Mapping chemical gradients within and along a fibrous structural tissue, mussel byssal threads

The byssal thread of a mussel is an extraorganismic connective tissue that exhibits a striking end-to-end gradient in mechanical properties and thus provides a unique opportunity for studying how gradients are made. Mfp-1 (Mytilus foot protein-1) is a conspicuous component of the protective outer cuticle of byssal threads given its high 3,4-dihydroxyphenylalanine (Dopa) content at 10-15 mol %. Amino acid analysis of mfp-1 extracted from successive foot sections of Mytilus galloprovincialis reveals a post-translationally mediated gradient with highest Dopa levels present in mfp-1 from the accessory gland near the tip of the foot decreasing gradually toward the base. The Dopa content of successive segments of byssal threads decreases from the distal to the proximal end and thus reflects the trend of mfp-1 in the foot. Inductively coupled plasma analysis indicates that certain metal ions including iron follow the trend in Dopa along the thread. Energy-dispersive x-ray spectrometry showed that iron, when present, was concentrated in the cuticle of the threads but sparse in the core. The axial iron gradient appears most closely correlated with the Dopa gradient. The direct incubation of mussels and byssal threads in Fe(3+) supplemented seawater showed that byssal threads are unable to sequester iron from the seawater. Instead, particulate/soluble iron is actively taken up by mussels during filter feeding and incorporated into byssal threads during their secretion. Our results suggest that mussels may exploit the interplay between Dopa and metals to tailor the different parts of threads for specific mechanical properties.     

Mussel collagen molecules with silk-like domains as load-bearing elements in distal byssal threads

Mechanically stressed biological materials like tendon, spider silk or mussel byssal threads are typically composite materials comprising multi-domain proteins, in which molecular building blocks contribute to overall material function. Mussel byssal threads are the anchorage of sessile mytilid mussels, which withstand recurring external loads from waves and tides. A single thread is elastic and ductile proximally, while the distal portion exhibits an extraordinary stiffness and toughness with a transient gradient of both mechanical features along the thread. The main components of byssal threads include a set of various collagen-like structural proteins (preCols) consisting of a collagenous core sequence flanked by globular domains. Here, structural analysis using polarized Fourier-transform infrared spectroscopy (FTIR) on stretched distal portions of mussel byssal threads determines the impact of external linear load on various molecular moieties. It is concluded that the preCol collagenous core domain is the main load-bearing element in distal byssal threads, while polyalanine beta-sheets in the flanking domains, similar to those found in spider silk proteins, provide high stiffness at low strains. Load dissipation is mediated by domain stretching of amorphous glycine-rich helical moieties followed by complete unfolding of the preCol flanking domains.     

pH-dependent locking of giant mesogens in fibers drawn from mussel byssal collagens

Byssal threads are tough collagenous fibers that mussels use to secure themselves against dislodgement by waves in the marine intertidal zone. Here, preCol, a family of hybrid collagens comprising up to 96% of the protein content in certain regions of byssal threads, was purified in mg amounts from mussel foot tissue for the first time. Conditions for drawing preCols into quality fibers ex vivo were investigated. The most important factor affecting fiber formation was the pH of the drawing solution. The morphology and tensile properties of drawn fibers were also characterized and suggest that a liquid crystal mesophase combined with cross-linking by His-metal coordination plays a role in the assembly/mechanics of drawn fibers and likely in native byssal threads as well.     

利用蛋白质组学手段鉴定新型贻贝足丝蛋白

贻贝通过足腺分泌特有的足丝并以此粘附于水下各种基质表面.贻贝足丝中富含各种粘附蛋白,其优异的水下粘附性能使其成为开发新型生物粘合剂的候选分子.厚壳贻贝足丝粘附能力强,本文采用尿素及盐酸胍抽提结合二维双向电泳技术（two-dimensional electrophoresis, 2-DE）,分别对厚壳贻贝足丝纤维和足丝盘的蛋白质进行分离及染色;采用串联质谱技术结合常规搜库和表达序列标签（EST) 数据库搜索,对分离获得的蛋白质点进行鉴定,从中获得了mfp-3、mfp-6、胶原蛋白以及3种未曾报道过的新型贻贝足丝蛋白成分.上述研究为深入了解厚壳贻贝足丝蛋白的分子多样性、探讨其粘附机理以及从中筛选具有应用前景的贻贝足丝蛋白奠定了基础.     

