Multiresponsive Liquid Crystal Collagen Guides Mussel Byssus Formation.

Marine mussels fabricate tough collagenous fibers known as byssal threads to anchor themselves. Threads are produced individually in minutes via secretion of liquid crystalline (LC) collagenous precursors (preCols); yet the physical and chemical parameters influencing thread formation remain unclear. Here, we characterized the structural anisotropy of native and artificially induced threads using quantitative polarized light microscopy and transmission electron microscopy to elucidate spontaneous vs regulated aspects of thread assembly, discovering that preCol LC phases form aligned domains of several hundred microns, but not the cm-level alignment of native threads. We then explored the hypothesized roles of mechanical shear, pH, and metal ions on thread formation through in vitro assembly studies employing a microfluidic flow focusing device using purified preCol secretory vesicles. Our results provide clear evidence for the role of all three parameters in modulating the structure and properties of the final product with relevance for fabrication of collagenous scaffolds for tissue engineering applications.

Fabrication of Tunable Mechanical Gradients by Mussels via Bottom-Up Self-Assembly of Collagenous Precursors.

Functionally graded interfaces are prominent in biological tissues and are used to mitigate stress concentrations at junctions between mechanically dissimilar components. Biological mechanical gradients serve as important role models for bioinspired design in technically and biomedically relevant applications. However, this necessitates elucidating exactly how natural gradients mitigate mechanical mismatch and how such gradients are fabricated. Here, we applied a cross-disciplinary experimental approach to understand structure, function, and formation of mechanical gradients in byssal threads─collagen-based fibers used by marine mussels to anchor on hard surfaces. The proximal end of threads is approximately 50-fold less stiff and twice as extensible as the distal end. However, the hierarchical structure of the distal-proximal junction is still not fully elucidated, and it is unclear how it is formed. Using tensile testing coupled with video extensometry, confocal Raman spectroscopy, and transmission electron microscopy on native threads, we identified a continuous graded transition in mechanics, composition, and nanofibrillar morphology, which extends several hundreds of microns and which can vary significantly between individual threads. Furthermore, we performed in vitro fiber assembly experiments using purified secretory vesicles from the proximal and distal regions of the secretory glands (which contain different precursor proteins), revealing spontaneous self-assembly of distinctive distal- and proximal-like fiber morphologies. Aside from providing fundamental insights into the byssus structure, function, and fabrication, our findings reveal key design principles for bioinspired design of functionally graded polymeric materials.

A strong quick-release biointerface in mussels mediated by serotonergic cilia-based adhesion.

The mussel byssus stem provides a strong and compact mechanically mismatched biointerface between living tissue and a nonliving biopolymer. Yet, in a poorly understood process, mussels can simply jettison their entire byssus, rebuilding a new one in just hours. We characterized the structure and composition of the byssus biointerface using histology, confocal Raman mapping, phase contrast-enhanced microcomputed tomography, and advanced electron microscopy, revealing a sophisticated junction consisting of abiotic biopolymer sheets interdigitated between living extracellular matrix. The sheet surfaces are in intimate adhesive contact with billions of motile epithelial cilia that control biointerface strength and stem release through their collective movement, which is regulated neurochemically. We posit that this may involve a complex sensory pathway by which sessile mussels respond to environmental stresses to release and relocate.

Mussels Fabricate Porous Glues via Multiphase Liquid-Liquid Phase Separation of Multiprotein Condensates.

Mussels (Mytilus edulis) adhere to hard surfaces in intertidal marine habitats with a porous underwater glue called the byssus plaque. The plaque is an established role model for bioinspired underwater glues and comprises at least six proteins, most of which are highly cationic and enriched in the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA). While much is known about the chemistry of plaque adhesion, less is understood about the natural plaque formation process. Here, we investigated plaque structure and formation using 3D electron microscopic imaging, revealing that micro- and nanopores form spontaneously during secretion of protein-filled secretory vesicles. To better understand this process, we developed a method to purify intact secretory vesicles for in vitro assembly studies. We discovered that each vesicle contains a sulfate-associated fluid condensate consisting of ∼9 histidine- and/or DOPA-rich proteins, which are presumably the required ingredients for building a plaque. Rupturing vesicles under specific buffering conditions relevant for natural assembly led to controlled multiphase liquid-liquid phase separation (LLPS) of different proteins, resulting in formation of a continuous phase with coexisting droplets. Rapid coarsening of the droplet phase was arrested through pH-dependent cross-linking of the continuous phase, producing native-like solid porous "microplaques" with droplet proteins remaining as fluid condensates within the pores. Results indicate that histidine deprotonation and sulfates figure prominently in condensate cross-linking. Distilled concepts suggest that combining phase separation with tunable cross-linking kinetics could be effective for microfabricating hierarchically porous materials via self-assembly.

