The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions.

Thread cell differentiation in the slime gland of the Pacific hagfish Eptatretus stouti has been studied using light microscopy and scanning and transmission electron microscopy. Thread cell differentiation is remarkable in that the life history of the cell is largely dedicated to the production of a single, tapered, cylindrical, highly coiled, and precisely packaged cytoplasmic thread that may attain lengths of 60 cm and diameters approaching 1.5 micron. Each tapered thread, in turn, is comprised almost entirely of large numbers of intermediate filaments (IFs) bundled in parallel. During differentiation of the thread, the IFs become progressively more tightly packed. Various numbers of microtubules (MTs) are found among the bundled IFs during differentiation of the thread but disappear during the latter stages of thread differentiation. Observations of regularly spaced dots in longitudinal bisections of developing threads, diagonal striations in tangential sections of developing threads, and circumferentially oriented, filament-like structures observed at the periphery of developing threads cut in cross section have led us to postulate a helically oriented component(s) wrapped around the periphery of the developing thread. The enormous size of the fully differentiated thread cell, its apparent singular dedication to the production of IFs, the ease of isolating and purifying the threads and IF subunits (see accompanying paper), and the unique position of the hagfish in the phylogenetic scheme of vertebrate evolution all contribute to the attractiveness of the hagfish slime gland thread cell as a potential model system for studying IF subunit synthesis, IF formation from IF subunits, aggregation of IFs into IF bundles and the interaction(s) of IFs and MTs.

Hagfish slime gland thread cells. II. Isolation and characterization of intermediate filament components associated with the thread.

The slime glands of hagfish have two major cell types, gland thread cells (GTCs) and gland mucous cells (GMCs), both of which upon contact with water contribute to the formation of an abundant quantity of viscous mucus. In previous studies we reported a method for the isolation of GTCs and showed that each ellipsoidal thread cell normally contains a single tapered thread which is uniquely coiled into a space-saving conformation and occupies most of the cell volume. Subsequently, the developing thread was found to consist mainly of intermediate filaments (IFs) aligned in parallel not only to one another but also to a far fewer number of interspersed microtubules (see accompanying paper). In the present report, urea extracts of GTCs were purified and characterized to establish the properties of thread components. One major (alpha) and two minor (beta, gamma) components prepared by anion exchange chromatography were shown to have similar apparent molecular weights of 63,500 +/- 500 daltons but different isoelectric pH values (alpha, 7.56; beta, 5.67; gamma, 5.31). Although the amino acid content of alpha differed significantly from beta and gamma, each of the three was highest in Gly, relatively high in Glx, Ser, Thr, Asx, Ala, Val, and Leu, and relatively low in Cys/2 and Trp. The amino acid compositions of beta and gamma were very similar, and only beta showed evidence of carbohydrate. The threonine content of the alpha component was higher than has been reported for IFs of different origin, and the high content of hydroxyamino acids (18, 19 residues per 100) in alpha, beta, and gamma has been approached only by several IF polypeptides from human or bovine epidermal keratins. Mixtures of the purified components formed 9-11-nm filaments in vitro. The results indicate that the hagfish thread cell is a rich source of IFs, which have a structure that facilitates formation of macrofibrils within the cell.

The hagfish slime gland: a model system for studying the biology of mucus.

The hagfish slime gland may provide a model system for studying certain aspects of the biology of mucus. Mucus is obtained in nonhydrated form by electrically stimulating the anesthetized hagfish and the secretions are stirred into ammonium sulfate. Centrifugation and filtration are than used to isolate the two major secretory products, mucous vesicles and threads. Specific advantages of the model and potential applications for research are discussed.

Threads in the hagfish slime gland thread cells: organization, biochemical features, and length.

Scanning electron microscopy in conjunction with cell isolation procedures revealed details of the packing of threads in hagfish slime gland thread cells. Biochemical studies indicate that the thread is largely composed of a protein subunit with a molecular weight of 63,500. Mathematical calculations suggest that the thread may attain lengths of 60 centimeters or more.

Cloning, expression, and regulation of a glucocorticoid-induced receptor in rat brain: effect of repetitive amphetamine.

