Byssal Plaques Research Articles

Mussel-Inspired Reversible Molecular Adhesion for Fabricating Self-Healing Materials.

Nature offers inspiration for the development of high-performance synthetic materials. Extensive studies on the universal adhesion and self-healing behavior of mussel byssus reveal that a series of reversible molecular interactions occurring in byssal plaques and threads play an essential role, and the mussel-inspired chemistry can serve as a versatile platform for the design of self-healing materials. In this Perspective, we provide an overview of the recent progress in the detection, quantification, and utilization of mussel-inspired reversible molecular interactions, which includes the elucidation of their binding mechanisms via force-measuring techniques and the development of self-healing materials based on these dynamic interactions. Both conventional catechol-medicated interactions and newly discovered chemistry beyond the catechol groups are discussed, providing insights into the design strategies of advanced self-healing materials via mussel-inspired chemistry.

Why mussel byssal plaques are tiny yet strong in attachment

<h2>Summary</h2> <i>Mytilus edulis</i>, or blue mussels, are known for producing byssal threads, allowing them to adhere to substrate in tidal zones. A byssal thread emerges from body tissue and terminates with an adhesive plaque. These byssal plaques are found to be tiny relative to the overall mussel size and do not vary much with different body sizes. In this study, we combine mechanical testing, 3D printing, and numerical modeling to investigate the effect plaque size has on adhesion strength. Our study reveals that the plaque structure governs the adhesion strength of the plaque, while plaques with a diameter three to five times that of the byssal thread yield optimized strength to resist detachment. Furthermore, bonding strength relies on the architecture of the byssal network rather than increased plaque size. Such knowledge helps to understand the design principle of byssal plaques and may shed light on designing reinforcement systems to secure engineering structures.

Attachment of zebra and quagga mussel adhesive plaques to diverse substrates

Like marine mussels, freshwater zebra and quagga mussels adhere via the byssus, a proteinaceous attachment apparatus. Attachment to various surfaces allows these invasive mussels to rapidly spread, however the adhesion mechanism is not fully understood. While marine mussel adhesion mechanics has been studied at the individual byssal-strand level, freshwater mussel adhesion has only been characterized through whole-mussel detachment, without direct interspecies comparisons on different substrates. Here, adhesive strength of individual quagga and zebra mussel byssal plaques were measured on smooth substrates with varying hydrophobicity—glass, PVC, and PDMS. With increased hydrophobicity of substrates, adhesive failures occurred more frequently, and mussel adhesion strength decreased. A new failure mode termed 'footprint failure' was identified, where failure appeared to be adhesive macroscopically, but a microscopic residue remained on the surface. Zebra mussels adhered stronger and more frequently on PDMS than quagga mussels. While their adhesion strengths were similar on PVC, there were differences in the failure mode and the plaque-substrate interface ultrastructure. Comparisons with previous marine mussel studies demonstrated that freshwater mussels adhere with comparable strength despite known differences in protein composition. An improved understanding of freshwater mussel adhesion mechanics may help explain spreading dynamics and will be important in developing effective antifouling surfaces.

Inter- and intramolecular adhesion mechanisms of mussel foot proteins

Mussel foot proteins (Mfps) secreted in the byssal plaque of marine mussels are widely researched for their relevance to mussel adhesion in water. As the abundant residue in the amino acid sequences of major adhesive proteins, 3,4-dihydroxyphenylalanine (Dopa) or its catecholic moiety plays a key role in both Mfp binding to surface and cohesive cross-linking of Mfps in byssal plaques. The binding performance of an Mfp significantly depends on the content and redox state of Dopa, whereas the types of interaction vary in line with different surface chemistries and pH conditions. Thorough understanding of mussel adhesion from a molecular perspective is crucial to promote the application of synthetic mussel-bionic adhesives. This article presents a brief review of the research progress on the adhesion mechanisms of Mfps, which further emphasizes the contributions of Dopa-mediated interactions and considers other amino acids and factors. The involved inter- and intramolecular interactions are responsible for not only the diverse adhesion capacities of an adhesive byssal plaque as mussel’s adhesion precursor but also the formation and properties of the plaque structure.

