Romana Santos

Adhesion of echinoderm tube feet to rough surfaces

SUMMARY Echinoderms attach strongly and temporarily to the substratum by means of specialized organs, the podia or tube feet. The latter consist of a basal extensible cylinder, the stem, which bears an apical flattened disc. The disc repeatedly attaches to and detaches from the substratum through adhesive and de-adhesive secretions. In their activities, echinoderms have to cope with substrata of varying degrees of roughness as well as with changing hydrodynamic conditions, and therefore their tube feet must adapt their attachment strength to these environmental constraints. This study is the first attempt to evaluate the influence of substratum roughness on the temporary adhesion of echinoderm tube feet and to investigate the material properties of their contact surface. It was demonstrated that tube foot discs are very soft (E-modulus of 6.0 and 8.1 kPa for sea stars and sea urchins, respectively), have viscoelastic properties and adapt their surface to the substratum profile. They also show increased adhesion on a rough substratum in comparison to its smooth counterpart, which is due mostly to an increase in the geometrical area of contact between the disc and the surface. Tenacity (force per unit area) increases with roughness [e.g. 0.18 and 0.34 MPa on smooth polymethyl-methacrylate (PMMA), 0.21 and 0.47 MPa on rough PMMA for sea stars and sea urchins, respectively] if only the projected surface area of the adhesive footprint is considered. However, if this tenacity is corrected to take into account the actual substratum 3-D profile, surface roughness no longer influences significantly the corrected adhesion strength (e.g. 0.18 and 0.34 MPa on smooth PMMA, 0.19 and 0.42 MPa on rough PMMA for sea stars and sea urchins, respectively). It can be hypothesized that, under slow self-imposed forces, disc material behaves viscously to adapt to substratum roughness while the adhesive fills out only very small surface irregularities (in the nanometer range). It is deposited as a thin film ideal for generation of strong adhesion. Under short pulses of wave-generated forces, attached discs probably behave elastically, distributing the stress along the entire contact area, in order to avoid crack generation and thus precluding disc peeling and tube foot detachment.

Morphology and tenacity of the tube foot disc of three common European sea urchin species: a comparative study

Abstract The variation in tenacity of single tube feet from three sea urchin species with contrasted habitats was assessed and correlated with the ultrastructure of their adhesive secretory granules. The tube feet of Arbacia lixula and Sphaerechinus granularis have larger discs and more complex adhesive granules than those of Paracentrotus lividus, but A. lixula attaches to glass with significantly lower tenacity (0.05 – 0.09 MPa) than the other two species (0.10 – 0.20 and 0.11 – 0.29 MPa, respectively). However, the estimated maximal attachment force one tube foot can produce is similar for all three species investigated. No clear relationship between tube foot size, tenacity, adhesive secretory granule ultrastructure and species habitat can therefore be established. For P. lividus the tenacity of single tube foot discs on four different smooth substrata was also compared, which showed that both the total surface energy and the ratio of polar to non-polar forces at the surface influence tube foot attachment strength. This influence of the surface characteristics of the substratum appears to affect the cohesiveness of the adhesive secretion more than its adhesiveness.

First Insights into the Biochemistry of Tube Foot Adhesive from the Sea Urchin Paracentrotus lividus (Echinoidea, Echinodermata)

Sea urchins are common inhabitants of wave-swept shores. To withstand the action of waves, they rely on highly specialized independent adhesive organs, the adoral tube feet. The latter are extremely well-designed for temporary adhesion being composed by two functional subunits: (1) an apical disc that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow the animal to move and (2) a stem that bears the tensions placed on the animal by hydrodynamism. Despite their technological potential for the development of new biomimetic underwater adhesives, very little is known about the biochemical composition of sea urchin adhesives. A characterization of sea urchin adhesives is presented using footprints. The latter contain inorganic residues (45.5%), proteins (6.4%), neutral sugars (1.2%), and lipids (2.5%). Moreover, the amino acid composition of the soluble protein fraction revealed a bias toward six amino acids: glycine, alanine, valine, serine, threonine, and asparagine/aspartic acid, which comprise 56.8% of the total residues. In addition, it also presents higher levels of proline (6.8%) and half-cystine (2.6%) than average eukaryotic proteins. Footprint insolubility was partially overcome using strong denaturing and reducing buffers, enabling the visualization of 13 proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugation of mass spectrometry with homology–database search allowed the identification of six proteins: alpha and beta tubulin, actin, and histones H2B, H3, H2A, and H4, whose location and function in the adhesive are discussed but require further investigation. For the remaining unidentified proteins, five de novo-generated peptide sequences were found that were not present in the available protein databases, suggesting that they might be novel or modified proteins.

