Biophysics and Biomimetics of Hagfish Slime

Abstract

Hagfish defend themselves with vast amounts of slime when provoked or attacked. The slime forms when so-called exudate, which consists of coiled up threads (‘skeins’) and mucin vesicles is released into the surrounding seawater. Skeins resemble a ‘ball of wool’ as they are made of a single coiled up intermediate filament (IF) protein thread that is up to 30 cm long and 1 3 μm in diameter. Skeins unravel and create an underwater fiber network. Simultaneously, the mucin vesicles swell and burst and release mucin-like glycoproteins, which interact with the threads and together form hagfish slime. The secreted slime is a unique biomaterial as it is the most dilute and fastest forming hydrogel known to date. Furthermore, the fibers provide high elasticity and cohesiveness to the otherwise soft gel and were found to have similar properties to spider’s silk. Intrigued by its fast, efficient, and cold gelation, hagfish slime was used as a model to characterize and mimic high-performance marine soft materials. By pursuing a holistic ‘from fish to fiber’ approach, we investigated how slime can be harvested, stabilized, regenerated, and eventually transformed into novel biomimetic materials. In a first part, harvesting and stabilization of hagfish exudate is investigated, whereby two stabilization methods immersion in MCT (medium chain triglycerides) oil and dispersion in a high osmolarity citrate/PIPES (CP) buffer were compared. Using water retention measurements to assess the functionality of hagfish slime, it was shown that for short storage times (< five hours) both stabilization methods produced slime networks equal to fresh exudate. Longer storage times caused the exudate samples to degrade, whereby MCT samples formed clumps after about seven days, probably due to osmotic and temperature driven rupture of mucin vesicles. CP buffer stabilized samples, in contrast showed a gradual loss of functionality due to reduced skein unraveling. Long buffer exposure times caused less skeins to unravel and therefore less water was retained. It is likely that a seawater soluble glue, which holds the threads together and mediates unraveling denatures during storage in the buffer and becomes insoluble and thus decreases slime functionality. Having stabilization guidelines at hand, we dealt with the dynamics of slime formation. Motivated by the fact that this fibrous polyelectrolyte hydrogel efficiently and rapidly forms in a high ionic strength environment, we demonstrate the crucial role of ionic strength and seawater cations especially Ca2+ for the formation dynamics and functionality of hagfish slime. We suggest that sufficient ionic strength controls the dynamics of skein unraveling and slime network formation. A low ionic strength caused a confined and narrow thread network in contrast to the widespread and expanded network formed in seawater. In MilliQ thread skeins swelled and unraveled uncontrolled from both sides, causing tangling of the threads and thus preventing a widespread network. The fast unraveling in ion-free water seems to originate in an excessive swelling of the intermediate filament slime thread, which would possess increased stored strain energy. Only in the presence of Ca2+ and Mg2+ a functional slime network is realized at seawater strength osmolarity. The presence of calcium allowed the formation of a functional slime network up to 3 M NaCl, corresponding to 45 times the ionic strength of seawater. These results show that a functional defensive slime that entraps and retains water can only be formed in the presence of divalent seawater cations Ca2+ or Mg2+ at a high ionic strength. With the boundaries of slime formation outlined, we tackled the question whether slime flow properties have implications on hagfish defense behavior. Oscillatory rheological measurements revealed that hagfish slime forms viscoelastic networks and that mucins alone do not contribute viscoelasticity at their natural concentration. However, in shear flow viscosity was observed. We propose that the threads provide extensibility and cohesiveness, prevent mucin washout, and allow the mucin to contribute to viscoelasticity by supplying anchoring points. When mucins were exposed to elongational stresses experienced by hagfish slime during suction feeding by predators mucin viscosity strongly increased. This increased resistance to flow could support clogging of an attacker’s gills. Shear flow, in contrast decreased the slime’s viscosity by mucin aggregation and lead to a collapse of the slime network. Hagfish may benefit from this collapse by tying a sliding knot with their body to shear off the slime when trapped in their own defensive weapon and facing suffocation. This removal could be facilitated by the apparent shear thinning behavior of the slime. Therefore hagfish slime, thickening in elongation and thinning in shear, possesses flow properties that seem beneficial for both, defense and escape. In a final step, we explored the potential to transform intermediate filaments (IFs) obtained from hagfish slime fibers into biomimetic films and fibers. Formic acid solubilized hagfish IFs were used to produce films by drop casting and coagulation on a MgCl2 buffer. Drop casting yielded self-supporting, smooth, and dense films rich in β-sheets (61%) whereas coagulation formed thin, porous films with a nano-rough surface and a lower βsheet content (51%). When immersed in water the films immediately swelled. X-ray scattering revealed that the β-crystallites remained stable upon hydration and that swelling presumably happens in the amorphous C-terminal tail-domains of the IFs. X-ray measurements further suggested a polyelectrolyte behavior of hagfish IFs as the average mesh-size of the IF network as well as the inter β-sheet distance decreased upon increase of salt concentration. Using AFM nanoindentation it was observed that hydration caused a roughly thousandfold decrease in apparent elastic modulus from roughly 10^9 to 10^6 Pa and that the hydrated films displayed distinct viscoelastic behavior, characteristic for soft-solid and tough hydrogels. Fitting a power-law rheology model directly to the force-distance curves yielded a power-law relaxation exponent α of roughly < 0.2 for both films, suggesting 80% of elastic storage and 20% of viscous loss in force measurements. We propose that hagfish IF films possess β-sheet clusters from an α→β transformed central part of the IFs embedded in an amorphous matrix constituted by the physically entangled C-terminal tail-domains, which determines cohesion and viscoelasticity in hydrated films. We further suggest that viscoelasticity and strong hydrogen bonding interactions of the coagulation film with the buffer surface are crucial for a successful fiber making process, in which a coagulation film is pulled from the buffer interface into a fiber. The combination of relaxing stresses within the film and strong hydrogen bonding of the film with the water interface allow for a continuous stretching yet prevent early removal of the film from the interface, thus creating long biomimetic fibers with high IF alignment similar to natural hagfish fibers. This last part shows that functional IF materials that immediately swell and soften in water without dissolving can be produced from hagfish slime fibers, which could be used in applications such as tissue implants, scaffolds for cell cultures, or contact lenses.