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THE SKIN | Hagfish Slime
Hagfish Slime DS Fudge, JE Herr, and TM Winegard, University of Guelph, Guelph, ON, Canada ª 2011 Elsevier Inc. All rights reserved.
Introduction The Hagfish Slime Gland GMCs and Mucin Vesicles Gland Thread Cells Volume and Concentration
Glossary Exocytosis The process by which neurotransmitters or hormones, which are stored in secretory vesicles or granules, are released from a nerve or endocrine cell when these storage granules fuse with the plasma membrane Gland mucus cell Cell within hagfish slime glands that give rise to the mucus component of hagfish slime. Gland thread cell Cell within hagfish slime glands that give rise to the fibrous component of hagfish slime. Hagfish slime The material that is formed when hagfishes forcefully release the glandular contents of their numerous slime glands into seawater. Hagfish slime exudate The thick white fluid that is ejected from hagfish slime glands and that interacts with
Introduction Hagfishes are best known for their ability to produce alarming amounts of slime when they are stressed or provoked (see also Hagfishes and Lamprey: Hagfishes). In fact, many of their common names include the word slime, such as slime eel and slime hag. The Atlantic hagfish, Myxine glutinosa, is twice named for its slime (myx ¼ slime, glutin ¼ glue). Many animals release slime under stress, but hagfish slime is unique because of the large volumes that are produced (up to several liters from a single hagfish) (Figure 1), the impressive speed with which it is made, and the fact that it contains tens of thousands of fine protein fibers (Figure 2). Hagfish slime can be defined as the material that is formed when hagfishes forcefully release the glandular contents of their numerous slime glands into seawater. Many marine animals, including hagfishes, make some sort of epidermal mucus or slime that coats their body, but hagfish slime is different from these secretions in many
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Mechanics and Function Slime Deployment and Maturation Structure and Function Further Reading
seawater to form hagfish slime. The exudate contains numerous mucin vesicles and thread skeins. Holocrine secretion Mode of secretion in which entire cells are ejected from a gland. Intermediate filaments The 10-nm-diameter cytoskeletal filaments that make up hagfish slime threads. Mucin vesicles Small, membrane-bound structures containing condensed mucus that are released when gland mucus cells are ejected from hagfish slime glands. Slime threads Fine protein fibers that permeate hagfish slime and arise from the deployment of ejected thread skeins. Thread skein Intricately coiled intermediate filament bundle that develops within gland thread cells and deploys after ejection from the slime glands to form the fibrous component of the hagfish slime.
ways. For one, epidermal mucus is secreted at a slow and relatively constant rate to replace the dispersal of mucus from the outer surface (see also The Skin: Functional Morphology of the Integumentary System in Fishes), whereas hagfish slime is released infrequently from specia lized glands. Another difference is that hagfish slime exudate is released via holocrine secretion in which entire cells are ejected from the glands, whereas epidermal mucus is secreted via exocytosis of mucin granules at the apical plasma membrane of goblet cells.
The Hagfish Slime Gland The slime originates within numerous slime glands located along both sides of the hagfish’s body (Figure 3). Eptatretus stoutii, the Pacific hagfish, typically possesses about 150–200 slime glands. The glands are dominated by two cell types: gland mucus cells (GMCs) and gland thread cells (GTCs), which are responsible for the mucus
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Figure 1 Hagfishes are able to produce vast amounts of slime when they are provoked. Here, a Pacific hagfish (Eptatretus stoutii) has produced liters of slime in response to being handled in an aquarium. Photo courtesy of Andra Zommers.
Figure 3 Hagfish slime is produced in numerous slime glands (arrows) that line both sides of the animal’s body. Here, slime exudate (arrowheads) has been expressed from several slime glands via mild electrical stimulation of the muscle layer that surrounds the gland capsule. Photo courtesy of Timothy Winegard.
GMCs and Mucin Vesicles
Figure 2 Hagfish slime contains tens of thousands of fine protein fibers that are over 10 cm long and about 2 mm in diameter. From Fudge DS, Winegard T, Ewoldt RH, et al. (2009) From ultra-soft slime to hard alpha-keratins: The many lives of intermediate filaments. Integrative and Comparative Biology 49: 32–39.
and fibrous components of the slime, respectively. GMCs and GTCs originate in the germinal layer of the gland and are pushed toward the gland lumen as they mature. The glands are approximately spherical in shape and are encased by a capsule of connective tissue, which is, in turn, surrounded by a thin layer of striated muscle fibers. When these muscle fibers contract, mature GMCs and GTCs are ejected from the gland via the holocrine mode into seawater through the gland pore.