Effect of condensed tannin ingestion in sheep and goat parotid saliva proteome

Saliva appears as a defence mechanism, against potential negative effects of tannins, in some species of animals which have to deal with these plant secondary metabolites in their regular diets. This study was carried out to investigate changes in parotid saliva protein profiles of sheep (Ovis aries) and goats (Capra hircus), induced by condensed tannin ingestion. Five Merino sheep and five Serpentina goats were maintained on a quebracho tannin enriched diet for 10 days. Saliva was collected through catheters inserted on parotid ducts and salivary proteins were separated by two-dimensional gel electrophoresis. Matrix-assisted Laser desorption ionization - time of flight (MALDI-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to identify the proteins whose expression levels changed after tannin consumption. Although no new proteins appeared, quebracho tannin consumption increased saliva total protein concentration and produced changes in the proteome of both species. While some proteins were similarly altered in both species parotid salivary protein profile, sheep and goats also presented species-specific differences in response to tannin consumption.

Serine Protease-mediated Host Invasion by the Parasitic Nematode Steinernema carpocapsae

Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 104 s−1 m−1 against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.

Echinoderm Adhesive Secretions: From Experimental Characterization to Biotechnological Applications

Adhesion is a way of life in echinoderms. Indeed, all the species belonging to this phylum use adhesive secretions extensively for various vital functions. According to the species or to the developmental stage considered, different adhesive systems may be recognized. (1) The tube feet or podia are organs involved in attachment to the substratum, locomotion, feeding or burrowing. Their temporary adhesion relies on a duo-gland adhesive system resorting to both adhesive and de-adhesive secretions. (2) The larval adhesive organs allow temporary attachment of larvae during settlement and strong fixation during metamorphosis. (3) The Cuvierian tubules are sticky defence organs occurring in some holothuroid species. Their efficacy is based on the instantaneous release of a quick-setting adhesive. All these systems rely on different types of adhesion and therefore differ in the way they operate, in their structure and in the composition of their adhesive. In addition to fundamental interests in echinoderm bioadhesives, a substantial impetus behind understanding these adhesives are the potential technological applications that can be derived from their knowledge. These applications cover two broad fields of applied research: design of water-resistant adhesives and development of new antifouling strategies. In this context, echinoderm adhesives could offer novel features or performance characteristics for biotechnological applications. For example, the rapidly attaching adhesive of Cuvierian tubules, the releasable adhesive of tube feet or the powerful adhesive of asteroid larvae could each be useful to address particular bioadhesion problems.

Exploring the proteome of an echinoderm nervous system: 2‐DE of the sea star radial nerve cord and the synaptosomal membranes subproteome

We describe the first proteomic characterization of the radial nerve cord (RNC) of an echinoderm, the sea star Marthasterias glacialis. The combination of 2‐DE with MS (MALDI‐TOF/TOF) resulted in the identification of 286 proteins in the RNC. Additionally, 158 proteins were identified in the synaptosomal membranes enriched fraction after 1‐DE separation. The 2‐DE RNC reference map is available via the WORLD‐2DPAGE Portal (http://www.expasy.ch/world‐2dpage/) along with the associated protein identification data which are also available in the PRIDE database. The identified proteins constitute the first high‐throughput evidence that seems to indicate that echinoderms nervous transmission relies primarily on chemical synapses which is similar to the synaptic activity in adult mammals spinal cord. Furthermore, several homologous proteins known to participate in the regeneration events of other organisms were also identified, and thus can be used as targets for future studies aiming to understand the poorly uncharacterized regeneration capability of echinoderms. This “echinoderm missing link” is also a contribution to unravel the mystery of deuterostomian CNS evolution.

Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences.

A proteomics study of the induction of somatic embryogenesis in Medicago truncatula using 2DE and MALDI-TOF/TOF.

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.

Proteome characterization of sea star coelomocytes - The innate immune effector cells of echinoderms

Sea star coelomic fluid is in contact with all internal organs, carrying signaling molecules and a large population of circulating cells, the coelomocytes. These cells, also known as echinoderm blood cells, are responsible for the innate immune responses and are also known to have an important role in the first stage of regeneration, i.e. wound closure, necessary to prevent disruption of the body fluid balance and to limit the invasion of pathogens. This study focuses on the proteome characterization of these multifunctional cells. The identification of 358 proteins was achieved using a combination of two techniques for protein separation (1‐D SDS‐PAGE followed by nanoLC and 2‐D SDS‐PAGE) and MALDI‐TOF/TOF MS for protein identification. To our knowledge, the present report represents the first comprehensive list of sea star coelomocyte proteins, constituting an important database to validate many echinoderm‐predicted proteins. Evidence for new pathways in these particular echinoderm cells are also described, and thus representing a valuable resource to stimulate future studies aiming to unravel the homology with vertebrate immune cells and particularly the origins of the immune system itself.