Physiology, biomechanics, and biomimetics of hagfish slime.

Hagfishes thwart attacks by fish predators by producing liters of defensive slime. The slime is produced when slime gland exudate is released into the predator's mouth, where it deploys in a fraction of a second and clogs the gills. Slime exudate is composed mainly of secretory products from two cell types, gland mucous cells and gland thread cells, which produce the mucous and fibrous components of the slime, respectively. Here, we review what is known about the composition of the slime, morphology of the slime gland, and physiology of the cells that produce the slime. We also discuss several of the mechanisms involved in the deployment of both mucous and thread cells during the transition from thick glandular exudate to ultradilute material. We review biomechanical aspects of the slime, along with recent efforts to produce biomimetic slime thread analogs, and end with a discussion of how hagfish slime may have evolved.

High concentrations of trimethylamines in slime glands inhibit skein unraveling in Pacific hagfish.

Hagfish defend themselves from fish predators by producing large volumes of gill-clogging slime when they are attacked. The slime consists of seawater and two major components that are ejected from the slime glands: mucus and threads. The threads are produced within specialized cells and packaged into intricately coiled bundles called skeins. Skeins are kept from unraveling via a protein adhesive that dissolves when the skeins are ejected from the slime glands. Previous work revealed that hagfish slime glands have high concentrations of methylamines including trimethylamine N-oxide (TMAO), trimethylglycine (betaine) and dimethylglycine (DMG); however, the function of these compounds in the slime glands is unknown. We hypothesized that methylamines have stabilizing effects on the skeins that prevent premature unraveling in the gland. To test this hypothesis, we quantified the effect of methylamines on skein unraveling in Pacific hagfish and found that TMAO and betaine have inhibitory effects on skein unraveling in vitro Furthermore, we found that TMAO is a more effective inhibitor of unraveling than betaine, but the presence of TMAO synergistically boosts the inhibitory action of betaine. Glycine and DMG were far less effective inhibitors of unraveling at natural concentrations. Our results support the hypothesis that high levels of trimethylamines in the slime glands may act to hold the coiled thread skeins together within gland thread cells, and they may do so by stabilizing adhesive proteins. These results advance our knowledge of skein stabilization and deployment and provide yet another example of trimethylamines functioning to stabilize proteins in a marine organism.

Structure and dynamics of hagfish mucin in different saline environments.

The defense mechanism of hagfish against predators is based on its ability to form slime within a few milliseconds. Hagfish slime consists of two main components, namely mucin-like glycoproteins and long protein threads, which together entrap vast amounts of water and thus form a highly dilute hydrogel. Here, we investigate the mucin part of this hydrogel, in particular the role of the saline marine environment on the viscoelasticity and structure. By means of dynamic light scattering (DLS), shear and extensional rheology we probe the diffusion dynamics, the flow behavior, and the longest filament breaking time of hagfish mucin solutions. Using DLS we find a concentration-independent diffusion coefficient - characteristic for polyelectrolytes - up to the entanglement regime of 0.2 mg ml-1, which is about ten times higher than the natural concentration of hagfish mucin in hagfish slime. We also observe a slow relaxation process associated with clustering, probably due to electrostatic interactions. Shear rheology further revealed that hagfish mucin possesses pronounced viscoelastic properties at high concentrations (3 mg ml-1), showing that mucin alone achieves mechanical properties similar to those of natural hagfish slime (mucins and protein threads). The main effects of added seawater salts, and predominantly CaCl2 is to reduce the intensity of the slow relaxation process, which suggests that calcium ions lead to an ionotropic gelation of hagfish mucins.

Emptying and refilling of slime glands in Atlantic (Myxine glutinosa) and Pacific (Eptatretus stoutii) hagfishes.

Hagfishes are known for their unique defensive slime, which they use to ward off gill-breathing predators. Although much is known about the slime cells (gland thread cells and gland mucous cells), little is known about how long slime gland refilling takes, or how slime composition changes with refilling or repeated stimulation of the same gland. Slime glands can be individually electrostimulated to release slime, and this technique was used to measure slime gland refilling times for Atlantic and Pacific hagfish. The amount of exudate produced, the composition of the exudate and the morphometrics of slime cells were analyzed during refilling, and as a function of stimulation number when full glands were stimulated in rapid succession. Complete refilling of slime glands for both species took 3-4 weeks, with Pacific hagfish achieving faster absolute rates of exudate recovery than Atlantic hagfish. We found significant changes in the composition of the exudate and in the morphometrics of slime cells from Pacific hagfish during refilling. Over successive stimulations of full Pacific hagfish glands, multiple boluses of exudate were released, with exudate composition, but not thread cell morphometrics, changing significantly. Finally, histological examination of slime glands revealed slime cells retained in glands after exhaustion. Discrepancies in the volume of cells released suggest that mechanisms other than contraction of the gland musculature alone may be involved in exudate ejection. Our results provide a first look at the process and timing of slime gland refilling in hagfishes, and raise new questions about how refilling is achieved at the cellular level.