Spatial distribution of proteins in the quagga mussel adhesive apparatus

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious ‘anchor’ (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

The staying power of adhesion-associated antioxidant activity in Mytilus californianus.

The California mussel, Mytilus californianus, adheres in the highly oxidizing intertidal zone with a fibrous holdfast called the byssus using 3, 4-dihydroxyphenyl-l-alanine (DOPA)-containing adhesive proteins. DOPA is susceptible to oxidation in seawater and, upon oxidation, loses adhesion. Successful mussel adhesion thus depends critically on controlling oxidation and reduction. To explore how mussels regulate redox during their functional adhesive lifetime, we tracked extractable protein concentration, DOPA content and antioxidant activity in byssal plaques over time. In seawater, DOPA content and antioxidant activity in the byssus persisted much longer than expected-50% of extractable DOPA and 30% of extractable antioxidant activity remained after 20 days. Antioxidant activity was located at the plaque-substrate interface, demonstrating that antioxidant activity keeps DOPA reduced for durable and dynamic adhesion. We also correlated antioxidant activity to cysteine and DOPA side chains of mussel foot proteins (mfps), suggesting that mussels use both cysteine and DOPA redox reservoirs for controlling interfacial chemistry. These data are discussed in the context of the biomaterial structure and properties of the marine mussel byssus.

Influence of Subtilisin on Mussel (&lt;i&gt;Mytilus edulis&lt;/i&gt;) Byssus Deposition and Diatom (&lt;i&gt;Navicula sp.&lt;/i&gt;) Settlement

Biofouling organisms secrete adhesive polymers to settle themselves on man-made substrata in the marine environment. Mussels (Mytilus edulis) and diatom (Navicula sp.) are both major marine biofoulers. They attach tenaciously to a wide variety of submerged surfaces by virtue of byssus and extracellular polymer substances, respectively. The aim of this study is to evaluate the antifouling potential of the serine protease subtilisin on mussels and benthic diatoms. The effects of enzyme concentration, treatment duration and application time were tested on byssus deposition. The results showed that subtilisin effectively prevented the byssus formation. The reduction rate of byssal plaques was dependent on enzyme concentration and treatment duration. The byssal plaques of Mytilus edulis became progressively less sensitive to hydrolysis as they cured. Attached diatoms on polystyrene substrate were effectively removed by subtilisin.

Purification, cDNA Clone and Recombinant Expression of Foot Protein-3 from Mytilus coruscus

Mussels Mytilus coruscus can adhere to various solid surface in the presence of moisture. Mussel foot protein-3 (mfp-3) has been suggested as the main adhesive protein in the plaques closest to the adhesion interface and been the focus of substantial biomaterials development research within the last decade. The byssal plaques of M. coruscus were accumulated and variants of a family known as mcofp3 (Mytilus coruscus foot protein 3) were purified from acetic acid/urea extracts of plaques, with their N-terminal sequences determined thereafter. The cDNA sequence coding for the mcofp3 precursor was obtained from M. coruscus foot cDNA library. These precursors contain a putative signal peptide of 24 residues, a mature peptide sequence of 41-56 amino acids rich in Tyr, Gly, Pro, and Asn. The recombinant mcofp3 fused with a hexa-histidine affinity ligand was successfully expressed through an Escherichia coli expression system, and the recombinant mcofp3 was purified using affinity chromatography followed by reverse phase high performance liquid chromatography (HPLC). The DOPA content and adhesive properties of purified recombinant mcofp3 with or without tyrosinase modification were compared with the native mcofp3. These assays showed that recombinant mcofp3 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.