Collagen Pentablock Copolymers Form Smectic Liquid Crystals as Precursors for Mussel Byssus Fabrication.

Protein-based biological materials are important role models for the design and fabrication of next generation advanced polymers. Marine mussels (Mytilus spp.) fabricate hierarchically structured collagenous fibers known as byssal threads via bottom-up supramolecular assembly of fluid protein precursors. The high degree of structural organization in byssal threads is intimately linked to their exceptional toughness and self-healing capacity. Here, we investigated the hypothesis that multidomain collagen precursor proteins, known as preCols, are stored in secretory vesicles as a colloidal liquid crystal (LC) phase prior to thread self-assembly. Using advanced electron microscopy methods, including scanning TEM and FIB-SEM, we visualized the detailed smectic preCol LC nanostructure in 3D, including various LC defects, confirming this hypothesis and providing quantitative insights into the mesophase structure. In light of these findings, we performed an in-depth comparative analysis of preCol protein sequences from multiple Mytilid species revealing that the smectic organization arises from an evolutionarily conserved ABCBA pentablock copolymer-like primary structure based on demarcations in hydropathy and charge distribution as well as terminal pH-responsive domains that trigger fiber formation. These distilled supramolecular assembly principles provide inspiration and strategies for sustainable assembly of nanostructured polymeric materials for potential applications in engineering and biomedical applications.

Microfluidic-like fabrication of metal ion-cured bioadhesives by mussels.

To anchor in seashore habitats, mussels fabricate adhesive byssus fibers that are mechanically reinforced by protein-metal coordination mediated by 3,4-dihydroxyphenylalanine (DOPA). The mechanism by which metal ions are integrated during byssus formation remains unknown. In this study, we investigated the byssus formation process in the blue mussel, Mytilus edulis, combining traditional and advanced methods to identify how and when metals are incorporated. Mussels store iron and vanadium ions in intracellular metal storage particles (MSPs) complexed with previously unknown catechol-based biomolecules. During adhesive formation, stockpiled secretory vesicles containing concentrated fluid proteins are mixed with MSPs within a microfluidic-like network of interconnected channels where they coalesce, forming protein-metal bonds within the nascent byssus. These findings advance our understanding of metal use in biological materials with implications for next-generation metallopolymers and adhesives.

Author Correction: Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases.

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.

Metal-Tunable Self-Assembly of Hierarchical Structure in Mussel-Inspired Peptide Films.

Bottom-up control over structural hierarchy from the nanoscale through the macroscale is a critical aspect of biological materials fabrication and function, which can inspire production of advanced materials. Mussel byssal threads are a prime example of protein-based biofibers in which hierarchical organization of protein building blocks coupled via metal complexation leads to notable mechanical behaviors, such as high toughness and self-healing. Using a natural amino acid sequence from byssal thread proteins, which functions as a pH-triggered self-assembly point, we created free-standing peptide films with complex hierarchical organization across multiple length scales that can be controlled by inclusion of metal ions (Zn2+ and Cu2+) during the assembly process. Additionally, analysis of film mechanical performance indicates that metal coordination bestows up to an order of magnitude increase in material stiffness, providing a paradigm for creating tunable polymeric materials with multiscale organizational structure.

Mussel Byssus Structure-Function and Fabrication as Inspiration for Biotechnological Production of Advanced Materials.

Biotechnology offers an exciting avenue toward the sustainable production of high performance proteinaceous polymeric materials. In particular, the mussel byssus-a high performance adhesive bio-fiber used by mussels to cling on hard surfaces-has become a veritable archetype for bio-inspired self-healing fibers, tough coatings, and versatile wet adhesives. However, successful translation of mussel-inspired design principles into man-made materials hinges upon elucidating structure-function relationships and biological fabrication processes. This review provides a detailed survey of the state-of-the-art understanding of the biochemical structure-function relationships defining byssus performance with a particular focus on structural hierarchy and metal coordination-based cross-linking. The efforts to mimic the byssus in man-made materials are then discussed. While there has been a strong push to mimic the byssus via synthetic chemistry taking a reductionist approach, herein the focus is specifically on recent progress of biotechnology-based strategies that more closely approximate the biochemical complexity of the natural material. As an outlook, an overview of recent research toward understanding the natural byssus assembly process is provided, as processing remains a critical factor in achieving native-like properties.