GMCs are distinct from the small and large mucus cells found in hagfish epidermis. The sole function of GMCs is to package condensed mucins into disk-shaped membrane-bound vesicles that are about 7 mm in diameter (Figure 4). When mature GMCs are ejected from the slime gland, they lose their plasma membrane (presumably via shearing within the gland duct) and release numerous vesicles that swell and rupture upon contact with seawater. Compared with GTCs, GMCs and their secretion products are poorly studied. The genes for the molecules that make up the mucus component of hagfish slime have yet to be characterized, but histological staining and immunolabeling suggest that they are mucins. Mucins are very large (0.5–30 MDa), heavily glycosylated proteins in which up to 85% of the dry weight is carbohydrate. Hagfish mucins are unique in that they comprise only 12% carbohydrate and contain less serine and more sulfate than typical mucins. Hagfish slime also has been shown to contain higher levels of lysozyme, alkaline phosphatase, cathepsin B, and proteases relative to hagfish epidermal mucus. Hagfish slime mucin vesicles are believed to have an internal osmolarity of about 897 mOsm based on the fact that they rupture in magnesium sulfate solutions with osmolarities lower than this, and are stable in magnesium sulfate solutions with higher osmolarities. The vesicles are stable in 1 M solutions containing the anions citrate, sulfate and phosphate, acetate, or tartrate, but rupture in similar solutions containing chloride, bicarbonate, or nitrate ions. These experiments suggest that the vesicle membrane is permeable to univalent anions but
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Figure 4 A small blob of exudate collected from a slime gland and pipetted into a drop of seawater. Hagfish slime gland exudate contains two main solid components – thread skeins (arrows) that consist of a single coiled intermediate filament bundle, and mucin vesicles (arrowheads), which appear as the granular material between the thread skeins. The material shown represents about 5 nl of exudate. From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
impermeable to polyvalent anions. Vesicle rupture likely occurs not via a simple osmotic influx of water (as the vesicle is about the same osmolarity as seawater if not slightly hypoosmotic), but rather via the influx of ions such as chloride across the vesicle membrane, which then causes a subsequent secondary osmotic influx of water molecules. The water causes the vesicle to swell and then rupture, (Figure 5), resulting in the release of the mucin molecules into the external environment, where they are then free to interact with the fibrous component of the slime. This model predicts the presence of anion channels in the vesicle membrane; however, at this point, there is no direct evidence for them. The fact that the mucin vesicles rupture readily in such a wide range of solutions raises the question of how they might be stabilized within the gland mucous cells prior to release from the slime gland. Chemical analysis of the fluid component of slime exudate has revealed high concentra tions of methylamines such as betaine, trimethylamine oxide (TMAO), and dimethylglycine (DMG) (Table 1). Although these compounds are known osmolytes in other organisms, neither TMAO nor betaine (the two most abundant compounds) is capable of stabilizing the vesicles,
0.38 s
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2.81 s
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Figure 5 A composite of stills from a video of isolated mucin vesicles in the slime of Myxine glutinosa rupturing on exposure to seawater. Timestamps in the lower right corner indicate time following contact with seawater. Scale ¼ 20 mm. Arrowheads indicate a single mucin vesicle as it changes over time. From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
Table 1 The concentration of identified inorganic and organic osmolytes in the fluid component (supernatant) of fresh slime exudate Osmolyte
Concentration (mmol L�1)
Cl� Kþ Naþ Mg2þ Ca2þ pH Total inorganic
191.5 � 6.6 143.0 � 3.0 41.2 � 2.6 2.15 � 0.76 0.000 45 � 0.000 09 7.31 � 0.02 379
Betaine TMAO Glycine Dimethylglycine Creatine Inositol b-alanine Taurine Glucose Total organic
218 � 7 101.3 � 4.8 79.9 � 7.5 68.6 � 6.0 15.0 � 1.4 2.30 � 0.68 2.17 � 0.68 2.13 � 0.42 1.23 � 0.22 490 � 10
The total osmolarity of the supernatant is approximately 888 mOsm.
Values are means � SEM, N ¼ 5.
Inorganic ions were measured using ion-specific electrodes.
Organic solutes were analyzed using HPLC, and TMAO was
analyzed using ferrous sulfate and EDTA.
TMAO, trimetheylamine oxide.
The Skin | Hagfish Slime 507 Betaine
Betaine and SW Percent of mucin vesicles ruptured after 60 s
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Betaine concentration (mM) Figure 6 The percentage of isolated mucin vesicles from the slime of Myxine glutinosa that ruptured after 60 s of exposure to varying concentrations of betaine in 5.0 mM Tris, pH 8.0 (solid line). The dotted line represents the effect of changing betaine concentration in the presence of sea salts (33‰). Values are means � SEM (N ¼ 6 in each treatment). Two-way ANOVA showed significant effects of betaine concentration and the presence or absence of sea salts (P < 0.05). From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
even at concentrations exceeding the internal osmolarity of the vesicle itself (Figure 6). Even more intriguing is the fact that the clear supernatant that is obtained when fresh slime exudate is centrifuged also is not stabilizing
100
a
(Figure 7). This suggests that stabilization of vesicles within the gland may depend upon the chemical microenvironment within GMCs or another mechanism such as elevated hydrostatic pressure.
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Figure 7 The percentage of mucin vesicles that ruptured after 60 s of exposure to different solutions. Values are means � SEM (N ¼ 6 in each treatment). Different lowercase letters denote significant differences between means (P < 0.05). From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
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Gland Thread Cells The fibrous component of hagfish slime originates in the GTCs. The function of these cells is to package 10-nm-diameter cytoskeletal elements known as ‘inter mediate filaments’ into a single, continuous, intricately coiled protein thread that, in mature cells, occupies the vast majority of the cell volume. GTCs are large and roughly ellipsoidal in shape, with distinctive blunt and pointed ends (Figure 8). Estimates in the literature of slime thread length vary between 6 and 60 cm, and this value certainly depends on the size of the GTC and presumably the species. More direct measurements of unraveled thread skeins suggest that slime threads are between 11 and 17 cm long, and can be stretched to 34 cm before they break. How the cell assembles a thread that is about 100 000 times longer than its width and 1000 times longer than the cell itself is not well understood. Some clues about the mechanisms of thread assembly and packaging have come from a detailed study of GTC maturation within the slime gland. This work suggests that a short primal thread arises near the blunt end of the cell near the nucleus. The primal thread is believed to be capped at one end and increases in length via the addition of intermediate filaments at the other. Thread diameter increases by the addition of intermediate filaments laterally. This model is consistent with the changes in thread mor phology that occur as immature GTCs develop into mature thread cells, but does not explain how the cell
regulates thread diameter and quality and achieves such exquisite packing. The mechanism of assembly aside, simply describ ing the anatomy of the thread within mature GTCs, is not trivial and has attracted the attention of several investigators. The majority of the thread is organized as if it were laid down in staggered loops within an egg-shaped barrel. Within these loops, the thread takes on three main trajectories – an ascending trajectory toward the pointed end of the cell, a curved trajectory in which the thread winds circumferentially around the cell periphery for about 60� , and a descending trajectory toward the blunt end. Because of the 60� peripheral runs, the loops are staggered around the longitudinal axis of the cell, and about six of these three-dimensional loops define a cone-like structure that points toward the pointed end of the cell. Although it is meaningless to delineate the beginning of one cone and the end of another, for the purpose of this description, successive cones are neatly nested on top of each other, and occupy the majority of the cell volume. The packing of the loop segments in the peripheral runs gives a cabled appearance to the sur face of intact slime thread skeins, but this cabling effect is an illusion, which is sensible because twisting of the thread around itself would hinder effective unraveling. At the blunt end of the cell, the hollow space inside the cones is filled by a series of loops that are oriented parallel to the longitudinal axis of the cell. This staggered loop packing of the thread within GTCs has been compared with the faking of a rope on the deck of a ship to prevent its fouling as it is paid out. Thread diameter within the GTC is not constant, with the largest diameter occurring in the middle of the thread (�3 mm) and the ends tapering down to less than 1 mm.