Effects of artificial epibionts on byssogenesis,attachment strength,and movement in two size classes of the blue mussel,Mytilus edulis

Blue mussels (Mytilus edulis) can alter the strength of byssal attachment and move between and within mussel aggregations on wave‐swept shores, but this movement ability may be limited by epibiont fouling. We quantified the effects of artificial epibiont fouling on the production of byssal threads, attachment strength, and movement in two size classes of blue mussels. In a factorial experiment, large epibiont‐covered mussels produced more functional byssal threads (i.e., those continuous from animal to substrate) after 24 h than large unfouled and small fouled mussels, but not more than small unfouled mussels. Small unfouled mussels formed and released more byssus bundles compared to any other treatment group, which indicates increased movement. Conversely, epibiont fouling resulted in decreased numbers of byssus bundles shed, and therefore reduced movement in small mussels. Epibiont‐covered mussels started producing byssal threads sooner than unfouled mussels, while small mussels began producing byssal threads earlier compared to large mussels. Mean attachment strength from both size classes increased by 9.5% when mussels were artificially fouled, and large mussels had a 34% stronger attachment compared to small mussels. On the other hand, a 2.3% decrease in attachment strength was found with increasing byssus bundles shed. Our results suggest that fouling by artificial epibionts influences byssal thread production and attachment strength in large mussels, whereas epibionts on small mussels impact their ability to move. Mussels are able to respond rapidly to fouling, which carries implications for the dynamics of mussel beds in their intertidal and subtidal habitats, especially in relation to movement of mussels within and among aggregations.     

Clumping behavior and byssus production as strategies for substrate competition in Mytilus edulis

Laboratory experiments showed that the mussel Mytilus edulis aggregated more intensely around living organisms (the bivalve Hiatella arctica and the solitary ascidian Styela rustica, which commonly co‐occur with mussels in fouling communities) than around inanimate objects. When exposed to an inanimate object, mussels attached their byssal threads primarily to the substrate, close to the object, but when exposed to a living organism, they attached their byssal threads directly to the organism. The ascidian was more intensely covered with byssal threads than was the bivalve. Mussel attachment to the ascidians was apparently determined by the physical characteristics of the tunic and to a lesser extent by the excretion‐secretion products released by S. rustica. This study indicates that mussels can use byssus threads as a means of entrapment of potential competitors for space. It remains unclear why mussels preferentially attached to ascidians compared to the bivalve. This can be explained either by competitive interactions, or by attractiveness of the ascidian tunic as an attachment substratum.     

Protein gradients in byssal threads of some marine bivalve molluscs

Many marine bivalve molluscs produce byssal threads for attachment to solid substrata. Small (less than 10 mm) consecutive sections of the byssal threads of Mytilus edulis, M. californianus, Geukensia demissa, Atrina vexillum, and A. rigida were analyzed by amino acid analysis to determine if chemical composition remains constant as a function of location in thread segments. Nonlinear longitudinal protein gradients, probably involving collagen and an elastic protein, were found in the Mytilus species. In these, collagen peaks in the distal third of the thread. In Geukensia and the Atrina species, although the two differed greatly in composition, there is a clear nonvariability in composition of the thread within each species as a function of location in the thread. The adhesive plaque at the tip of the thread of all species examined differs substantially in composition from the remainder of the thread. Protein gradients in the threads of some bivalves may reflect specific adaptations evolved to respond to exposed habitats in high-energy environments.     

Elastomeric gradients: a hedge against stress concentration in marine holdfasts?

The byssal threads of marine mussels are elastomeric fibres with a great capacity for absorbing and dissipating energy. Up to 70% of the total absorbed energy can be dissipated in the byssus. Because byssal threads attach the mussel to hard inert surfaces in its habitat, they must combine the need to be good shock absorbers with appropriate matching of Young's modulus between living tissue and a hard sub-stratum such as stone - stiffnesses that can differ by five orders of magnitude. Recent data suggest that improved modulus matching and decreased stress concentration between different portions of the byssus is achieved by the use of protein gradients. Protein gradients in byssal threads are constructed using natural macromolecular chimeras having a central collagenous domain, variable flanking modules and histidine-rich amino and carboxy termini. Stiff silk-like flanking modules prevail distally, while at the animal end, rubbery modules resembling elastin predominate. In between the two thread ends there is a mix of both module types. The histidine-rich termini provide metal binding/cross-linking sites, while collagen domains may confer self-assembly on all parts of the structure. A graded axial distribution of flanking modules is expected to moderate stress concentration in joined materials having disparate moduli.     

