A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin

BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.     

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha- internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.     

Schwann cells of the myelin-forming phenotype express neurofilament protein NF-M

Immature Schwann cells of the rat sciatic nerve can differentiate into myelin-forming or non-myelin-forming cells. The factors that influence this divergent development are unknown but certain markers such as galactocerebroside distinguish the two cell populations at an early stage of Schwann cell differentiation. Because myelination requires extensive changes in cell morphology, we have investigated the composition and structure of the Schwann cell cytoskeleton at a time when these cells become committed to myelination. Here we show that Schwann cells express a cytoskeletal protein of M(r) 145 before diverging into the myelin-forming path, i.e., before they acquire cell-surface galactocerobroside. The p145 protein has the characteristics of an intermediate filament (IF) protein and immunoelectron microscopy shows that it colocalizes with vimentin, which suggests that these two proteins can coassemble into IFs. Elevated intracellular cAMP levels, which can mimic some of the early effects of axons on Schwann cell differentiation, induced p145 synthesis, therefore, we conclude that myelin-forming Schwann cells express this protein at a very early stage in their development. Immunological comparisons with other IF proteins revealed a close similarity between p145 and the neurofilament protein NF-M; the identification of p145 as NF-M was confirmed by isolating and sequencing a full-length clone from a Schwann cell cDNA library. These data demonstrate that Schwann cells remodel their IFs by expressing NF-M before acquiring the myelin-forming phenotype and that IF proteins of the neurofilament-type are not restricted to neurons in the vertebrate nervous system.     

Functions of intermediate filaments in neuronal development and disease

Five major types of intermediate filament (IF) proteins are expressed in mature neurons: the three neurofilament proteins (NF-L, NF-M, and NF-H), alpha-internexin, and peripherin. While the differential expression of IF genes during embryonic development suggests potential functions of these proteins in axogenesis, none of the IF gene knockout experiments in mice caused gross developmental defects of the nervous system. Yet, deficiencies in neuronal IF proteins are not completely innocuous. Substantial developmental loss of motor axons was detected in mice lacking NF-L and in double knockout NF-M;NF-H mice, supporting the view of a role for IFs in axon stabilization. Moreover, the absence of peripherin resulted in approximately 30% loss of small sensory axons. Mice lacking NF-L had a scarcity of IF structures and exhibited a severe axonal hypotrophy, causing up to 50% reduction in conduction velocity, a feature that would be very detrimental for large animal species. Unexpectedly, the NF-M rather than NF-H protein turned out to be required for proper radial growth of large myelinated axons. Studies with transgenic mice suggest that some types of IF accumulations, reminiscent of those found in amyotrophic lateral sclerosis (ALS), can have deleterious effects and even cause neurodegeneration. Additional evidence for the involvement of IFs in pathogenesis came from the recent discovery of neurofilament gene mutations linked to ALS and Charcot-Marie-Tooth disease (CMT2E). Conversely, we discuss how certain types of perikaryal neurofilament aggregates might confer protection in motor neuron disease.     

Development and Maintenance of the Neuronal Cytoskeleton in Aggregated Cell Cultures of Fetal Rat Telencephalon and Influence of Elevated K+ Concentrations

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development-dependent expression of several cytoskeletal proteins, including the heavy- and medium-sized neurofilament subunits (H-NF and M-NF, respectively); brain spectrin; synapsin I; beta-tubulin; and the microtubule-associated proteins (MAPs) 1, 2, and 5 and tau protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular beta-tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M-NF preceded that of H-NF, with the latter being detectable only after approximately 3 weeks in culture. Furthermore, MAP 2 and tau protein showed a development-dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron-enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M-NF, H-NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145-kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron-enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton.     

Phosphorylation of neurofilament proteins by endogenous calcium/calmodulin-dependent protein kinase

A protein fraction containing neurofilaments was prepared from rat brain cytosol by differential centrifugation and gel filtration chromatography. These preparations were enriched for a calcium/calmodulin-dependent kinase activity that phosphorylated endogenous neurofilament proteins. The enzyme incorporated approximately 1 mol PO4/mol of each neurofilament triplet polypeptide. These data suggest that a calmodulin-dependent kinase may mediate some of the effects of calcium on cytoskeletal function by phosphorylation of neurofilament proteins.     