Volume and Concentration
Figure 8 Scanning electron micrograph of gland thread cells that have lost their plasma membranes during ejection from the slime gland. These ball-of-yarn-like structures are referred to as thread skeins. Scale ¼ 50 mm. Image courtesy of Douglas Fudge.
Estimates of the sliming capacity of a single hagfish vary widely in the literature, but most agree that, in a typical sliming event, a hagfish can produce a mass of slime that is many times larger than itself. For a Pacific hagfish, a typical sliming event induced by a pinch on the tail produces about 900 mL of slime. As for the total amount of slime that a hagfish can produce, estimates in the literature vary from 7 to 100 L. The most recent estimate puts this number at about 25 L for the Pacific hagfish. This number is based in part on the mass of stored slime exudate in the glands, which is about 3–4% of body weight. One reason that hagfishes are able to produce so much of the slime is that it is remarkably dilute, containing only
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Recent work has begun to characterize the mechanical properties of hagfish slime and its components. One aspect of the behavior of the slime that is fairly well documented is its tendency to contract, especially when it is agitated. Slime volume decreases by a factor of 50 when it is stirred, and this presumably occurs due to a collapsing of the mucin and thread components of the slime and egress of the bulk seawater that makes up so much of the slime volume just after it is formed. Isolated slime threads are soft and extensible in water, but ulti mately quite strong (Figure 9). Their high extensibility means that they can stretch to a length greater than 3 times their original length before they break. At low strains, the threads are elastic, but when they are stretched to strains greater than 0.35, they become stiffer and recover less (Figure 10). This change in properties has been demonstrated to occur due to a conformational change in the constituent proteins in which �-helices are stretched out to form �-sheets (Figure 11). Although there is little information on the mechanical properties of the mucus component of the slime, hagfish slime mucin solutions have been shown to exhibit low
Engineering stress (MPa)
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about 20 mg of threads and 15 mg of mucins per liter, which is about 1000� more dilute than typical mucus such as gastric mucus. These measurements suggest that the vast majority of the volume of hagfish slime is seawater.
0.1 Strain (ΔL/Lo)
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Figure 9 Stress–strain curve for a slime thread tested in water compared to a wool fiber in water. Note the low stiffness of the slime thread at low strains, the extreme strain stiffening that happens at higher strains, and the high breaking stress. From Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
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Figure 10 Recovery behavior of hagfish threads in seawater. (a) Typical load cycle at low strains, showing completely reversible deformation. (b) Typical load cycle at higher strains, showing that deformation past the yield point is mostly plastic. (c) Results from trials in which threads were extended to a given strain, held, and allowed to recover. Note that deformation is elastic up to a strain of 0.35 and plastic thereafter. From Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
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Slime Deployment and Maturation One of the more remarkable aspects of hagfish slime is the speed at which it expands from a thick glandular exudate into an ultra-dilute material with a volume over a 1000 times greater. High-speed video has revealed two behavioral phenomena that aid in the rapid deployment of the slime. The first is the fact that the exudate is forcefully ejected from the slime glands (Figure 12). The second is the convective mixing that occurs as the hagfish struggles to escape whatever has provoked it (Figure 13). Exudate transferred into still seawater in a beaker does not form competent slime, but simply sinks as a coherent blob, whereas modest amounts of mixing allow the slime to fully deploy (Figure 14). Perhaps the most impressive aspect about deployment of the slime is that it involves the unraveling of slime threads from a condensed form within GTCs to an unra veled form that is 1000� longer (150 mm GTC!150 mm slime thread). Early hagfish slime researchers hypothe sized that the thread skein was assembled under significant pressure within the GTC, with deployment triggered by osmotic rupture of the GTC membrane upon contact with seawater. Later it was shown that the thread skeins lose their plasma membrane during ejection from the slime gland, which precludes any mechanism involving osmotic swelling across a membrane. More recent work has shown that disruption of the mucin
Figure 11 X-ray diffraction patterns for bundles of hagfish threads as a function of prior strain. (a) Unstrained threads exhibit a typical �-pattern, whereas threads extended to a strain of 1.0 exhibit a typical �-pattern (c). Threads extended to a strain of 0.60 exhibit a mixed pattern, suggesting the presence of both �-helix and �-sheet structure (b). Diffraction maxima (dark spots) are labeled according to the molecular spacings (in Angstroms, A˚) to which they correspond. From Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
viscosity similar to seawater, even at mucin concentra tions many times higher than natural concentrations. Recent work on the mechanical response of fresh slime suggests that it is an ultra-soft material with material properties that are unlike any other polymer.
t = 0 ms
t = 16 ms
t = 32 ms
t = 48 ms
Figure 12 Close-up of slime release from a single slime gland of a hagfish constrained in a tube with a window cut in it. Note that the slime is released as a coherent jet. These events were filmed at 125 frames s�1, and the mean jet velocity was 0.17 m s�1. Scale ¼ 5 mm. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209:702–710.
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t = 0 ms
t = 12 ms
t = 24 ms
t = 36 ms
t = 48 ms
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Figure 13 High-speed video (125 frames s�1) of a sliming event demonstrating that released slime rarely envelops the hagfish and often is dispersed by an evasive maneuver that mixes the exudate with seawater. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710.
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t = 0.12 s
t = 0.23 s
t = 0.35 s
t = 0.47 s
t = 0.58 s
t = 0.70 s
t = 0.82 s
Figure 14 Slime exudate introduced into still seawater from a syringe fails to hydrate as it does in vivo. From Lim J, Fudge DS,
Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology
209: 702–710.
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network with dithiothreitol (DTT) has an inhibitory effect on whole slime formation, and these effects are mediated at least in part by an inhibition of thread skein unraveling (Figure 15). Further exploration of this phenomenon revealed that mucin vesicles shear into long mucus strands (Figure 16) that attach to the thread skeins and initiate unraveling via the transmission of hydrodynamic mixing forces to them (Figure 17; Video Clip 1). The subsequent
entanglement between threads and mucin strands further unravels the thread skeins until the slime has fully deployed. These findings provide evidence for a unique biological process in which the thread skeins, which are immune to turbulent mixing forces because of their small size, are critically dependent on the mucin vesicles for their deployment. This suggests that mucins preceded the thread skeins in the evolution of the slime in hagfishes.
