Douglas S. Fudge

The Mechanical Properties of Hydrated Intermediate Filaments: Insights from Hagfish Slime Threads

Intermediate filaments (IFs) impart mechanical integrity to cells, yet IF mechanics are poorly understood. It is assumed that IFs in cells are as stiff as hard alpha-keratin, F-actin, and microtubules, but the high bending flexibility of IFs and the low stiffness of soft alpha-keratins suggest that hydrated IFs may be quite soft. To test this hypothesis, we measured the tensile mechanics of the keratin-like threads from hagfish slime, which are an ideal model for exploring the mechanics of IF bundles and IFs because they consist of tightly packed and aligned IFs. Tensile tests suggest that hydrated IF bundles possess low initial stiffness (E(i) = 6.4 MPa) and remarkable elasticity (up to strains of 0.34), which we attribute to soft elastomeric IF protein terminal domains in series with stiffer coiled coils. The high tensile strength (180 MPa) and toughness (130 MJ/m(3)) of IF bundles support the notion that IFs lend mechanical integrity to cells. Their long-range elasticity suggests that IFs may also allow cells to recover from large deformations. X-ray diffraction and congo-red staining indicate that post-yield deformation leads to an irreversible alpha-->beta conformational transition in IFs, which leads to plastic deformation, and may be used by cells as a mechanosensory cue.

Molecular design of the α–keratin composite: insights from a matrix–free model, hagfish slime threads

We performed mechanical tests on a matrix–free keratin model—hagfish slime threads—to test the hypothesis that intermediate filaments (IFs) in hydrated hard α–keratins are maintained in a partly dehydrated state. This hypothesis predicts that dry IFs should possess mechanical properties similar to the properties of hydrated hard α–keratins, and should swell more than hard α–keratins in water. Mechanical and swelling measurements of hagfish threads were consistent with both of these predictions, suggesting that an elastomeric keratin matrix resists IF swelling and keeps IF stiffness and yield stress high. The elastomeric nature of the matrix is indirectly supported by the inability of matrix–free IFs (i.e. slime threads) to recover from post–yield deformation. We propose a general conceptual model of the structural mechanics of IF–based materials that predicts the effects of hydration and cross–linking on stiffness, yield stress and extensibility.

Composition, morphology and mechanics of hagfish slime

SUMMARY Hagfish slime consists of mucins and protein threads that are released from slime glands and mix with seawater to produce an ephemeral material with intriguing physical properties. We recently characterized the mechanics of the slimes fibrous component, and here we report the first mechanical properties of the mucin component and the slime as a whole. Our results suggest that hagfishes can produce remarkable quantities of the slime because it is almost three orders of magnitude more dilute than typical mucus secretions. Mechanical experiments using whole slime produced in vitro demonstrate that the slime threads dominate the slimes material properties and impart elasticity. Mucins impart viscosity at the strain rates tested and are important for rapid deployment of the slime. We also found that slime threads are tapered at both ends, which suggested to us that hagfish slime might best be modeled as a discontinuous fibre-reinforced composite. Our measurements demonstrate that the mucins are not capable of providing shear linkage between threads, but this is not necessary because the threads are long enough to span an entire slime mass. Our findings suggest that hagfish slime consists mainly of bulk seawater entrained between mucin-coated threads, and in this way functions more like a fine sieve than coherent mucus. These results are consistent with the hypothesis that the slime has evolved as a defense against gill-breathing predators.

A Fish Out of Water: Gill and Skin Remodeling Promotes Osmo- and Ionoregulation in the Mangrove Killifish Kryptolebias marmoratus

The euryhaline, amphibious mangrove killifish Kryptolebias marmoratus is known to survive weeks out of water in moist environments. We tested the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus. We predicted that under terrestrial conditions, gill and skin remodeling would result in an enhanced role for skin and a diminished role for the gills in osmo- and ionoregulation. Fish were exposed to water—either freshwater (FW, 1‰) or hypersaline water (saltwater [SW], 45‰)—or air over a moist surface of FW or SW for 9 d and then recovered in water. When fish were emersed for 9 d, 22Na and 3H-H2O were exchanged across the cutaneous surface. Homeostasis of whole-body Cl− and water levels but not of Na+ levels was maintained over 9 d in air. In air-exposed fish, there was a significant increase in the size of skin ionocytes (in SW), a decrease in the number of skin mucous cells (in SW), and an increase in the gill interlamellar cell mass relative to those of fish in water. Gill ionocytes were mostly embedded away from the external surface in air-exposed fish, but the number and size of ionocytes increased (in FW). Interestingly, skin ionocytes formed distinct clusters of 20–30 cells. The estimated number of ionocytes over the whole skin surface was comparable to that in the gills. Overall, the findings support the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus in aquatic and terrestrial environments. Reversible cellular and morphological changes to the skin and gills during air exposure probably enhanced the cutaneous contribution to ion and water balance.

The intermediate filament network in cultured human keratinocytes is remarkably extensible and resilient.

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes.