Unraveling inter-species differences in hagfish slime skein deployment.

Hagfishes defend themselves from fish predators by producing defensive slime consisting of mucous and thread components that interact synergistically with seawater to pose a suffocation risk to their attackers. Deployment of the slime occurs in a fraction of a second and involves hydration of mucous vesicles as well as unraveling of the coiled threads to their full length of ∼150 mm. Previous work showed that unraveling of coiled threads (or 'skeins') in Atlantic hagfish requires vigorous mixing with seawater as well as the presence of mucus, whereas skeins from Pacific hagfish tend to unravel spontaneously in seawater. Here, we explored the mechanisms that underlie these different unraveling modes, and focused on the molecules that make up the skein glue, a material that must be disrupted for unraveling to proceed. We found that Atlantic hagfish skeins are also held together with a protein glue, but compared with Pacific hagfish glue, it is less soluble in seawater. Using SDS-PAGE, we identified several soluble proteins and glycoproteins that are liberated from skeins under conditions that drive unraveling in vitro Peptides generated by mass spectrometry of five of these proteins and glycoproteins mapped strongly to 14 sequences assembled from Pacific hagfish slime gland transcriptomes, with all but one of these sequences possessing homologs in the Atlantic hagfish. Two of these sequences encode unusual acidic proteins that we propose are the structural glycoproteins that make up the skein glue. These sequences have no known homologs in other species and are likely to be unique to hagfishes. Although the ecological significance of the two modes of skein unraveling described here are unknown, they may reflect differences in predation pressure, with selection for faster skein unraveling in the Eptatretus lineage leading to the evolution of a glue that is more soluble.

Cellular mechanisms of slime gland refilling in Pacific hagfish (Eptatretus stoutii).

Hagfishes use their defensive slime to ward off gill-breathing predators. Slime gland refilling is a surprisingly slow process, and previous research has shown that the composition of the slime exudate changes significantly during refilling, which likely has consequences for the functionality of the slime. This study set out to expand our understanding of slime gland refilling by examining the cellular processes involved in refilling of the glands, as well as determining where in the gland the main slime cells - the gland thread cells and gland mucous cells - arise. Slime glands were electro-stimulated to exhaust their slime stores, left to refill for set periods of time, and harvested for histological and immunohistochemical examination. Whole slime glands, gland thread cell morphometrics and slime cell proportions were examined over the refilling cycle. Slime glands decreased significantly in size after exhaustion, but steadily increased in size over refilling. Gland thread cells were the limiting factor in slime gland refilling, taking longer to replenish and mature than gland mucous cells. Newly produced gland thread cells underwent most of their growth near the edge of the gland, and larger cells were found farthest from the edge of the gland. Immunohistochemical analysis also revealed proliferating cells only within the epithelial lining of the slime gland, suggesting that new slime cells originate from undifferentiated cells lining the gland. Our results provide an in-depth look at the cellular dynamics of slime gland refilling in Pacific hagfish, and provide a model for how slime glands refill at the cellular level.

Self-Assembly of Recombinant Hagfish Thread Keratins Amenable to a Strain-Induced α-Helix to ß-Sheet Transition.

Hagfish slime threads are assembled from protein-based bundles of intermediate filaments (IFs) that undergo a strain-induced α-helical coiled-coil to ß-sheet transition. Draw processing of native fibers enables the creation of mechanically tuned materials, and under optimized conditions this process results in mechanical properties similar to spider dragline silk. In this study, we develop the foundation for the engineering of biomimetic recombinant hagfish thread keratin (TK)-based materials. The two protein constituents from the hagfish Eptatretus stoutii thread, named EsTKα and EsTKγ, were expressed in Escherichia coli and purified. Individual (rec)EsTKs and mixtures thereof were subjected to stepwise dialysis to evaluate their protein solubility, folding, and self-assembly propensities. Conditions were identified that resulted in the self-assembly of coiled-coil rich IF-like filaments, as determined by circular dichroism (CD) and transmission electron microscopy (TEM). Rheology experiments indicated that the concentrated filaments assembled into gel-like networks exhibiting a rheological response reminiscent to that of IFs. Notably, the self-assembled filaments underwent an α-helical coiled-coil to ß-sheet transition when subjected to oscillatory shear, thus mimicking the critical characteristic responsible for mechanical strengthening of native hagfish threads. We propose that our data establish the foundation to create robust and tunable recombinant TK-based materials whose mechanical properties are controlled by a strain-induced α-helical coiled-coil to ß-sheet transition.