Pelleted thread skeins (% of number at 0 rpm)
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DTT
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300
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rpm Figure 15 Mean (�SEM) number of condensed thread skeins as a function of mixing rate expressed as the percentage of the number of skeins at 0 rpm. These data provide an inverse measure of the degree of thread skein unraveling in the whole slime. Solid line indicates data for trials in which mucins were disrupted with 10 mM dithiothreitol (DTT). The data demonstrate that the effects of DTT on whole slime formation are mediated at least in part by an inhibition of thread skein unraveling. These data also suggest that mucins play a critical role in the unraveling of the threads. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
50 μm Figure 16 Fluorescent tagging of mucins using a fluorescein-labeled lectin dye illustrates the formation of mucin strands from elongated mucin vesicles exposed to shear in a flow-through chamber. Arrow indicates an aggregation of ruptured mucin vesicles that have begun elongating to form strands. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
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(a)
(b)
(c)
(d)
(e)
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Figure 17 Slime exudate exposed to flow created by a syringe pump. (a) Condensed exudate puddle prior to flow. Note the condensed thread skeins on the far right and the mucin vesicle boundary layer to the left. (b, c) Mucin strands and chains begin to form as flow is initiated. (d, e) Mucin strand attachment to thread skeins and movement of thread skeins. (f, g) Unraveling of thread skeins initiated. Arrowheads indicate an unraveling thread skein; arrows indicate aggregations of mucin strands. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
Structure and Function Early models of slime structure hypothesized that swollen mucin vesicles decorated deployed slime threads. However, visualization of the mucin network with fluorescently labeled lectins disproved this hypothesis and suggested that mucins form a separate network of fibers that interacts with the thousands of deployed slime threads in mature slime (Figure 18). The current model of slime structure suggests that the vast majority of the volume of mature slime must be bulk seawater. This idea is supported by the fact that hagfish slime that is lifted out of seawater into air will decrease in volume as water runs out of it, leaving only about 5% of its original volume in a matter of minutes. These observations suggest that the slime is adapted not to bind water irreversibly, but rather to entrain a large volume of seawater
within countless microchannels defined by the inter mingling networks of slime threads and mucin strands. Many functions have been proposed for the slime, including defense against predators, feeding, localization of eggs, and avoidance of competition while scavenging, but few of these hypotheses have been tested experi mentally. Videos of hagfishes scavenging on baited traps show that they sometimes release slime while feeding, which may serve to discourage competitors from approaching too closely. One group of researchers tested the hypothesis that hagfish slime functions as a defense against gill-breathing predators by measuring the hydro dynamic resistance across the gills in isolated fish heads with and without slime. They found that the slime is superbly suited to catching on the gills and increasing the resistance to flow through them (Figures 19 and 20). Regardless of the exact function of hagfish slime, it is a
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Figure 18 Whole slime formed in seawater containing a fluorescent lectin dye that binds to hagfish slime mucins. (a) Differential interference contrast (DIC) image of whole slime network depicting unraveled threads and mucin strand network. Arrowhead indicates mucin strands connecting slime threads, and the arrow indicates a slime thread. (b) Same image viewed in fluorescence highlights the complexity of the mucin network. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
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Figure 19 The effects of hagfish slime on (a) water flow rates and (b) gill resistance in the gills of an isolated rockfish head. Slime release occurred at �95 s; results from two fish heads are shown separately, and the data have been normalized to their pre-slime values. Note the log scale for normalized resistance. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710.
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Figure 20 Hagfish slime is very effective at catching on the gills of fishes like this rockfish. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710.
complex and fascinating material that has likely played an important role in the evolutionary staying power of the hagfishes. See also: Hagfishes and Lamprey: Hagfishes. The Skin: Functional Morphology of the Integumentary System in Fishes.
Further Reading Downing SW, Spitzer RH, Koch EA, and Salo WL (1984) The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament–microtubule interactions. Journal of Cell Biology 98: 653–669. Downing SW, Spitzer RH, Salo WL, et al. (1981) Hagfish slime gland thread cells: Organization, biochemical features, and length. Science 212: 326–328. Fernholm B (1981) Thread cells from the slime glands of hagfish (Myxinidae). Acta Zoologica 62: 137–145. Ferry JD (1941) A fibrous protein from the slime of the hagfish. Journal of Biological Chemistry 138: 263–268. Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
Fudge DS, Levy N, Chiu S, and Gosline JM (2005) Composition, morphology and mechanics of hagfish slime. Journal of Experimental Biology 208: 4613–4625. Fudge DS, Winegard T, Ewoldt RH, et al. (2009) From ultra-soft slime to hard alpha-keratins: The many lives of intermediate filaments. Integrative and Comparative Biology 49: 32–39. Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099. Koch EA, Spitzer RH, and Pithawalla RB (1991) Structural forms and possible roles of aligned cytoskeletal biopolymers in hagfish (slime eel) mucus. Journal of Structural Biology 106: 205–210. Koch EA, Spitzer RH, Pithawalla RB, Castillos FA, 3rd, and Parry DA (1995) Hagfish biopolymer: A type I/type II homologue of epidermal keratin intermediate filaments. International Journal of Biological Macromolecules 17: 283–292. Koch EA, Spitzer RH, Pithawalla RB, and Downing SW (1991) Keratinlike components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti). Cell and Tissue Research 264: 79–86. Koch EA, Spitzer RH, Pithawalla RB, and Parry DA (1994) An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stoutii). Journal of Cell Science 107: 3133–3144. Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710. Luchtel DL, Martin AW, and Deyrup-Olson I (1991) Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish. Tissue and Cell 23: 939–948. Newby WW (1946) The slime glands and thread cells of the hagfish Polistotrema stouti. Journal of Morphology 78: 397–409. Salo WL, Downing SW, Lidinsky WA, et al. (1983) Fractionation of hagfish slime gland secretions: Partial characterization of the mucous vesicle fraction. Preparative Biochemistry 13: 103–135. Spitzer RH, Downing SW, and Koch EA (1979) Metabolic–morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii). Cell and Tissue Research 197: 235–255. Subramanian S, Ross NW, and MacKinnon SL (2008) Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.). Fish and Shellfish Immunology 25: 625–632. Verdugo P, Deyrupolsen I, Aitken M, Villalon M, and Johnson D (1987) Molecular mechanism of mucin secretion. 1. The role of intragranular charge shielding. Journal of Dental Research 66: 506–508. Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240. For supplementary material please also visit the companion site: http://www.elsevierdirect.com/companions/9780123745453
Introduction The Hagfish Slime Gland GMCs and Mucin Vesicles Gland Thread Cells Volume and Concentration
Glossary Exocytosis The process by which neurotransmitters or hormones, which are stored in secretory vesicles or granules, are released from a nerve or endocrine cell when these storage granules fuse with the plasma membrane Gland mucus cell Cell within hagfish slime glands that give rise to the mucus component of hagfish slime. Gland thread cell Cell within hagfish slime glands that give rise to the fibrous component of hagfish slime. Hagfish slime The material that is formed when hagfishes forcefully release the glandular contents of their numerous slime glands into seawater. Hagfish slime exudate The thick white fluid that is ejected from hagfish slime glands and that interacts with