Behavioral sensitization to psychostimulants involves neuroadaptation of stress-responsive systems. We have identified and sequenced a glucocorticoid-induced receptor (GIR) cDNA from rat prefrontal cortex. The full-length GIR cDNA encodes a 422 amino acid protein belonging to G-protein-coupled receptor superfamily. Although the ligand for GIR is still unknown, the dendrogram construction indicates that GIR may belong to peptide receptor subfamily (e.g., substance P receptor), with more distant relationship to subfamilies of glycoprotein hormone receptors (e.g., thyrotropin receptor) and biogenic amine receptors (e.g., dopamine receptor). GIR shares 31-34% amino acid identity to the tachykinin receptors (substance P receptor, neurokinin A receptor, and neurokinin B receptor). GIR mRNA is expressed preferentially in brain, and its neuronal expression is relegated to limbic brain regions, particularly in forebrain. GIR transcript levels are increased significantly and persistently in prefrontal cortex for 7 d after discontinuation of chronic amphetamine exposure. The induction of GIR expression by amphetamine is associated with augmented behavioral activation. These findings suggest that modulation of GIR expression may be involved in behavioral sensitization, and GIR may play a role at the interface between stress and neuroadaptation to psychostimulants.

The dopamine D3 receptor antagonist nafadotride inhibits development of locomotor sensitization to amphetamine.

Behavioral sensitization is a well-studied model of behavioral plasticity mediated at least in part by dopaminergic systems believed to play an important role in several psychiatric conditions. In the rodent, locomotion is regulated by the opposing balance of D3 and D2 receptors, with D2 activation increasing and D3 stimulation inhibiting locomotion. However, receptor occupancy of D3 dopamine receptors is far greater than D2 or D1 occupancy at typical post-stimulant dopamine concentrations. We therefore hypothesized that tolerance of D3 receptor inhibition of locomotion contributes to the development of sensitization. To test this hypothesis, we examined the effect of the D3 receptor antagonist nafadotride on sensitization. As predicted, nafadotride inhibits augmentation of the locomotion response to repetitive amphetamine. This finding is consistent with the proposed model of adaptive down-regulation of D3 dopamine receptor function contributing to the development of behavioral sensitization.

Quantification of dopamine D3 receptor mRNA level associated with the development of amphetamine-induced behavioral sensitization in the rat brain.

We hypothesized that changes in expression of dopamine (DA) D3 receptor gene in the rat brain would correlate with the behavioral sensitization induced by amphetamine (AMPH). In order to test this hypothesis, we measured D3 receptor mRNA levels in the striatum, nucleus accumbens and prefrontal cortex, in individual rats following AMPH treatment (2.5 mg/kg s.c., for 5 consecutive days) using a ribonuclease protection assay method. We observed similar levels of D3 receptor mRNA in saline and AMPH treated animals in each brain region examined. These results suggest behavioral sensitization to AMPH is not mediated through postsynaptic transcriptional regulation of D3 receptor.

The hagfish oocyte at late stages of oogenesis: structural and metabolic events at the micropylar region.

The hagfishes (Class Agnatha), primitive vertebrates and massive secretors of mucus, occupy a unique niche in a marine benthic environment and exists in abundance despite an infrequent ovulation of a low number (12-45) of notably large, prolate ellipsoidal (approximately 8 mm x 28 mm, width x length, respectively) eggs. To establish factors that might contribute to survivability of one species (Eptatretus stouti), we examined three groups of mature females and document structural events characteristic of oocytes at late developmental stages: attached to the gonad, after ovulation into the body cavity, and after deposition on aquaria substrata. Quantitative autoradiographic analyses following administration in vivo of [(3)H]-leucine was useful to confirm post-vitellogenic stages and late metabolic events at the micropylar region located at one end of each egg. After egg disassembly, a combination of light microscopy and scanning electron microscopy was used to delineate structural features and 3-D relationships in juxtaposition to the micropylar cup and canal. A thick (approximately 200 microm) complex tripartite chorion surrounds a large micropylar cup (approximately 300 microm, width at base) consisting of fused polygonal (mainly hexagonal) substructural units (3-4 microm, width) and collectively serves to stabilize the micropylar canal (of size to prevent polyspermy) that penetrates the ooplasm near the germinal vesicle. A tuft composed of numerous chorionic appendages ('anchor filaments', approximately 2-3 mm length) is enmeshed in a gel matrix and surrounds the cup. When the eggs are deposited, intermediate filament aggregates (IFA) from slime gland mucus become interspersed in the tuft/gel complex. A similar IFA/tuft/gel region found on the vegetal pole-end facilitates firm egg-end attachments and possibly assists localization of egg clutches to a favorable substratum. Although the mode of fertilization is as yet unknown, the collective structural characteristics of the hagfish egg arc indicative of physical strength and are markedly different from eggs of the other group of cyclostomes (lampreys) and from teleosts. The hagfish produces fewer but larger eggs which are prolate ellipsoidal rather than spherical and have a thicker chorion, a greater chorion to egg width ratio, a much larger micropylar cup with a unique substructure, larger and more localized chorionic appendages, and appendage tufts interspersed with adhesive substances derived not only from the follicular epithelium but also from the slime glands.

Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments.

In contrast to most intermediate filaments (IF) which function intracellularly or constitute epidermal appendages, the single massive (approximately 60 cm length, approximately 3 microns width) IF-rich 'thread' biopolymer synthesized by the specialized hagfish gland thread cell is released extracellularly via holocrine secretion to interact with mucins and seawater, thereby modifying the viscoelastic properties of the copious mucous exudate. Recently, using the Pacific hagfish (Eptatretus stouti, class Agnatha), a jawless scaleless marine vertebrate of ancient lineage, we determined that the deduced amino acid sequence of one thread IF chain (alpha, 66.6 kDa, native pI 7.5) contained an atypical, threonine-rich central rod domain of low identity (< 30%) with other vertebrate IF types, but that the N- and C-terminal domains exhibited several keratin-like features. From these and other unexpected characteristics, it was concluded that hagfish alpha is best categorized as a type II homologue of an epidermal keratin. We now report the deduced sequence of a second thread IF subunit (gamma, 62.7 kDa, native pI 5.3) which is co-expressed and co-assembles in vitro with alpha in a 1:1 ratio. As was found for alpha, the N- and C-terminal domains of gamma have keratin-like parameters, but the central rod has low identity to IFs of types I-V (< 31%), a cephalochordate IF (< 29%) and invertebrate IFs (< 20%) and no particular homology to type I or type II keratins. Central rod identity between gamma and alpha is also low (approximately 23%), as is typical of comparisons between different rod types but atypical of similar rod types (> 50%). The central rods of both gamma and alpha lack the 42-residue insert of helix 1B present in lamins and invertebrate IFs, have unusually high threonine contents (gamma, 10%; alpha, 13%) compared to other IF types (2-5%), contain a number of unexpected residues in consensus conserved sites, and employ a L12 segment of 21 residues rather than the 16 or 17 residues found in keratins. Theoretical analyses indicate that the hagfish molecules exist as coiled coil heterodimers (alpha/gamma) in which the chains are parallel, in axial register, and stabilized by significant numbers of ionic interactions. Fast Fourier-transform analyses revealed that the linear distribution period of approximately 9.55 for basic and acidic residues in other IF chains is not completely maintained, partly due to the high threonine content. The threonine residues occupy mainly outer sites b, c, f in the heptad substructure, possibly abetting parallel alignment of thousands of IFs within the thread, interactions with mucins at the thread periphery, and hierarchical IF chain assembly. It is suggested that the gamma and alpha chains from this most primitive extant vertebrate are type I and type II homologues of epidermal keratin chains, possibly related to early specialized keratins.

Autoradiographic studies of protein and polysaccharide synthesis during vitellogenesis in Drosophila.