Vimentin in the Central Nervous System

Intermediate filament proteins were identified by two-dimensional gel electrophoresis in urea extracts of rat optic nerves undergoing Wallerian degeneration and in cytoskeletal preparations of rat brain and spinal cord during postnatal development. The glial fibrillary acidic (GFA) protein and vimentin were the major optic nerve proteins following Wallerian degeneration. Vimentin was a major cytoskeletal component of newborn central nervous system (CNS) and then progressively decreased until it became barely identifiable in mature brain and spinal cord. The decrease of vimentin occurred concomitantly with an increase in GFA protein. A protein with the apparent molecular weight of 61,000 and isoelectric point of 5.6 was identified in both cytoskeletal preparations of brain and spinal cord, and in urea extracts of normal optic nerves. The protein disappeared together with the polypeptides forming the neurofilament triplet in degenerated optic nerves.     

Protein 4.1 in forebrain postsynaptic density preparations: enrichment of 4.1 gene products and detection of 4.1R binding proteins.

4.1 Proteins are a family of multifunctional cytoskeletal components (4.1R, 4.1G, 4.1N and 4.1B) derived from four related genes, each of which is expressed in the nervous system. Using subcellular fractionation, we have investigated the possibility that 4.1 proteins are components of forebrain postsynaptic densities, cellular compartments enriched in spectrin and actin, whose interaction is regulated by 4.1R. Antibodies to each of 4.1R, 4.1G, 4.1N and 4.1B recognize polypeptides in postsynaptic density preparations. Of these, an 80-kDa 4.1R polypeptide is enriched 11-fold in postsynaptic density preparations relative to brain homogenate. Polypeptides of 150 and 125 kDa represent 4.1B; of these, only the 125 kDa species is enriched (threefold). Antibodies to 4.1N recognize polypeptides of approximately 115, 100, 90 and 65 kDa, each enriched in postsynaptic density preparations relative to brain homogenate. Minor 225 and 200 kDa polypeptides are recognized selectively by specific anti-4.1G antibodies; the 200 kDa species is enriched 2.5-fold. These data indicate that specific isoforms of all four 4.1 proteins are components of postsynaptic densities. Blot overlay analyses indicate that, in addition to spectrin and actin, postsynaptic density polypeptides of 140, 115, 72 and 66 kDa are likely to be 4.1R-interactive. Of these, 72 kDa and 66 kDa polypeptides were identified as neurofilament L and alpha-internexin, respectively. A complex containing 80 kDa 4.1R, alpha-internexin and neurofilament L was immunoprecipitated with anti-4.1R antibodies from brain extract. We conclude that 4.1R interacts with the characteristic intermediate filament proteins of postsynaptic densities, and that the 4.1 proteins have the potential to mediate the interactions of diverse components of postsynaptic densities.     

Characterization of dimer subunits of intermediate filament proteins

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.     

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with³H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of³²P into purified neurofilament proteins when they were incubated with³²P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.     

Characterization of mammalian neurofilament triplet proteins. Subunit stoichiometry and morphology of native and reconstituted filaments

The three major proteins of mammalian neurofilaments of molecular weights 179,000 (NF1), 129,000 (NF2), and 66,500 (NF3) have been purified to homogeneity by multiple anion-exchange and hydroxylapatite absorption chromatography in 8 M urea. Silver staining of polyacrylamide gels of the purified proteins show single bands. In order to gain further insight into the molecular organization of the neurofilament triplet proteins, the molar stoichiometries and morphologies of native and reconstituted filaments and those isolated from developing brain were studied. Denaturing polyacrylamide gel electrophoresis followed by quantitative dye-binding analysis shows that the molar ratio of the three components in neurofilaments isolated from bovine spinal cord myelinated nerve is 4:2:1 (NF3:NF2:NF1). Comparison of the molar ratios of each component in neurofilaments isolated from rat, bovine, and human brain shows a variation in the ratio of each of these polypeptides and raises questions about the physiological uniqueness of the molar composition of the neurofilament triplet. Reconstitution of the three bovine polypeptides into 10-nm filaments was accomplished under conditions in which the NF3 protein was limiting. Reassembly of 10-nm filaments with varying amounts of NF2 and NF1 indicate that the NF3 homopolymer has a limiting capacity to bind NF2 and NF1 and is saturated at a molar ratio of 2:2:1 (NF3:NF2:NF1). Isolation of the neurofilament complex at various stages of rat brain maturation indicates that NF3 and NF2 are integrated into the neurofilament complex as early as embryonic day 17, while NF1 copurifies with these proteins at postnatal day 16, eventually reaching a molar stoichiometry of 2:2:1 in the adult rat. The molecular stoichiometry of the neurofilament proteins, the differential integration of these proteins during brain development, and the variation of the molar composition between mammalian species suggest accessory roles for the NF2 and NF1 proteins in the neurofilament complex.     

Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co- expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.     