Introduction Hagfishes are best known for their ability to produce alarming amounts of slime when they are stressed or provoked (see also Hagfishes and Lamprey: Hagfishes). In fact, many of their common names include the word slime, such as slime eel and slime hag. The Atlantic hagfish, Myxine glutinosa, is twice named for its slime (myx ¼ slime, glutin ¼ glue). Many animals release slime under stress, but hagfish slime is unique because of the large volumes that are produced (up to several liters from a single hagfish) (Figure 1), the impressive speed with which it is made, and the fact that it contains tens of thousands of fine protein fibers (Figure 2). Hagfish slime can be defined as the material that is formed when hagfishes forcefully release the glandular contents of their numerous slime glands into seawater. Many marine animals, including hagfishes, make some sort of epidermal mucus or slime that coats their body, but hagfish slime is different from these secretions in many
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Mechanics and Function Slime Deployment and Maturation Structure and Function Further Reading
seawater to form hagfish slime. The exudate contains numerous mucin vesicles and thread skeins. Holocrine secretion Mode of secretion in which entire cells are ejected from a gland. Intermediate filaments The 10-nm-diameter cytoskeletal filaments that make up hagfish slime threads. Mucin vesicles Small, membrane-bound structures containing condensed mucus that are released when gland mucus cells are ejected from hagfish slime glands. Slime threads Fine protein fibers that permeate hagfish slime and arise from the deployment of ejected thread skeins. Thread skein Intricately coiled intermediate filament bundle that develops within gland thread cells and deploys after ejection from the slime glands to form the fibrous component of the hagfish slime.
ways. For one, epidermal mucus is secreted at a slow and relatively constant rate to replace the dispersal of mucus from the outer surface (see also The Skin: Functional Morphology of the Integumentary System in Fishes), whereas hagfish slime is released infrequently from specia lized glands. Another difference is that hagfish slime exudate is released via holocrine secretion in which entire cells are ejected from the glands, whereas epidermal mucus is secreted via exocytosis of mucin granules at the apical plasma membrane of goblet cells.
The Hagfish Slime Gland The slime originates within numerous slime glands located along both sides of the hagfish’s body (Figure 3). Eptatretus stoutii, the Pacific hagfish, typically possesses about 150–200 slime glands. The glands are dominated by two cell types: gland mucus cells (GMCs) and gland thread cells (GTCs), which are responsible for the mucus
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Figure 1 Hagfishes are able to produce vast amounts of slime when they are provoked. Here, a Pacific hagfish (Eptatretus stoutii) has produced liters of slime in response to being handled in an aquarium. Photo courtesy of Andra Zommers.
Figure 3 Hagfish slime is produced in numerous slime glands (arrows) that line both sides of the animal’s body. Here, slime exudate (arrowheads) has been expressed from several slime glands via mild electrical stimulation of the muscle layer that surrounds the gland capsule. Photo courtesy of Timothy Winegard.
GMCs and Mucin Vesicles
Figure 2 Hagfish slime contains tens of thousands of fine protein fibers that are over 10 cm long and about 2 mm in diameter. From Fudge DS, Winegard T, Ewoldt RH, et al. (2009) From ultra-soft slime to hard alpha-keratins: The many lives of intermediate filaments. Integrative and Comparative Biology 49: 32–39.
and fibrous components of the slime, respectively. GMCs and GTCs originate in the germinal layer of the gland and are pushed toward the gland lumen as they mature. The glands are approximately spherical in shape and are encased by a capsule of connective tissue, which is, in turn, surrounded by a thin layer of striated muscle fibers. When these muscle fibers contract, mature GMCs and GTCs are ejected from the gland via the holocrine mode into seawater through the gland pore.
GMCs are distinct from the small and large mucus cells found in hagfish epidermis. The sole function of GMCs is to package condensed mucins into disk-shaped membrane-bound vesicles that are about 7 mm in diameter (Figure 4). When mature GMCs are ejected from the slime gland, they lose their plasma membrane (presumably via shearing within the gland duct) and release numerous vesicles that swell and rupture upon contact with seawater. Compared with GTCs, GMCs and their secretion products are poorly studied. The genes for the molecules that make up the mucus component of hagfish slime have yet to be characterized, but histological staining and immunolabeling suggest that they are mucins. Mucins are very large (0.5–30 MDa), heavily glycosylated proteins in which up to 85% of the dry weight is carbohydrate. Hagfish mucins are unique in that they comprise only 12% carbohydrate and contain less serine and more sulfate than typical mucins. Hagfish slime also has been shown to contain higher levels of lysozyme, alkaline phosphatase, cathepsin B, and proteases relative to hagfish epidermal mucus. Hagfish slime mucin vesicles are believed to have an internal osmolarity of about 897 mOsm based on the fact that they rupture in magnesium sulfate solutions with osmolarities lower than this, and are stable in magnesium sulfate solutions with higher osmolarities. The vesicles are stable in 1 M solutions containing the anions citrate, sulfate and phosphate, acetate, or tartrate, but rupture in similar solutions containing chloride, bicarbonate, or nitrate ions. These experiments suggest that the vesicle membrane is permeable to univalent anions but
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Figure 4 A small blob of exudate collected from a slime gland and pipetted into a drop of seawater. Hagfish slime gland exudate contains two main solid components – thread skeins (arrows) that consist of a single coiled intermediate filament bundle, and mucin vesicles (arrowheads), which appear as the granular material between the thread skeins. The material shown represents about 5 nl of exudate. From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
impermeable to polyvalent anions. Vesicle rupture likely occurs not via a simple osmotic influx of water (as the vesicle is about the same osmolarity as seawater if not slightly hypoosmotic), but rather via the influx of ions such as chloride across the vesicle membrane, which then causes a subsequent secondary osmotic influx of water molecules. The water causes the vesicle to swell and then rupture, (Figure 5), resulting in the release of the mucin molecules into the external environment, where they are then free to interact with the fibrous component of the slime. This model predicts the presence of anion channels in the vesicle membrane; however, at this point, there is no direct evidence for them. The fact that the mucin vesicles rupture readily in such a wide range of solutions raises the question of how they might be stabilized within the gland mucous cells prior to release from the slime gland. Chemical analysis of the fluid component of slime exudate has revealed high concentra tions of methylamines such as betaine, trimethylamine oxide (TMAO), and dimethylglycine (DMG) (Table 1). Although these compounds are known osmolytes in other organisms, neither TMAO nor betaine (the two most abundant compounds) is capable of stabilizing the vesicles,
0.38 s
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Figure 5 A composite of stills from a video of isolated mucin vesicles in the slime of Myxine glutinosa rupturing on exposure to seawater. Timestamps in the lower right corner indicate time following contact with seawater. Scale ¼ 20 mm. Arrowheads indicate a single mucin vesicle as it changes over time. From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
Table 1 The concentration of identified inorganic and organic osmolytes in the fluid component (supernatant) of fresh slime exudate Osmolyte
Concentration (mmol L�1)
Cl� Kþ Naþ Mg2þ Ca2þ pH Total inorganic
191.5 � 6.6 143.0 � 3.0 41.2 � 2.6 2.15 � 0.76 0.000 45 � 0.000 09 7.31 � 0.02 379
Betaine TMAO Glycine Dimethylglycine Creatine Inositol b-alanine Taurine Glucose Total organic
218 � 7 101.3 � 4.8 79.9 � 7.5 68.6 � 6.0 15.0 � 1.4 2.30 � 0.68 2.17 � 0.68 2.13 � 0.42 1.23 � 0.22 490 � 10
The total osmolarity of the supernatant is approximately 888 mOsm.