Quantitative light- and electron-microscopic autoradiography was used to evaluate metabolic processes that occur during late developmental stages (10-14) of oogenesis in Drosophila melanogaster. Major differences in radiolabelling patterns were found after in vivo (10-45 min) uptake of [3H]-monosaccharides and [3H]-L-lysine. Several different methods of data analysis were required to facilitate interpretation of these patterns. [3H]-L-lysine produced extensive cytoplasmic labelling at all developmental stages. In addition, about 15% of alpha yolk spheres were intensely labelled at stage 10, reflecting the incorporation of radiolabelled vitellogenins synthesized during the incubation period. Subsequent stages showed low silver grain density over alpha yolk spheres until stage 14, when a burst of [3H]-L-lysine incorporation by most alpha spheres was observed, possibly indicative of a maturation process for embryogenesis. [3H]-D-glucose and [3H]-D-galactose (10 min, in vivo) both induced intense labelling of the beta yolk spheres in a manner suggesting in situ assembly beginning at early stage 13. Inasmuch as the polysaccharide of beta yolk spheres has the properties of glycogen (e.g., rosette structure digested by alpha-amylase) and the radiolabelled monosaccharides were introduced intra-abdominally, it is evident that transport systems as well as enzymes utilizing glucose and galactose for glycogenesis must be readily available. It is notable that wide-spread labelling of egg chambers was elicited by [3H]-D-glucose and [3H]-D-galactose (e.g., nurse cells, follicle cells, chorion, vitelline membrane), but the labelling induced by [3H]-N-acetylmannosamine was restricted mainly to the endochorion. A possible role of microtubules in distribution and assembly of yolk spheres was inferred when colchicine, admixed to the culture medium (2-5 ppm), produced abnormal distribution and diminution in number of both alpha and beta yolk spheres. In addition to revealing previously unknown metabolic events of vitellogenesis, the results provide additional criteria for stage characterization as well as a means to specifically label certain macromolecules for purposes of isolation.

Multiple effects of colchicine on oogenesis in Drosophila: induced sterility and switch of potential oocyte to nurse-cell developmental pathway.

Adult female fruitflies exposed to colchicine admixed to the culture medium show a series of dosage-related abnormalities that affect oogenesis and may induce sterility. Among the effects observed were decreased fecundity and hatchability of laid eggs, formation of oocytes lacking chorionic appendages, abnormal distribution and diminution in number of yolk spheres, inhibition of oocyte growth and abnormally located oocyte nuclei. Potentially the most significant effect was the development of egg chambers which contained the normal complement of 16 cells but in which all the cells had the nuclear morphology of nurse cells. The approach provides for the first time an experimental means to divert a potential oocyte into the developmental pathway of the nurse cell in a wild-type fly, and hence should be helpful in the elucidation of factors which control oocyte and nurse cell differentiation. In addition, the results serve to expand the usefulness of oogenesis in Drosophila as a model system for the evaluation of drug-induced metabolic-morphologic abnormalities.

Maturation of hagfish gland thread cells: composition and characterization of intermediate filament polypeptides.

Previous studies with the hagfish, a primitive vertebrate, have shown that the gland thread cells (GTCs) each contain a single thread (approximately 60 cm long in average-sized cells) in the form of a concisely coiled cytoskeletal entity destined for export by holocrine secretion. The thread in relatively immature GTCs consists almost entirely of intermediate filaments (IFs) bundled in parallel alignment with far fewer microtubules (MTs). The three thread polypeptides described earlier (alpha, basic; beta, acidic; gamma, most acidic; each with a Mr of 63-64 kD) are now further evaluated with respect to in vitro assembly, cross-reactivity with IF polypeptides from higher vertebrates, and peptide sequence homology with known IF polypeptides. The overall results mainly suggest that the hagfish polypeptides are keratinlike substances but lamins or a new type of IF is not ruled out. However, cross-reactivity is weak with mammalian keratins; the 8-11-nm filaments formed from mixtures of alpha and gamma in vitro are generally linear rather than the curvilinear structures usually formed by keratin and nonkeratin IFs; and mixtures of alpha and beta tend to yield 9-12-nm granules or granular strings. Polypeptide analyses on GTCs segregated on the basis of maturational stage show a progressive increase in beta/gamma values which correlates with cell maturation, but the alpha/(beta + gamma) ratios remain near 1. Inasmuch as beta and gamma have many similar properties, the documented increase in the amount of the beta component in aging GTCs might in part be the result of a failure in a posttranslational modification system and may contribute to the ultrastructural changes that accompany thread maturation in preparation for holocrine secretion and subsequent modulation of the viscoelastic properties of mucus.

Metabolic-morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii).