Structure-activity relations of myxinidin, an antibacterial peptide derived from the epidermal mucus of hagfish.

The structure-activity relations of myxinidin, a peptide derived from epidermal mucus of hagfish, Myxine glutinosa L., were investigated. Analysis of key residues allowed us to design new peptides with increased efficiency. Antimicrobial activity of native and modified peptides demonstrated the key role of uncharged residues in the sequence; the loss of these residues reduces almost entirely myxinidin antimicrobial activity, while insertion of arginine at charged and uncharged position increases antimicrobial activity compared with that of native myxinidin. Particularly, we designed a peptide capable of achieving a high inhibitory effect on bacterial growth. Experiments were conducted using both Gram-negative and Gram-positive bacteria. Nuclear magnetic resonance (NMR) studies showed that myxinidin is able to form an amphipathic α-helical structure at the N terminus and a random coil region at the C terminus.

Mechanisms of gill-clogging by hagfish slime.

Hagfishes defend themselves from gill-breathing predators by producing large volumes of fibrous slime when attacked. The slime's effectiveness comes from its ability to clog predators' gills, but the mechanisms by which hagfish slime clogs are uncertain, especially given its remarkably dilute concentration of solids. We quantified the clogging performance of hagfish slime over a range of concentrations, measured the contributions of its mucous and thread components, and measured the effect of turbulent mixing on clogging. To assess the porous structure of hagfish slime, we used a custom device to measure its Darcy permeability. We show that hagfish slime clogs at extremely dilute concentrations like those found in native hagfish slime and displays clogging performance that is superior to three thickening agents. We report an extremely low Darcy permeability for hagfish slime, and an effective pore size of 10-300 nm. We also show that the mucous and thread components play distinct yet crucial roles, with mucus being responsible for effective clogging and low permeability and the threads imparting mechanical strength and retaining clogging function over time. Our results provide new insights into the mechanisms by which hagfish slime clogs gills and may inspire the development of ultra-soft materials with novel properties.

From reductionism to synthesis: The case of hagfish slime.

Reductionist strategies aim to understand the mechanisms of complex systems by studying individual parts and their interactions. In this review, we discuss how reductionist approaches have shed light on the structure, function, and production of a complex biomaterial - hagfish defensive slime. Hagfish slime is an extremely dilute hydrogel-like material composed of seawater, mucus, and silk-like proteins that can deploy rapidly. Despite being composed almost entirely of water, hagfish slime has remarkable physical properties, including high strength and toughness. While hagfish slime has a promising future in biomimetics, including the development of eco-friendly high-performance fibers, recreating hagfish slime in the lab has been a difficult challenge. Over the past two decades, reductionist experiments have provided a wealth of information about the individual components of hagfish slime. However, a reductionist approach provides a limited understanding because hagfish defensive slime, like most biological phenomena, is more than just the sum of its parts. We end by providing some thoughts about how the knowledge generated in the last few decades might be synthesized into a working model that can explain hagfish slime structure and function.

Biomimetic nuclear lamin fibers with remarkable toughness and stiffness.

The native hagfish slime threads, which are made up of two intermediate filament (IF)-like proteins, exhibit mechanical properties comparable to dragline spider silk fiber, the toughest fiber in nature. However, unlike silk, the design of artificial IF-protein-based fibers has been rarely studied, possibly because the unique hierarchical organization of the keratin-like proteins within these threads is challenging to mimic, and consequently, extraordinary fiber mechanics has not been shown in slime threads from recombinant IF-protein-based system. Here, we have reported the synthesis and properties of recombinant type V IF-protein, based on the Caenorhabditis elegans (Ce) lamin gene. The protein was solubilized and wet-spun into aqueous solutions to prepare Ce-lamin fibers by varying injection flow rates and Ca+2 ion concentrations in the coagulation buffer. At specific set of conditions, Ce-lamin fibers demonstrated remarkable toughness and stiffness, comparable to hagfish slime threads and natural dragline spider silk. Transmission electron microscopy analysis showed that paracrystals were the main nanometric structure within the fibers. This study demonstrates that outstanding mechanical properties can be achieved with recombinant IF-proteins through self-organization. Thus, these results have broadened the pool of fibrous proteins that can be used in functional materials for a diverse range of applications.

An active domain HF-18 derived from hagfish intestinal peptide effectively inhibited drug-resistant bacteria in vitro/vivo.