Values are means � SEM, N ¼ 5.
Inorganic ions were measured using ion-specific electrodes.
Organic solutes were analyzed using HPLC, and TMAO was
analyzed using ferrous sulfate and EDTA.
TMAO, trimetheylamine oxide.
The Skin | Hagfish Slime 507 Betaine
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Betaine concentration (mM) Figure 6 The percentage of isolated mucin vesicles from the slime of Myxine glutinosa that ruptured after 60 s of exposure to varying concentrations of betaine in 5.0 mM Tris, pH 8.0 (solid line). The dotted line represents the effect of changing betaine concentration in the presence of sea salts (33‰). Values are means � SEM (N ¼ 6 in each treatment). Two-way ANOVA showed significant effects of betaine concentration and the presence or absence of sea salts (P < 0.05). From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
even at concentrations exceeding the internal osmolarity of the vesicle itself (Figure 6). Even more intriguing is the fact that the clear supernatant that is obtained when fresh slime exudate is centrifuged also is not stabilizing
100
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(Figure 7). This suggests that stabilization of vesicles within the gland may depend upon the chemical microenvironment within GMCs or another mechanism such as elevated hydrostatic pressure.
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Figure 7 The percentage of mucin vesicles that ruptured after 60 s of exposure to different solutions. Values are means � SEM (N ¼ 6 in each treatment). Different lowercase letters denote significant differences between means (P < 0.05). From Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099.
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Gland Thread Cells The fibrous component of hagfish slime originates in the GTCs. The function of these cells is to package 10-nm-diameter cytoskeletal elements known as ‘inter mediate filaments’ into a single, continuous, intricately coiled protein thread that, in mature cells, occupies the vast majority of the cell volume. GTCs are large and roughly ellipsoidal in shape, with distinctive blunt and pointed ends (Figure 8). Estimates in the literature of slime thread length vary between 6 and 60 cm, and this value certainly depends on the size of the GTC and presumably the species. More direct measurements of unraveled thread skeins suggest that slime threads are between 11 and 17 cm long, and can be stretched to 34 cm before they break. How the cell assembles a thread that is about 100 000 times longer than its width and 1000 times longer than the cell itself is not well understood. Some clues about the mechanisms of thread assembly and packaging have come from a detailed study of GTC maturation within the slime gland. This work suggests that a short primal thread arises near the blunt end of the cell near the nucleus. The primal thread is believed to be capped at one end and increases in length via the addition of intermediate filaments at the other. Thread diameter increases by the addition of intermediate filaments laterally. This model is consistent with the changes in thread mor phology that occur as immature GTCs develop into mature thread cells, but does not explain how the cell
regulates thread diameter and quality and achieves such exquisite packing. The mechanism of assembly aside, simply describ ing the anatomy of the thread within mature GTCs, is not trivial and has attracted the attention of several investigators. The majority of the thread is organized as if it were laid down in staggered loops within an egg-shaped barrel. Within these loops, the thread takes on three main trajectories – an ascending trajectory toward the pointed end of the cell, a curved trajectory in which the thread winds circumferentially around the cell periphery for about 60� , and a descending trajectory toward the blunt end. Because of the 60� peripheral runs, the loops are staggered around the longitudinal axis of the cell, and about six of these three-dimensional loops define a cone-like structure that points toward the pointed end of the cell. Although it is meaningless to delineate the beginning of one cone and the end of another, for the purpose of this description, successive cones are neatly nested on top of each other, and occupy the majority of the cell volume. The packing of the loop segments in the peripheral runs gives a cabled appearance to the sur face of intact slime thread skeins, but this cabling effect is an illusion, which is sensible because twisting of the thread around itself would hinder effective unraveling. At the blunt end of the cell, the hollow space inside the cones is filled by a series of loops that are oriented parallel to the longitudinal axis of the cell. This staggered loop packing of the thread within GTCs has been compared with the faking of a rope on the deck of a ship to prevent its fouling as it is paid out. Thread diameter within the GTC is not constant, with the largest diameter occurring in the middle of the thread (�3 mm) and the ends tapering down to less than 1 mm.
Volume and Concentration
Figure 8 Scanning electron micrograph of gland thread cells that have lost their plasma membranes during ejection from the slime gland. These ball-of-yarn-like structures are referred to as thread skeins. Scale ¼ 50 mm. Image courtesy of Douglas Fudge.
