The intermediate filament architecture as determined by X-ray diffraction modeling of hard alpha-keratin

Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard alpha-keratin intermediate filaments under in vivo conditions. In short, the alpha-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.     

Distribution of intermediate filaments in amphibian oxyntic cells

Summary Intermediate filaments of toad oxyntic cells were isolated and analysed by SDS-PAGE. The major proteins of the residue were identified as actin and a 51,000 dalton polypeptide. Immunological crossreactivity between toad oxyntic cell intermediate filament components and anti-prekeratin, was shown by double immunodiffusion tests and indirect immunofluorescence. The immunofluorescent decoration of oxyntic cells and the electron microscope images are coincident in locating the intermediate filaments mainly at the cortical and perinuclear basal zones. Furthermore, the cortical zone appears especially rich in prekeratin-like material at its adluminal third. This results in a cup-like structure that encloses the cell portion occupied by the tubulovesicular system, which does not contain intermediate filaments. The translocation of membranes occurring during the secretory cycle of the oxyntic cell, has been attributed to a system of contractile proteins. The disposition of the prekeratin-like material suggests a role for intermediate filaments in the generation of movement, produced by actin and myosin interaction, by providing a fixed plane for the anchoring of actin microfilaments.     

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography

Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion.     

Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography

Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.     

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2–4 nm diameter, and intermediate filament protein is included in numerous, variously sized (2–15 μm) spheroidal aggregates containing densely stained granular particles of 5–16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.     

The structure of the chorion and associated surface filaments in Oryzias--evidence for the presence of extracellular tubules

The structure of the chorion with its associated surface filaments has been examined in Oryzias latipes using several techniques, including scanning and transmission electron microscopy, enzymatic digestion, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The chorion of the recently fertilized egg was found to be organized into three zones: an outer, fuzzy electron-lucent zone that was continuous over the surface of filaments, a middle, homogeneous electron-dense zone, and an inner zone of ten to 12 horizontal, fibrous lamellae. Two topographically distinct types of filaments were found on the chorionic surface: nonattaching and attaching. Nonattaching filaments showed a regular spatial distribution over the chorion with an interfilament distance of about 60-70 microns. Attaching filaments originated from a localized portion of the chorion and united with those of neighboring eggs to anchor the egg cluster to the gonoduct of the female. Both nonattaching and attaching filaments were morphologically regionalized into basal and distal segments. Internally, nonattaching and attaching filaments were constructed of unbranched, packed tubules with an average outside diameter of approximately 19.5 and 18.8 nm, respectively. Using the attaching filament for further study, it was determined by rotational analysis (Markham et al., '63) that the wall of each tubule was a cylinder composed of 14 globular subunits. Two structural types of attaching filaments were identified. The type I attaching filament was similar in internal organization to the nonattaching filament and consisted of only tubules. The type II attaching filament, however, showed a highly osmiophilic, electron-dense bar surrounded by packed tubules. Tubules of attaching filaments of the adult were resistant to the action of Triton X-100 and colchicine, but sensitive to a 0.1% protease solution. However, colchicine-treated ovary tissue showed an absence and pattern of disorganization of tubules at the periphery of developing filaments. Solubilized attaching filament samples electrophoresed on 7.5% polyacrylamide-SDS gels were resolved into a pair of Coomassie-blue-positive bands that comigrated with purified porcine brain tubulin. The apparent molecular weight of the attaching filament polypeptide was determined to be approximately 55,000 daltons. These data suggest that the extracellular, tubular components of attaching filaments (as well as nonattaching filaments) are proteinaceous and show properties similar to those of cytoplasmic microtubules. Tubular precursor material was electron-dense and appeared to originate in the cisternae of the rough endoplasmic reticulum of ovarian foll     

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

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

A structural comparison of tryptic fragments of three types of intermediate filaments

We have compared tryptic fragments of three types of intermediate filaments, emphasizing structural characteristics as seen in the electron microscope. Variable, long alpha-helical rod fragments were found to be similar for keratin, neurofilaments and desmin filaments. Short rod fragments from keratin and neurofilaments appeared similar when observed by electron microscopy. Short rod fragments were not seen in desmin filament digests. In addition to these elongated particles, globular fragments, which have not been described previously, were obtained from all three types of intermediate filaments. These globular fragments were characterized by gel filtration and electron microscopy, and compared to globular proteins of known size using both methods. The diameter was about 6 nm and the molecular weight was estimated to be 50 000-60 000. These globular particles may comprise the short, nonhelical regions from several IF protein subunits, which are clustered into an interface in the intact filament or protofilaments.     