Antibiotic resistance is spreading faster than the development of new antibiotics into clinical practice. Currently, the design of antimicrobial peptides (AMPs), potential new antibacterial agents with rare antimicrobial resistance, is the available strategy to enhance the antimicrobial activity and lower the toxicity of AMPs. In this study, a peptide derived from hagfish intestinal peptide was designed and termed as HF-18 (GFFKKAWRKVKKAFRRVL). After antimicrobial/bactericidal test in vitro, we found that HF-18 exhibited a potent antimicrobial activity with MIC of only 4 µg/ml against drug-resistant Staphylococcus aureus (S. aureus). Meanwhile, it eliminated the test bacteria within 1 h, suggesting its rapid bactericidal effect. Importantly, this peptide had no obvious hemolytic activity and cytotoxicity to mammalian cells. Furthermore, its notable antimicrobial effects in vivo was confirmed again in S. aureus induced mouse bacteremia and skin wound infection, reflecting as the decrease in bacterial counts in mouse lung or skin (up to 1.9 or 3.5 log CFU respectively), and including the inhibitory activity on inflammatory cytokines secretion. The possible mechanisms underlying HF-18 against drug-resistant S. aureus may attribute that HF-18 neutralized the negative charge in S. aureus surface and then disrupted the integrity of cell membranes to enhance the permeation of bacterial membrane, showing as the increased uptake of NPN and PI and the obvious morphology changes of S. aureus. In addition, this peptide bound to bacterial genomic DNA to suppress the expression of Panton-Valentine leukocidin (pvl) and nuclease (nuc) genes, which play major roles in S. aureus virulence. The properties of HF-18 suggest a path towards developing antibacterial agents that has stronger antibacterial activity and greater security for clinical treatment of infection induced by S. aureus, especially drug-resistant S. aureus.

Unravelling hagfish slime.

Hagfish slime is a unique predator defence material containing a network of long fibrous threads each ∼10 cm in length. Hagfish release the threads in a condensed coiled state known as skeins (∼100 µm), which must unravel within a fraction of a second to thwart a predator attack. Here we consider the hypothesis that viscous hydrodynamics can be responsible for this rapid unravelling, as opposed to chemical reaction kinetics alone. Our main conclusion is that, under reasonable physiological conditions, unravelling due to viscous drag can occur within a few hundred milliseconds, and is accelerated if the skein is pinned at a surface such as the mouth of a predator. We model a single skein unspooling as the fibre peels away due to viscous drag. We capture essential features by considering simplified cases of physiologically relevant flows and one-dimensional scenarios where the fibre is aligned with streamlines in either uniform or uniaxial extensional flow. The peeling resistance is modelled with a power-law dependence on peeling velocity. A dimensionless ratio of viscous drag to peeling resistance appears in the dynamical equations and determines the unraveling time scale. Our modelling approach is general and can be refined with future experimental measurements of peel strength for skein unravelling. It provides key insights into the unravelling process, offers potential answers to lingering questions about slime formation from threads and mucous vesicles, and will aid the growing interest in engineering similar bioinspired material systems.

Concentration-independent mechanics and structure of hagfish slime.

The defense mechanism of hagfish slime is remarkable considering that hagfish cannot control the concentration of the resulting gel directly; they simply exude a concentrated material into a comparably "infinite" sea of water to form a dilute, sticky, cohesive elastic gel. This raises questions about the robustness of gel formation and rheological properties across a range of concentrations, which we study here for the first time. Across a nearly 100-fold change in concentration, we discover that the gel has similar viscoelastic time-dependent properties with constant power-law exponent (α=0.18±0.01), constant relative damping tanδ=G''/G'≈0.2-0.3, and varying overall stiffness that scales linearly with the concentration (∼c0.99±0.05). The power-law viscoelasticity (fit by a fractional Kelvin-Voigt model) is persistent at all concentrations with nearly constant fractal dimension. This is unlike other materials and suggests that the underlying material structure of slime remains self-similar irrespective of concentration. This interpretation is consistent with our microscopy studies of the fiber network. We derive a structure-rheology model to test the hypothesis that the origins of ultra-soft elasticity are based on bending of the fibers. The model predictions show an excellent agreement with the experiments. Our findings illustrate the unusual and robust properties of slime which may be vital in its physiological use and provide inspiration for the design of new engineered materials. STATEMENT OF SIGNIFICANCE: Hagfish produce a unique gel-like material to defend themselves against predator attacks. The successful use of the defense gel is remarkable considering that hagfish cannot control the concentration of the resulting gel directly; they simply exude a small quantity of biomaterial which then expands by a factor of 10,000 (by volume) into an "infinite" sea of water. This raises questions about the robustness of gel formation and properties across a range of concentrations. This study provides the first ever understanding of the mechanics of hagfish slime over a very wide range of concentration. We discover that some viscoelastic properties of slime are remarkably constant regardless of its concentration. Such a characteristic is uncommon in most known materials.