Estimates of the sliming capacity of a single hagfish vary widely in the literature, but most agree that, in a typical sliming event, a hagfish can produce a mass of slime that is many times larger than itself. For a Pacific hagfish, a typical sliming event induced by a pinch on the tail produces about 900 mL of slime. As for the total amount of slime that a hagfish can produce, estimates in the literature vary from 7 to 100 L. The most recent estimate puts this number at about 25 L for the Pacific hagfish. This number is based in part on the mass of stored slime exudate in the glands, which is about 3–4% of body weight. One reason that hagfishes are able to produce so much of the slime is that it is remarkably dilute, containing only
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Recent work has begun to characterize the mechanical properties of hagfish slime and its components. One aspect of the behavior of the slime that is fairly well documented is its tendency to contract, especially when it is agitated. Slime volume decreases by a factor of 50 when it is stirred, and this presumably occurs due to a collapsing of the mucin and thread components of the slime and egress of the bulk seawater that makes up so much of the slime volume just after it is formed. Isolated slime threads are soft and extensible in water, but ulti mately quite strong (Figure 9). Their high extensibility means that they can stretch to a length greater than 3 times their original length before they break. At low strains, the threads are elastic, but when they are stretched to strains greater than 0.35, they become stiffer and recover less (Figure 10). This change in properties has been demonstrated to occur due to a conformational change in the constituent proteins in which �-helices are stretched out to form �-sheets (Figure 11). Although there is little information on the mechanical properties of the mucus component of the slime, hagfish slime mucin solutions have been shown to exhibit low
Engineering stress (MPa)
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about 20 mg of threads and 15 mg of mucins per liter, which is about 1000� more dilute than typical mucus such as gastric mucus. These measurements suggest that the vast majority of the volume of hagfish slime is seawater.
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Figure 9 Stress–strain curve for a slime thread tested in water compared to a wool fiber in water. Note the low stiffness of the slime thread at low strains, the extreme strain stiffening that happens at higher strains, and the high breaking stress. From Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
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Figure 10 Recovery behavior of hagfish threads in seawater. (a) Typical load cycle at low strains, showing completely reversible deformation. (b) Typical load cycle at higher strains, showing that deformation past the yield point is mostly plastic. (c) Results from trials in which threads were extended to a given strain, held, and allowed to recover. Note that deformation is elastic up to a strain of 0.35 and plastic thereafter. From Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
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Slime Deployment and Maturation One of the more remarkable aspects of hagfish slime is the speed at which it expands from a thick glandular exudate into an ultra-dilute material with a volume over a 1000 times greater. High-speed video has revealed two behavioral phenomena that aid in the rapid deployment of the slime. The first is the fact that the exudate is forcefully ejected from the slime glands (Figure 12). The second is the convective mixing that occurs as the hagfish struggles to escape whatever has provoked it (Figure 13). Exudate transferred into still seawater in a beaker does not form competent slime, but simply sinks as a coherent blob, whereas modest amounts of mixing allow the slime to fully deploy (Figure 14). Perhaps the most impressive aspect about deployment of the slime is that it involves the unraveling of slime threads from a condensed form within GTCs to an unra veled form that is 1000� longer (150 mm GTC!150 mm slime thread). Early hagfish slime researchers hypothe sized that the thread skein was assembled under significant pressure within the GTC, with deployment triggered by osmotic rupture of the GTC membrane upon contact with seawater. Later it was shown that the thread skeins lose their plasma membrane during ejection from the slime gland, which precludes any mechanism involving osmotic swelling across a membrane. More recent work has shown that disruption of the mucin
Figure 11 X-ray diffraction patterns for bundles of hagfish threads as a function of prior strain. (a) Unstrained threads exhibit a typical �-pattern, whereas threads extended to a strain of 1.0 exhibit a typical �-pattern (c). Threads extended to a strain of 0.60 exhibit a mixed pattern, suggesting the presence of both �-helix and �-sheet structure (b). Diffraction maxima (dark spots) are labeled according to the molecular spacings (in Angstroms, A˚) to which they correspond. From Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
viscosity similar to seawater, even at mucin concentra tions many times higher than natural concentrations. Recent work on the mechanical response of fresh slime suggests that it is an ultra-soft material with material properties that are unlike any other polymer.
t = 0 ms
t = 16 ms
t = 32 ms
t = 48 ms
Figure 12 Close-up of slime release from a single slime gland of a hagfish constrained in a tube with a window cut in it. Note that the slime is released as a coherent jet. These events were filmed at 125 frames s�1, and the mean jet velocity was 0.17 m s�1. Scale ¼ 5 mm. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209:702–710.
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t = 0 ms
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Figure 13 High-speed video (125 frames s�1) of a sliming event demonstrating that released slime rarely envelops the hagfish and often is dispersed by an evasive maneuver that mixes the exudate with seawater. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710.
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Figure 14 Slime exudate introduced into still seawater from a syringe fails to hydrate as it does in vivo. From Lim J, Fudge DS,
Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology
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network with dithiothreitol (DTT) has an inhibitory effect on whole slime formation, and these effects are mediated at least in part by an inhibition of thread skein unraveling (Figure 15). Further exploration of this phenomenon revealed that mucin vesicles shear into long mucus strands (Figure 16) that attach to the thread skeins and initiate unraveling via the transmission of hydrodynamic mixing forces to them (Figure 17; Video Clip 1). The subsequent
entanglement between threads and mucin strands further unravels the thread skeins until the slime has fully deployed. These findings provide evidence for a unique biological process in which the thread skeins, which are immune to turbulent mixing forces because of their small size, are critically dependent on the mucin vesicles for their deployment. This suggests that mucins preceded the thread skeins in the evolution of the slime in hagfishes.
