies in invertebrate pathology.     

Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties

Summary The problematic ligament of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in catch. In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the sliding fibril hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.     

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified beta gamma-subunits of alpha beta gamma-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the alpha- and beta-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems.     

Transversely isotropic distribution of sulfated glycosaminoglycans in human medial collateral ligament: A quantitative analysis

Decorin and its associated glycosaminoglycan (GAG) side chain, dermatan sulfate (DS), play diverse roles in soft tissue formation and potentially aid in the mechanical integrity of the tissue. Deeper understanding of the distribution and orientation of the GAGs on a microscopic level may help elucidate the structure/function relationship of these important molecules. The hypothesis of the present study was that sulfated GAGs are aligned with transversely isotropic material symmetry in human medial collateral ligament (MCL) with the collagen acting as the axis of symmetry. To test the hypothesis, sulfated GAGs were visualized using transmission electron microscopy (TEM). Three orthogonal anatomical planes were examined to evaluate GAG distributions against symmetry criteria. GAG populations were differentiated using targeted enzyme digestion. Results suggest that sulfated GAGs including DS, chondroitin sulfates A and C, as well as other sub-populations assume transversely isotropic distributions in human MCL. Sulfated GAGs in the plane normal to the collagen axis were found to be isotropic with no preferred orientation. GAGs in the two planes along the collagen axis did not statistically differ and exhibited apparent bimodal distributions, favoring orthogonal distributions with over half at other angles with respect to collagen. A previously developed model, GAGSim3D, was used to interpret potential TEM artifacts. The data collected herein provide refined inputs to micro-scale models of the structure/function relationship of sulfated GAGs in soft tissues.     

Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.     

Matrix Metalloproteinases in a Sea Urchin Ligament with Adaptable Mechanical Properties

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states (“standard”, “compliant” and “stiff”) was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability.     

Metallothioneins: new functional and structural insights

In the past few years, tissue-specific mammalian metallothioneins (i. e. metallothionein-3 and metallothionein-4) have been discovered that possess distinct functional properties. Other recent developments include an insight into the role of the widely expressed mammalian metallothionein-1 and metallothionein-2 isoforms in zinc homeostasis and apoptosis. The three-dimensional structure of the evolutionary distant sea urchin Cd(7)-metallothionein A, which contains two metal-thiolate clusters, supports previous conclusions regarding the functional importance of this structural motif. Despite correlated efforts involving techniques of structural biochemistry and molecular biology, the primary function of metallothioneins remains enigmatic.     

Spatial distribution and orientation of dermatan sulfate in human medial collateral ligament

The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been suggested to contribute to the mechanical stability and material properties of connective tissues. The spatial distribution and orientation of DS within the tissue are relevant to these mechanical roles, but measurements of length and orientation from 2D transmission electron microscopy (TEM) are prone to errors from projection. The objectives of this study were to construct a 3D geometric model of DS GAGs and collagen fibrils, and to use the model to interpret TEM measurements of the spatial orientation and length of DS GAGs in the medial collateral ligament of the human knee. DS was distinguished from other sulfated GAGs by treating tissue with chondroitinase B, an enzyme that selectively degrades DS. An image processing pipeline was developed to analyze the TEM micrographs. The 3D model of collagen and GAGs quantified the projection error in the 2D TEM measurements. Model predictions of 3D GAG orientation were highly sensitive to the assumed GAG length distribution, with the baseline input distribution of 69+/-23 nm providing the best predictions of the angle measurements from TEM micrographs. The corresponding orientation distribution for DS GAGs was maximal at orientations orthogonal to the collagen fibrils, tapering to near zero with axial alignment. Sulfated GAGs that remained after chondroitinase B treatment were preferentially aligned along the collagen fibril. DS therefore appears more likely to bridge the interfibrillar gap than non-DS GAGs. In addition to providing quantitative data for DS GAG length and orientation in the human MCL, this study demonstrates how a 3D geometric model can be used to provide a priori information for interpretation of geometric measurements from 2D micrographs.     

Structural features associated with movement and ‘catch’ of sea-urchin spines

Spine catch ligaments of a sea urchin Arbacia punctulata were extended under constant load. Ligaments from an undisturbed animal may show any extension rate from zero (catch state) to rapid extension to failure. Replacing the preparation bath with Ca²⁺- and Mg²⁺-free sea water reversibly abolishes the catch state. The fine structure of the outer muscle layer and inner ligament cone associated with the spine base is described. The unstriated paramyosin muscles bear thin flanges and form compact interlocking rows. Subsurface cisternae are associated with the plasma membrane. The muscles are innervated by glia-free axons ending in bulbous terminals containing lucent synaptic vesicles. The ligament comprises cylindrical bundles of collagen fibrils: one or more minute muscle fibers (paramyosin) lie parallel with and closely adjoining each bundle. The mean diameter of these muscles is 0.3 μg and they occupy 2–3 % of the ligament's cross-sectional area. Axons containing electronopaque secretory droplets accompany the muscles between the collagen bundles: the cell bodies of these neurones generally lie on the outer surface of the ligament. When an urchin points a spine, the ligament on the side of the contracting spine muscle shortens but does not buckle. A function of the intraligamental muscles is to effect this non-buckled shortening. The catch mechanism (which resides entirely within the ligament) may be due either to the intraligamental muscles and/or to a locked polymer mechanism in which matrix molecules between collagen fibrils are reversibly crosslinked by divalent cations.