Interspecific comparison of the mechanical properties of mussel byssus

Byssally tethered mussels are found in a variety of habitats, including rocky intertidal, salt marsh, subtidal, and hydrothermal vents. One key to the survival of mussels in these communities is a secure attachment, achieved by the production of byssal threads. Although many studies have detailed the unique biomechanical properties of byssal threads, only a few prevalent species have been examined. This study assesses the variation in the mechanical properties of byssus in a broad range of mussel species from diverse environments, including intertidal and subtidal Mytilus edulis, Modiolus modiolus, Geukensia demissa, Bathymodiolus thermophilus, and Dreissena polymorpha. A tensometer was used to measure quasi-static and dynamic mechanical properties of individual threads, and several aspects of morphology were quantified. The results indicate that thread mechanical properties vary among mussel species, and several novel properties were observed. For example, of the species examined, D. polymorpha threads were the strongest, stiffest, least resilient, and fastest to recover after partial deformation. Threads of M. modiolus were characterized by the presence of two distinct yield regions prior to tensile failure. This comparative study not only provides insight into the ecological limitations and evolution of mussels, but also suggests new models for the design of novel biomimetic polymers.     

Morphology of the Bivalve Mollusks Crenomytilus grayanus and Mytilus coruscus in Relation to Their Spatial Distribution in the Upper Subtidal Zone

The morphology of the shell and byssus threads was studied in two closely related mussel species Crenomytilus grayanus and Mytilus coruscus. The two species differ significantly from each other in the shell shape and in the degrees of development and deformation of byssus threads. These differences, in turn, determine (either directly or indirectly) the differences in strength of the byssal attachment and are discussed in terms of their functional morphology with respect to the spatial distribution of the mussels in marine coastal zones.     

利用质谱技术结合EST库搜索鉴定厚壳贻贝新型足丝蛋白

贻贝利用足丝粘附于水下各种基质表面.作为一种具有优异粘附性能的生物材料,贻贝足丝蛋白在新型水下粘附剂及表面保护涂层的研制与开发中具有重要的仿生学意义.目前,已报道的贻贝足丝蛋白分子达11种,但是仍然有更多的足丝蛋白分子不为人知.为进一步探讨贻贝足丝蛋白的分子多样性,并从中筛选具有特殊生物学功能的足丝蛋白分子,本文采用鸟枪法-液相色谱-质谱/质谱技术（shotgun-LC-MS/MS）对厚壳贻贝足丝蛋白进行了蛋白质组学分析.将厚壳贻贝足丝分为足丝纤维和足丝盘两部分,每一部分均采用醋酸-尿素溶液,以及醋酸-盐酸胍溶液进行蛋白质抽提;抽提后的足丝蛋白经胰蛋白酶酶解,利用线性离子阱四级杆质谱（LTQ）进行鸟枪法质谱分析.二级质谱图（MS/MS）用以搜索公共数据库中的贻贝表达序列标签（expressed sequence tag,EST）数据库.采用上述方法,获得14种贻贝新型足丝蛋白的高可信度结果及其所匹配的部分或全长cDNA序列;经结构域分析,发现上述新型贻贝足丝蛋白分子的序列中多数包含各种类型的结构域,包括胶原蛋白结构域、C1Q结构域、C1Q结合结构域、微管蛋白辅助折叠结构域、蛋白酶拮抗结构域、VWA结构域、几丁质酶结构域等.在此基础上,对上述新型足丝蛋白在贻贝足丝形成以及粘附方面的功能进行了推测.上述结果对进一步了解贻贝足丝的分子组成以及粘附机理奠定了基础.     

Collagen-binding matrix proteins from elastomeric extraorganismic byssal fibers

The byssal threads of marine mussels represent a peculiar case of extraorganismic extracellular material. The threads consist of fibrous chimeric collagens such as preCol-P (with collagenous, elastin-like and histidine-rich domains) embedded in a microfibrillar matrix. We report here on the extraction, purification, and characterization of water-soluble proximal thread matrix protein 1 (PTMP1), which is preferentially located in the proximal portion of each byssal thread and decreases in a proximal to distal direction. PTMP1 has a mass of about 50 kDa as determined by matrix-assisted laser desorption-ionization with time-of-flight (MALDI-TOF) mass spectrometry. Glycine is the most common residue at 12.2 mol %, followed by asparagine/aspartic acid and glutamine/glutamic acid at 11.4 and 9.9 mol %, respectively. Glycosylation has been detected by Western blotting with biotinylated concanavalin A and neutral sugar analysis. With degenerate primers designed from the N-terminal sequence and an additional internal peptide derived by Lys-C endopeptidase digestion, a complete cDNA sequence for this protein was obtained by polymerase chain reaction (PCR) amplification of a Mytilus edulis foot cDNA library. Two variants with minor sequence differences limited to the N-terminus were found. The cDNA-deduced protein sequence reveals two symmetric internal repeats that together account for >85% of the protein. Sequence and epitope similarity of PTMP1 to the A domains of von Willebrand factor and integrin alpha(1)I suggest a capacity for collagen binding. Enzyme-linked immunosorbent assay (ELISA)-based measurement of PTMP1 binding to immobilized type I collagen shows high affinity (apparent K(D) = 0.25 microM), but the binding exhibits no dependence on metals. Using primers designed from M. edulis, we also found a PTMP1-like cDNA in a related species, M. galloprovincialis, with a deduced protein sequence having 97% identity with one M. edulis variant and 99% identity with the other. The corresponding cDNA sequences have 94% and 96% identity, respectively.     