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rpm Figure 15 Mean (�SEM) number of condensed thread skeins as a function of mixing rate expressed as the percentage of the number of skeins at 0 rpm. These data provide an inverse measure of the degree of thread skein unraveling in the whole slime. Solid line indicates data for trials in which mucins were disrupted with 10 mM dithiothreitol (DTT). The data demonstrate that the effects of DTT on whole slime formation are mediated at least in part by an inhibition of thread skein unraveling. These data also suggest that mucins play a critical role in the unraveling of the threads. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
50 μm Figure 16 Fluorescent tagging of mucins using a fluorescein-labeled lectin dye illustrates the formation of mucin strands from elongated mucin vesicles exposed to shear in a flow-through chamber. Arrow indicates an aggregation of ruptured mucin vesicles that have begun elongating to form strands. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
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Figure 17 Slime exudate exposed to flow created by a syringe pump. (a) Condensed exudate puddle prior to flow. Note the condensed thread skeins on the far right and the mucin vesicle boundary layer to the left. (b, c) Mucin strands and chains begin to form as flow is initiated. (d, e) Mucin strand attachment to thread skeins and movement of thread skeins. (f, g) Unraveling of thread skeins initiated. Arrowheads indicate an unraveling thread skein; arrows indicate aggregations of mucin strands. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
Structure and Function Early models of slime structure hypothesized that swollen mucin vesicles decorated deployed slime threads. However, visualization of the mucin network with fluorescently labeled lectins disproved this hypothesis and suggested that mucins form a separate network of fibers that interacts with the thousands of deployed slime threads in mature slime (Figure 18). The current model of slime structure suggests that the vast majority of the volume of mature slime must be bulk seawater. This idea is supported by the fact that hagfish slime that is lifted out of seawater into air will decrease in volume as water runs out of it, leaving only about 5% of its original volume in a matter of minutes. These observations suggest that the slime is adapted not to bind water irreversibly, but rather to entrain a large volume of seawater
within countless microchannels defined by the inter mingling networks of slime threads and mucin strands. Many functions have been proposed for the slime, including defense against predators, feeding, localization of eggs, and avoidance of competition while scavenging, but few of these hypotheses have been tested experi mentally. Videos of hagfishes scavenging on baited traps show that they sometimes release slime while feeding, which may serve to discourage competitors from approaching too closely. One group of researchers tested the hypothesis that hagfish slime functions as a defense against gill-breathing predators by measuring the hydro dynamic resistance across the gills in isolated fish heads with and without slime. They found that the slime is superbly suited to catching on the gills and increasing the resistance to flow through them (Figures 19 and 20). Regardless of the exact function of hagfish slime, it is a
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Figure 18 Whole slime formed in seawater containing a fluorescent lectin dye that binds to hagfish slime mucins. (a) Differential interference contrast (DIC) image of whole slime network depicting unraveled threads and mucin strand network. Arrowhead indicates mucin strands connecting slime threads, and the arrow indicates a slime thread. (b) Same image viewed in fluorescence highlights the complexity of the mucin network. From Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240.
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Time (s)
Figure 19 The effects of hagfish slime on (a) water flow rates and (b) gill resistance in the gills of an isolated rockfish head. Slime release occurred at �95 s; results from two fish heads are shown separately, and the data have been normalized to their pre-slime values. Note the log scale for normalized resistance. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710.
The Skin | Hagfish Slime 515
Figure 20 Hagfish slime is very effective at catching on the gills of fishes like this rockfish. From Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710.
complex and fascinating material that has likely played an important role in the evolutionary staying power of the hagfishes. See also: Hagfishes and Lamprey: Hagfishes. The Skin: Functional Morphology of the Integumentary System in Fishes.
Further Reading Downing SW, Spitzer RH, Koch EA, and Salo WL (1984) The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament–microtubule interactions. Journal of Cell Biology 98: 653–669. Downing SW, Spitzer RH, Salo WL, et al. (1981) Hagfish slime gland thread cells: Organization, biochemical features, and length. Science 212: 326–328. Fernholm B (1981) Thread cells from the slime glands of hagfish (Myxinidae). Acta Zoologica 62: 137–145. Ferry JD (1941) A fibrous protein from the slime of the hagfish. Journal of Biological Chemistry 138: 263–268. Fudge DS, Gardner KH, Forsyth VT, Riekel C, and Gosline JM (2003) The mechanical properties of hydrated intermediate filaments: Insights from hagfish slime threads. Biophysical Journal 85: 2015–2027.
Fudge DS, Levy N, Chiu S, and Gosline JM (2005) Composition, morphology and mechanics of hagfish slime. Journal of Experimental Biology 208: 4613–4625. Fudge DS, Winegard T, Ewoldt RH, et al. (2009) From ultra-soft slime to hard alpha-keratins: The many lives of intermediate filaments. Integrative and Comparative Biology 49: 32–39. Herr JE, Winegard TM, O’Donnell MJ, Yancey PH, and Fudge DS (2010) Stabilization and swelling of hagfish slime mucin vesicles. Journal of Experimental Biology 213: 1092–1099. Koch EA, Spitzer RH, and Pithawalla RB (1991) Structural forms and possible roles of aligned cytoskeletal biopolymers in hagfish (slime eel) mucus. Journal of Structural Biology 106: 205–210. Koch EA, Spitzer RH, Pithawalla RB, Castillos FA, 3rd, and Parry DA (1995) Hagfish biopolymer: A type I/type II homologue of epidermal keratin intermediate filaments. International Journal of Biological Macromolecules 17: 283–292. Koch EA, Spitzer RH, Pithawalla RB, and Downing SW (1991) Keratinlike components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti). Cell and Tissue Research 264: 79–86. Koch EA, Spitzer RH, Pithawalla RB, and Parry DA (1994) An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stoutii). Journal of Cell Science 107: 3133–3144. Lim J, Fudge DS, Levy N, and Gosline JM (2006) Hagfish slime ecomechanics: Testing the gill-clogging hypothesis. Journal of Experimental Biology 209: 702–710. Luchtel DL, Martin AW, and Deyrup-Olson I (1991) Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish. Tissue and Cell 23: 939–948. Newby WW (1946) The slime glands and thread cells of the hagfish Polistotrema stouti. Journal of Morphology 78: 397–409. Salo WL, Downing SW, Lidinsky WA, et al. (1983) Fractionation of hagfish slime gland secretions: Partial characterization of the mucous vesicle fraction. Preparative Biochemistry 13: 103–135. Spitzer RH, Downing SW, and Koch EA (1979) Metabolic–morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii). Cell and Tissue Research 197: 235–255. Subramanian S, Ross NW, and MacKinnon SL (2008) Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.). Fish and Shellfish Immunology 25: 625–632. Verdugo P, Deyrupolsen I, Aitken M, Villalon M, and Johnson D (1987) Molecular mechanism of mucin secretion. 1. The role of intragranular charge shielding. Journal of Dental Research 66: 506–508. Winegard TM and Fudge DS (2010) Deployment of hagfish slime thread skeins requires the transmission of mixing forces via mucin strands. Journal of Experimental Biology 213: 1235–1240. For supplementary material please also visit the companion site: http://www.elsevierdirect.com/companions/9780123745453