Light- and electron-microscopic autoradiography were used to obtain a coordinated metabolic-morphologic view of some of the events of cellular differentiation that occur across the epidermis of the Pacific hagfish (Eptatretus stoutii) and which enable this animal to secrete copious amounts of mucus. As judged by epidermal incorporation of [3H]-thymidine in vivo, about 98% of DNA replication is confined to the basal three layers of the total of 6--8 layers of cells. Small mucous cells (SMC), the most numerous of the three major cell types involved in mucigenesis, show in vitro and in vivo radioincorporation profiles of [3H]-L-lysine and [3H]-D-glucosamine which differ markedly from those of [3H]-L-fucose and [3H]-D-galactose. Time-course incorporation profiles (mean silver grains/cell and percentage of cells with at least one cluster of silver grains) of [3H]-L-lysine and [3H]-D-glucosamine not only reflected the metabolic activities of cell renewal and differentiation in basally-located cells but also the high mucigenic activity in cells near the epidermal surface. By contrast, [3H]-L-fucose and [3H]-D-galactose were mainly incorporated by the more mature SMC in juxtanuclear regions near Golgi complexes and newly formed secretory vesicles. The intensity of [3H]-fucose labeling appeared proportional to the intensity of histochemical staining of the apical cytoplasm. The prominent capsule, within SMC in basal and lateral regions, which arises from a tight intermingling of tonofilaments, appears to restrict secretory vesicles to apical regions while the cell progressively differentiates and migrates to the epidermal surface. The other mucigenic cell types, large mucous cells and thread cells, each show distinctive differentiation and radioincorporation patterns.

An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stouti).

Each slime gland thread cell from the primitive Pacific hagfish (Eptatretus stouti) contains a massive, conical, intermediate filament (IF)-rich biopolymer ('thread,' approximately 60 cm length, approximately 3 microns width). In view of the unusual ultrastructure of the thread, its extracellular role in modulation of the viscoelastic properties of mucus, and the ancient lineage of this primitive vertebrate, we report the nucleotide and deduced amino acid sequences of one major thread IF subunit, alpha (pI 7.5), which is coexpressed with a second polypeptide, gamma (pI 5.3). These two polypeptides coassemble in vitro into approximately 10 nm filaments. The alpha-thread chain, a 66.6 kDa polypeptide, has an unusual central rod domain containing 318 residues flanked by N- and C-terminal domains of 192 and 133 residues, respectively. Each peripheral region exhibits some epidermal keratin-like features including peptide repeats and a high total content of glycine and serine residues. The terminal domains, however, lack the H1 and H2 subdomains characteristic of known keratins. Moreover, when the central rod is aligned either in relation to established homology profiles (J. F. Conway and D. A. D. Parry (1988) Int. J. Biol. Macromol. 10, 79-98) of other IF subunits (types I-V, nestin, non-neuronal invertebrate), or by computer-based homology searches of the GenBank/EMBL Data Bank, a low identity (< 30%) is evident, with no preferred identity to keratins or other known IF types. Although the central rod of 318 residues consists of the canonical apolar heptad repeats interspersed with three linker regions, a discontinuity in phasing of the heptad substructure in rod 2B, and conserved sequences at either end of the rod domain, other collective characteristics are atypical: overall high threonine content (13.2% vs 2.3-5.4% for other IFs), high threonine content in rod 1B (18.8% vs 1-6%), five Thr-Thr repeats in coiled coil segments, L12 of length greater than in keratins, substitution of phenylalanine for a highly conserved glutamate in the sixth position of L2, and a glycine-proline sequence in segment 2B. Possibly as a result of the high threonine content, the percentage of both acidic and basic residues in most helical subdomains is reduced relative to type I and II chains. Fast Fourier transform analyses show that only the acidic residues in segment 1B and basic residues in segment 2 have near typical IF periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Postulated developmental forms in the life cycle of the lymphocystis virus.

Electron micrographs of lymphocystis sk8n lesions obtained from the walleye pike, Stizostedion vitreum, show viral particles of differing sizes and morphologies. Earlier studies have described only particles ranging in size from 200-250 mmu in diameter. We show, in addition, new forms (65-100 mmu in diameter) intermingled among the large particles and also contained within the latter. Both forms are associated with a single inclusion body. The possible role of the small particle in the life cycle of the virus is discussed.

Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti).

The hagfishes (cyclostomes) are known to secrete copious amounts of mucus mainly by the holocrine mode from the slime glands. Stressed animals release two types of cells (gland thread cells, GTCs; gland mucous cells. GMCs) which rupture on contact with water and rapidly form a mass of viscous mucus. Herein we report some key sequential events of this process and document a novel role for cytoskeletal polymers. After electrostimulation of Pacific hagfish (Eptatretus stouti), the exudate was collected in a stabilization buffer and GTCs segregated from GMC vesicles. Water was added progressively to mixtures of known quantities of these entities. The changing mucous composition and properties were monitored by light- and electron microscopy, viscometry and immunogold assay. Sequentially, the threads uncoil from GTCs, aggregate with the vesicles, the vesicles rupture and release mucin-like substances, at least some of which adhere to the thread. It was found that the intermediate filament (IF)-rich threads markedly facilitate hydration and modulate the viscoelastic and cohesive properties of the resultant mucus. It was speculated that the thread abets localization of mucus in an aqueous environment and promotes adhesion of mucus to surfaces such as the fish integument. As judged by immunostaining in situ, GTCs, as well as several cell-types in the epidermis, contain keratin-like components. The role of biopolymers on the properties of teleost and mammalian mucus is discussed.

Metabolic-morphologic characteristics of the integument of teleost fish with mature lymphocystis nodules.

Normal and virus-infected (lymphocystis disease) integument from five species of teleosts was examined by light and TEM autoradiography and SEM to establish metabolic-morphologic characteristics of integument with mature lymphocystis cells (LC's). LC's with numerous morphologic attributes of a late developmental stage showed highest incorporation of [3H]-thymidine in vivo (1-91 h) above the intracytoplasmic inclusion body (ci) with little radiolabel in nuclei, cytoplasmic icosahedral deoxyriboviruses (ICDV's) or capsule. Analysis by quantitative autoradiography revealed that the % total cell label in ci and cytoplasm did not vary appreciably from 1-91 h and was corroborative with morphologic criteria of maturity. A possibly phylogenetic difference was noted between teleosts, wherein normal integument showed uptake of [3H]-thymidine in vivo (1 h) by cells at all levels of the epidermis, and cyclostomes (Spitzer et al. 1979) wherein labeling was confined to the basal third of the epidermis. Among four infected teleost species, the mean diameters of the ICDV's measured under the same conditions, ranged from 259.5 nm to 290.0 nm with the mean for each species differing significantly (p less than 0.01) from each of the other means. Ruptured LC's were shown by TEM and SEM to have released ICDV's onto the lesions and integument. Various stages of LC degeneration, host response, and integumental repair processes were documented. An evaluation of labeling in vivo of the capsular matrix was compatible ([3H]-D-galactose greater than [3H]-L-lysine much greater than [3H]-L-fucose) with a glycosaminoglycan-protein structure.

Subcellular localization of monoamine oxidase in bovine thalamus tissue using immunoferritin conjugates.

A combination of discontinuous sucrose gradient analysis and polyacrylamide electrophoresis was used to isolate one of the multiple forms of monoamine oxidase (MAO) from bovine thalamus. This substance (the principle form and the most anodic of the five MAO forms observed) was used as a basis for an immunoferritin-electron microscope approach to determine the subcellular localization of MAO in thalamus. This form of MAO, as well as antigenically related forms, was found to reside mainly on the outer mitochondrial membrane. In addition, the action of SDS on solubilization and interconversion of MAO forms was studied and found to be dependent on the concentration and time of reaction of SDS with thalamus tissue.

Fractionation of hagfish slime gland secretions: partial characterization of the mucous vesicle fraction.

This paper deals with the collection, fractionation and partial characterization of the slime gland secretion of the Pacific hagfish (Eptatretus stouti) with emphasis on the mucous fraction. Secretions were collected by electrical stimulation of the glands of anesthetized hagfish and, using three different methods, separated into three fractions: 1) the thread cells, 2) the mucous vesicles of the mucous cells, and 3) the soluble fraction. The methods take advantage of the stabilization of the thread cells and mucous vesicles by ammonium sulfate and sodium citrate.