The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons

Studies on the axonal transport of neurofilament proteins in cultured neurons have shown they move at fast rates, but their overall rate of movement is slow because they spend most of their time not moving. Using correlative light and electron microscopy, we have shown that these proteins move in the form of assembled neurofilament polymers. However, the polypeptide composition of these moving polymers is not known. To address this, we visualized neurofilaments in cultured neonatal mouse sympathetic neurons using GFP-tagged neurofilament protein M and performed time-lapse fluorescence microscopy of naturally occurring gaps in the axonal neurofilament array. When neurofilaments entered the gaps, we stopped them in their tracks using a rapid perfusion and permeabilization technique and then processed them for immunofluorescence microscopy. To compare moving neurofilaments to the total neurofilament population, most of which are stationary at any point in time, we also performed immunofluorescence microscopy on neurofilaments in detergent-splayed axonal cytoskeletons. All neurofilaments, both moving and stationary, contained NFL, NFM, peripherin and alpha-internexin along>85% of their length. NFH was absent due to low expression levels in these neonatal neurons. These data indicate that peripherin and alpha-internexin are integral and abundant components of neurofilament polymers in these neurons and that both moving and stationary neurofilaments in these neurons are complex heteropolymers of at least four different neuronal intermediate filament proteins.     

Novel insights into intermediate-filament function from studies of transgenic and knockout mice

Summary The function of intermediate-filament (IF) proteins has been a matter of speculation for a long time. Now, the analysis of genetically altered mice is contributing to the understanding of their function. While the initial analysis of knockout mice supports the global view that keratins in epidermis and desmin in muscle serve an important structural function by protecting these tissues against mechanical stress, the detailed examination of these and other mice suggests that IF are more than passive cytoskeletal proteins. This is highlighted by mice with deficiencies for keratins in internal epithelia, vimentin, GFAP, or neurofilament proteins. These lack overt phenotypes expected as a result of cytoskeletal deficiency but show defects compatible with a role of IF in protecting tissues against toxic and other forms of stress. Moreover, the first round of gene replacement experiments suggests that keratins from internal epithelia are unable to take the place of their epidermal counterparts. The development of mice with point mutations, paralleled by the mutation analysis of human diseases and the characterization of IF-associated proteins will be instrumental to understand why evolution has produced such a diverse gene family to encode simple 10 nm diameter filaments.     

Peripherin-mediated death of motor neurons rescued by overexpression of neurofilament NF-H proteins

In previous studies, we showed that overexpression of peripherin, a neuronal intermediate filament (IF) protein, in mice deficient for neurofilament light (NF-L) subunits induced a progressive adult-onset degeneration of spinal motor neurons characterized by the presence of IF inclusion bodies reminiscent of axonal spheroids found in amyotrophic lateral sclerosis (ALS). In contrast, the overexpression of human neurofilament heavy (NF-H) proteins provoked the formation of massive perikaryal IF protein accumulations with no loss of motor neurons. To further investigate the toxic properties of IF protein inclusions, we generated NF-L null mice that co-express both peripherin and NF-H transgenes. The axonal count in L5 ventral roots from 6 and 8-month-old transgenic mice showed that NF-H overexpression rescued the peripherin-mediated degeneration of motor neurons. Our analysis suggests that the protective effect of extra NF-H proteins is related to the sequestration of peripherin into the perikaryon of motor neurons, thereby abolishing the development of axonal IF inclusions that might block transport. These findings illustrate the importance of IF protein stoichiometry in formation, localization and toxicity of neuronal inclusion bodies.     

Intermediate filament proteins in the developing chick spinal cord

The distribution of different intermediate filament (IF) proteins in the embryonic chick spinal cord was examined at several stages of development using immunohistochemical techniques, analytic gel electrophoresis, and electron microscopy. We have found that: (1) the fibroblast-type IF protein (vimentin) is present in virtually all of the replicating neuroepithelial cells of the early neural tube, as well as in radial glia, astrocytes, and Schwann cells in later stages of development; (2) the fibroblast-type IF protein is not detectable in definitive neurons; (3) the neurofilament proteins are first detectable in postmitotic neuroblasts at about the time of initial axon formation and they are restricted to neurons; (4) the astrocyte-type IF protein (glial fibrillary acidic protein) is in definitive astrocytes, but not in radial glia; (5) the prekeratin proteins are restricted to cells of the leptomeninges; and (6) the muscle-type IF protein (desmin) is restricted to vascular tissue in and around the developing spinal cord. These findings suggest that the fibroblast-type IF protein is the only IF protein in the early neuroepithelial cells and that the progeny of these cells will follow one of three different patterns of IF protein expression: (1) continued expression of only the fibroblast-type IF protein (radial glia); (2) expression of both the fibroblast-type IF protein and the astrocyte-type IF protein (astrocytes); or (3) expression of only the neurofilament proteins (neurons).     

58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.