Structure and Nanomechanics of Dry and Hydrated Intermediate Filament Films and Fibers Produced from Hagfish Slime Fibers.

Intermediate filaments (IFs) are known for their extensibility, flexibility, toughness, and their ability to hydrate. Using keratin-like IFs obtained from slime fibers from the invertebrate Atlantic hagfish ( Myxine glutinosa), films were produced by drop-casting and coagulation on the surface of a MgCl2 buffer. Drop-casting produced self-supporting, smooth, and dense films rich in ß-sheets (61%), whereas coagulation formed thin, porous films with a nanorough surface and a lower ß-sheet content (51%). The films hydrated and swelled immediately when immersed in water and did not dissolve. X-ray diffraction showed that the ß-crystallites remained stable upon hydration, that swelling presumably happens in the amorphous C-terminal tail-domains of the IFs, and that high salt conditions caused a denser network mesh size, suggesting polyelectrolyte behavior. Hydration resulted in a roughly 1000-fold decrease in apparent Young's modulus from 109 to 106 Pa as revealed by atomic force microscopy nanoindentation. Nanoindentation-based power-law rheology and stress-relaxation measurements indicated viscoelasticity and a soft-solid hydrogel character for hydrated films, where roughly 80% of energy is elastically stored and 20% is dissipated. By pulling coagulation films from the buffer interface, macroscopic fibers with highly aligned IF ß-crystals similar to natural hagfish fibers were produced. We propose that viscoelasticity and strong hydrogen bonding interactions with the buffer interface are crucial for the production of such long biomimetic fibers with aligned ß-sheets. This study demonstrates that hagfish fiber IFs can be reconstituted into functional biomimetic materials that are stiff when dry and retain the ability to hydrate to become soft and viscoelastic when in water.

Gelation of Soy Milk with Hagfish Exudate Creates a Flocculated and Fibrous Emulsion- and Particle Gel.

Hagfish slime is an ultra dilute, elastic and cohesive hydrogel that deploys within milliseconds in cold seawater from a glandularly secreted exudate. The slime is made of long keratin-like fibers and mucin-like glycoproteins that span a network which entraps water and acts as a defense mechanism against predators. Unlike other hydrogels, the slime only confines water physically and is very susceptible to mechanical stress, which makes it unsuitable for many processing operations and potential applications. Despite its huge potential, little work has been done to improve and functionalize the properties of this hydrogel. To address this shortcoming, hagfish exudate was mixed with a soy protein isolate suspension (4% w/v) and with a soy emulsion (commercial soy milk) to form a more stable structure and combine the functionalities of a suspension and emulsion with those of the hydrogel. Hagfish exudate interacted strongly with the soy systems, showing a markedly increased viscoelasticity and water retention. Hagfish mucin was found to induce a depletion and bridging mechanism, which caused the emulsion and suspension to flocculate, making "soy slime", a cohesive and cold-set emulsion- and particle gel. The flocculation network increases viscoelasticity and substantially contributes to liquid retention by entrapping liquid in the additional confinements between aggregated particles and protein fibers. Because the mucin-induced flocculation resembles the salt- or acid-induced flocculation in tofu curd production, the soy slime was cooked for comparison. The cooked soy slime was similar to conventional cooked tofu, but possessed a long-range cohesiveness from the fibers. The fibrous, cold-set, and curd-like structure of the soy slime represents a novel way for a cold coagulation and fiber incorporation into a suspension or emulsion. This mechanism could be used to efficiently gel functionalized emulsions or produce novel tofu-like structured food products.

Two forms of adaptive immunity in vertebrates: similarities and differences.

Unlike jawed vertebrates that use T-cell and B-cell receptors for antigen recognition, jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors. VLRs generate diversity comparable to that of gnathostome antigen receptors by assembling variable leucine-rich repeat modules. The discovery of VLR has revolutionized our understanding of how adaptive immunity emerged and highlighted the differences between the adaptive immune systems (AISs) of jawed and jawless vertebrates. However, emerging evidence also indicates that their AISs have much in common. Particularly striking is the conservation of lymphocyte lineages. The basic architecture of the AIS including the dichotomy of lymphocytes appears to have been established in a common ancestor of jawed and jawless vertebrates. We review here the current knowledge on the AIS of jawless vertebrates, emphasizing both the similarities to and differences from the AIS of jawed vertebrates.