Hagfish slime ecomechanics : testing the gill-clogging hypothesis

SUMMARY Hagfish are able to produce substantial amounts of slime when harassed, but the precise ecological function of the slime is unclear. One possibility is that the slime acts as a defence against gill-breathing predators, whose gills may become entangled with the slimes mixture of mucins and fibrous threads during an attack. We previously demonstrated that hagfish slime does not bind water tightly, but instead behaves like a fine sieve that slows water down via viscous entrainment. These properties are consistent with the gill-clogging hypothesis, which we tested here by quantifying the effects of hagfish slime on water flow through an artificial gill model and real fish gills. Our results indicate that the slime is capable of clogging gills and increasing the resistance that they present to the flow of water. We also characterized the behaviour of slime release from live hagfish and the effect of convective mixing on the formation of slime in vitro. Our observations show that exudate is locally released from the slime glands as a coherent jet and that hagfish do not appear to use their slime as a protective envelope. We found that convective mixing between the exudate and seawater is necessary for proper slime formation, but excessive mixing leads to the slimes collapse. We suggest that the loose binding of water by the slime may be an optimal solution to the problem of delivering an expanding jet of flow-inhibiting material to the gills of would-be predators.

Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands

SUMMARY Hagfishes are benthic marine protovertebrates that secrete copious quantities of slime when threatened. The slime originates as a two-component glandular exudate comprised of coiled bundles of cytoskeletal intermediate filaments (thread skeins) and mucin vesicles. Holocrine secretion of the slime into seawater results in the rapid deployment of both fibrous and mucin components, resulting in about a liter of dilute slime. Deployment of the thread skeins involves their unraveling in a fraction of a second from a 150 μm-long ellipsoid bundle to a thread that is 100× longer. We hypothesized that thread skein deployment requires both vigorous hydrodynamic mixing and the presence of mucin vesicles, both of which are required for whole slime deployment. Here we provide evidence that mixing and mucin vesicles are indeed crucial for skein unraveling. Specifically, we show that mucin vesicles mixed into seawater swell and elongate into high-aspect ratio mucin strands that attach to the thread skeins, transmit hydrodynamic forces to them and effect their unraveling by loading them in tension. Our discovery of mucin strands in hagfish slime not only provides a mechanism for the rapid deployment of thread skeins in vivo, it also helps explain how hagfish slime is able to trap such impressive volumes of seawater via viscous entrainment. We believe that the deployment of thread skeins via their interaction with shear-elongated mucins represents a unique mechanism in biology and may lead to novel technologies for transmitting hydrodynamic forces to microscale particles that would typically be immune to such forces.

From ultra-soft slime to hard α-keratins: The many lives of intermediate filaments

Intermediate filaments are filaments 10 nm in diameter that make up an important component of the cytoskeleton in most metazoan taxa. They are most familiar for their role as the fibrous component of α-keratins such as skin, hair, nail, and horn but are also abundant within living cells. Although they are almost exclusively intracellular in their distribution, in the case of the defensive slime produced by hagfishes, they are secreted. This article surveys the impressive diversity of biomaterials that animals construct from intermediate filaments and will focus on the mechanisms by which the mechanical properties of these materials are achieved. Hagfish slime is a dilute network of hydrated mucus and compliant intermediate filament bundles with ultrasoft material properties. Within the cytoplasm of living cells, networks of intermediate filaments form soft gels whose elasticity arises via entropic mechanisms. Single intermediate filaments or bundles are also elastic, but substantially stiffer, exhibiting modulus values similar to that of rubber. Hard α-keratins like wool are stiffer still, an effect that is likely achieved via dehydration of the intermediate filaments in these epidermal appendages. The diverse mechanisms described here have been employed by animals to generate materials with stiffness values that span an impressive eleven orders of magnitude.

Morphology and Development of Blue Whale Baleen: An Annotated Translation of Tycho Tullberg's Classic 1883 Paper

Herein we present an annotated translation of the classic paper by Tycho Tullberg on the structure and development of baleen in blue whales. The three blue whale fetuses on which this study was based were obtained from a whaling station in Norway during a time when blue whales were still abundant enough to support a whaling industry. The value of this text for the modern reader is that it provides a glimpse into the mechanisms of development of baleen in the largest rorqual whale, which is something that modern biologists are unlikely to be able to replicate for a long time. Tullberg’s careful morphology, histology, and developmental thinking provide a coherent account of how the elaborate baleen racks develop from simple epidermal and dermal origins. The figures, which we have reproduced here, are superb and provide a rare window into the morphology of blue whale baleen at three fetal stages. The histology is excellent for its time and provides insights into the various keratin tissue phases that make up the baleen plates and bristles as well as the enigmatic Zwischensubstanz that acts as a spacer and possible shock-absorber between plates.

The Mechanical Behavior of Mutant K14-R125P Keratin Bundles and Networks in NEB-1 Keratinocytes

Epidermolysis bullosa simplex (EBS) is an inherited skin-blistering disease that is caused by dominant mutations in the genes for keratin K5 or K14 proteins. While the link between keratin mutations and keratinocyte fragility in EBS patients is clear, the exact biophysical mechanisms underlying cell fragility are not known. In this study, we tested the hypotheses that mutant K14-R125P filaments and/or networks in human keratinocytes are mechanically defective in their response to large-scale deformations. We found that mutant filaments and networks exhibit no obvious defects when subjected to large uniaxial strains and have no negative effects on the ability of human keratinocytes to survive large strains. We also found that the expression of mutant K14-R125P protein has no effect on the morphology of the F-actin or microtubule networks or their responses to large strains. Disassembly of the F-actin network with Latrunculin A unexpectedly led to a marked decrease in stretch-induced necrosis in both WT and mutant cells. Overall, our results contradict the hypotheses that EBS mutant keratin filaments and/or networks are mechanically defective. We suggest that future studies should test the alternative hypothesis that keratinocytes in EBS cells are fragile because they possess a sparser keratin network.