Along the silk road, spiders make way for mussels

A novel strategy for coating extensible fibers is revealed from the study of the 'silk' tethers produced by marine mussels. The tethers, known as byssal threads, are molded collagenous fibers coated with a thin (2-4 microm) cuticle that protects the fibrillar core from abrasion and bacterial attack. One mussel species infuses the cuticle with nanoscale granules, which increase the extensibility of the hard coating by to 70%, making it seven times stretchier than any synthetic polymer coating. The mussel cuticle could therefore inspire new strategies for the design and manufacture of thin composite coatings that are both hard and extensible.     

Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima

An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.     

Effects of Environmental Factors on Byssal Thread Formation in Some Members of the Family Mytilidae from the Sea of Japan

The ability to attach repeatedly to a substrate (glass, boulders, sand) in three common mussel species of the upper sublittoral zone of the Sea of Japan, Grayan's mussel Crenomytilus grayanus, the Korean mussel Mytilus coruscus, and northern horse mussel Modiolus modiolus, was studied under experimental conditions. It was found that during 120 h of the experiment C. grayanus and M. modiolus produced more byssal threads than M. coruscus. A decrease in the water temperature from 20 to 0°C slowed the rate of production of byssal threads down to full passivity in some experimental mollusks. This was more typical of M. coruscus and less typical of C. grayanus. Renewed threads differed in their length, thick, size of the adhesive plate, and strength. M. coruscus formed the shortest, thickest, and strongest threads with rather a large adhesive disk. The observed differences are discussed from the position of morphophysiological adaptations of species for colonization of different natural substrata under contrasting conditions of the upper sublittoral zone.     

基于illumina测序的厚壳贻贝足组织转录组研究

贻贝足丝是贻贝足组织分泌的足丝蛋白形成的非细胞组织,具有在水环境下的极强粘附性能,是当前生物粘附剂及抗腐蚀材料的研发热点．为进一步了解贻贝足丝蛋白的分子多样性特征,采用新一代Illumina高通量测序平台对厚壳贻贝(Mytilus coruscus)足组织进行转录组测序,首次构建了厚壳贻贝足组织的转录组数据库．共计获得7 199 799 840 nt的碱基数据经过序列拼接和组装,获得88 825条unigene．对上述unigene开展了序列注释,共计37 007条unigene获得注释．在此基础上,经序列检索和比对,从中筛选出与目前已知的11种足丝蛋白同源的56条unigene序列并进行分析．结果表明,厚壳贻贝足丝蛋白具有明显的氨基酸偏好性,部分足丝蛋白具有重复序列,且厚壳贻贝足丝蛋白与其他种类的贻贝足丝蛋白具有较高的序列相似性．上述结果为后续贻贝足丝蛋白的批量鉴定以及在此基础上的贻贝足丝形成、固化以及粘附机制相关研究奠定了基础．     

Byssus thread strength in the mussel, Mytilus edulis

Mytilus edulis attaches to the substratum by means of a proteinaceous byssus complex. This consists of three portions: a root, embedded in the pedal tissues, a stem, continuous with the root but external to the body and a number of byssus threads attached proximally to the stem and distally to the substratum via adhesive discs. Byssus strength varies seasonally on the shore, in response to changes in wave action (Price, in press). As a decline in byssal attachment strength implies a decline in strength of the constituent threads, a study was undertaken to establish the extent to which byssus thread strength is determined by age. The ultimate tensile stress, ultimate tensile strain and Young's Modulus were measured in threads of known age and length and a stepped regression performed on the results. It was found that age and length correlate significantly with tensile stress and Young's Modulus. Length is a less important influence than age on tensile stress but has a greater effect than age on Young's Modulus. Tensile strain is independent of both length and age.     

Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned by various treatments, particularly agitation in aerated or nitrogen-sparged seawater. Stiffness can be permanently increased by more than two times, e.g., from 25 MPa to a maximum of 65 MPa, by simple agitation in aerated seawater. Much but not all of this stiffening can be prevented by agitation under nitrogen. Reversible strain stiffening would seem to be a useful adaptation to lower residual stresses arising from the deformation of two joined materials, i.e., distal and proximal portions with rather different elastic moduli. The permanent strain stiffening that characterizes proximal byssal threads subjected to oxidative stress is probably due to protein cross-linking. In the short term, this results in a stronger thread but at the expense of dynamic interactions between the molecules in the 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.