Ultrastructural and molecular evidence for potentially symbiotic bacteria within the byssal plaques of the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study reports on the presence of a putatively symbiotic bacterial flora within the byssus plaque of the deep sea hydrothermal mussel Bathymodiolus azoricus, contributing to metal sequestration/deposition and testing positive to methane oxidizing symbiont-specific fluorescent probes. Combining an array of approaches including histology, electron microscopy, X-ray microanalysis, analytical chemistry, and microbiology we provide evidence for the frequently assumed, but rarely shown influence of prokaryotes on the biogeochemical cycling of metals as well as inorganic C sources (i.e., methane) at deep sea hydrothermal vents. Our results indicate that in spite of its antibacterial protective sheath, the byssus plaque gives access to a whole range of prokaryotic organisms which may be responsible for the extremely high concentration of metallic elements (Fe, Cu, Zn, Mn, Co, Mo, Cd, Pb and Hg) measured in this attachment organ. The very high levels of metals in byssus, together with its frequent renewal rate due to the dynamic nature of the habitat, suggest that intra-byssal bacteria may have a major influence on biomineralisation/deposition of metals. The presence of a methanotroph morphotype within the byssus plaque was confirmed by FISH and TEM. The implications of the biogeochemical cycling of metals and methane at hydrothermal vents are discussed.

Linking Adhesive and Structural Proteins in the Attachment Plaque of Mytilus californianus

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090-11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-L-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.

Rotational Echo Double Resonance Detection of Cross-links Formed in Mussel Byssus under High-Flow Stress

13C2H rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13C]tyrosine and L-[ring-d4]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5'-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.

Reverse engineering of bioadhesion in marine mussels.

Marine mussels (Mytilus) are experts at bonding to a variety of solid surfaces in a wet, saline and turbulent environment. Bonding is rapid, permanent, versatile and protein-based. In mussels, adhesive bonding takes the form of a byssus--a bundle of extracorporeal threads--each connected to living tissues of the animal at one end and secured by an adhesive plaque at the other. We have investigated the composition and formation of byssal plaques and threads with the hope of discovering technologically relevant innovations in chemistry and materials science. All proteins isolated from the byssus to date share the quality of containing the unusual amino acid, 3,4-dihydroxyphenylalanine. This residue appears to have a dual functionality with significant consequences for adsorption and cohesion. On the one hand, it forms a diverse array of weaker molecular interactions such as metal chelates, H-bonds, and pi-cations: these appear to dominate in surface behavior (adsorption). On the other hand, 3,4-dihydroxyphenylalanine and its redox couple, dopaquinone, can mediate formation of covalent cross-links among byssal proteins (cohesion). One of the challenges in making functional biomimetic versions of byssal adhesion is to understand how these two reactivities are balanced.

Modes of Byssal Failure in Forced Detachment of Zebra Mussels

Microscopic observations of the detached byssal apparatus have confirmed that the substrate plays a direct role in determining the strength of adhesion of zebra mussels. Depending on the material, 20–95% of byssal plaques detached intact due to failure of the adhesive bond with the substrate. The forces required to remove individual mussels from plates of stainless steel, mild steel, polyvinylchloride (PVC), Teflon and concrete were measured using both a direct tensile pull-off test and a wall-jet apparatus in which mussels are swept from the substrate using a jet of water. In each case the detached mussels were subsequently dissected in order to determine the mode of failure of the byssal apparatus, i.e., thread rupture, plaque adhesive failure, and root failure. It was found that the force required to detach the mussels varied significantly among the different materials and was directly proportional to the percentage of broken byssal threads.

A multiconfiguration relativistic Dirac-Fock program

Mytilus edulis, or blue mussels, are known for producing byssal threads, allowing them to adhere to substrate in tidal zones. A byssal thread emerges from body tissue and terminates with an adhesive plaque. These byssal plaques are found to be tiny relative to the overall mussel size and do not vary much with different body sizes. In this study, we combine mechanical testing, 3D printing, and numerical modeling to investigate the effect plaque size has on adhesion strength. Our study reveals that the plaque structure governs the adhesion strength of the plaque, while plaques with a diameter three to five times that of the byssal thread yield optimized strength to resist detachment. Furthermore, bonding strength relies on the architecture of the byssal network rather than increased plaque size. Such knowledge helps to understand the design principle of byssal plaques and may shed light on designing reinforcement systems to secure engineering structures.