ULTRASTRUCTURE OF THE RESTING AND CONTRACTED STRIATED MUSCLE FIBER AT DIFFERENT DEGREES OF STRETCH

Passive stretch, isometric contraction, and shortening were studied in electron micrographs of striated, non-glycerinated frog muscle fibers. The artifacts due to the different steps of preparation were evaluated by comparing sarcomere length and fiber diameter before, during, and after fixation and after sectioning. Tension and length were recorded in the resting and contracted fiber before and during fixation. The I filaments could be traced to enter the A band between the A filaments on both sides of the I band, creating a zone of overlap which decreased linearly with stretch and increased with shortening. This is consistent with a sliding filament model. The decrease in the length of the A and I filaments during isometric contraction and the finding that fibers stretched to a sarcomere length of 3.7 µ still developed 30 per cent of the maximum tetanic tension could not be explained in terms of the sliding filament model. Shortening of the sarcomeres near the myotendinous junctions which still have overlap could account for only one-sixth of this tension, indicating that even those sarcomeres stretched to such a degree that there is a gap between A and I filaments are activated during isometric contraction (increase in stiffness). Shortening, too, was associated with changes in filament length. The diameter of A filaments remained unaltered with stretch and with isometric contraction. Shortening of 50 per cent was associated with a 13 per cent increase in A filament diameter. The area occupied by the fibrils and by the interfibrillar space increased with shortening, indicating a 20 per cent reduction in the volume of the fibrils when shortening amounted to 40 per cent.     

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): a comparison with their in situ counterparts

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.     

The Pathway of Assembly of Intermediate Filaments from Recombinant α-Internexin

The pathway of filament assembly from the neuronal intermediate filament α-intermexin was investigated. Optimal assembly occurred in solutions of pH 6.5 to 7 and moderate ionic strength at 37°C. Short filaments formed upon dialysis at 24°C, which elongated further when incubated at 37°C. Soluble forms of α-internexin were characterized by analytical ultracentrifugation and electron microscopy. In 10 mM Tris, pH 8, conditions that favor formation of tetramers and other small oligomers for other intermediate filament proteins, α-internexin formed 10.5 S particles, apparently unit-length half-filaments in the form of rods 10.6 nm in diameter and 68 nm long. Dialysis vs the same buffer with added 10 mM NaCl yielded 16 S rods, probably unit-length filaments, of the same length but 13.0 nm in diameter. At 50 mM NaCl, rods about 13 nm in diameter and heterogeneous in length were observed in electron micrographs, apparently formed from longitudinal annealing of unit-length rods. The results favor a model of assembly in which coiled coil dimers aggregate laterally to form first “unit-length half-filaments” (Herrmann, H., and Aebi, U. (1998)Curr. Opin. Struct. Biol.8, 177–185) and then “unit-length filaments,” which subsequently elongate by annealing.     

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.     

Isolation of intermediate filament assemblies from human hair follicles

We used developing human hair follicle cells for the isolation of hard alpha-keratin structural components. Intracellular dispersions examined by electron microscopy contained both individual alpha-keratin filaments and the tactoid-like filament assemblies observed in situ organized along subfibrillar arms of macrofibrils. The assemblies of average width 47 nm were composed of closely packed alpha-keratin filaments and originated from the initial filament arrays observed in sections of developing mammalian hair follicles. We have distinguished two types of assemblies: the para-like or hexagonally packed and the ortho-like spiral or whorl type. Axial banding extended across the width of filament assemblies, which suggested that hard alpha-keratin filaments pack in lateral register and form a lattice that contains interfilamentous bridges. We observed axial banding patterns with periods ranging from 20 to 22 nm, consistent with the 22-nm periodic structure deduced from x-ray diffraction studies and present in models proposed for hard alpha-keratin and other intermediate filaments. Preliminary biochemical studies of filaments and filament assemblies indicate that they consist of the closely related group of proteins (low-sulfur proteins) ubiquitous among extracts of hard mammalian keratins. Isolated hard alpha-keratin filament assemblies provide a new and valuable structural entity for investigating the assembly mechanisms involved in the formation of the filament-matrix framework found in hard mammalian keratin appendages.     

Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.     

Intermediate filaments: structure, regulation of expression and possible function

New data are reviewed on intermediate filaments, i.e. on one of the cytoskeleton components. Structural proteins of intermediate filaments, their enzymatic modification, filament-associated proteins and the peculiarities of filament assembly are dealt with. The regularities of expression of intermediate filament proteins in normal tissues are analysed, as well as during differentiation and cultured cell growth. In the final part of the paper possible functions of intermediate filaments are discussed.     

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.     

Immobilization effects of anticell and antiaxial filament sera on Treponema zuelzerae

Rabbit antisera were produced against whole cells and against highly purified axial filaments of Treponema zuelzerae. Both types of sera react with axial filaments. Antisera against whole cells immobilize the organism; antisera against axial filaments do not. The immobilizing activity of anticell sera could be neutralized by preabsorption with whole cells but not by preabsorption with axial filaments. Preabsorption with axial filaments did, however, abolish the ability of anticell sera to react with axial filaments. Anticell sera also react with the outer cell envelope; this ability was not affected by preabsorption with axial filaments. The data show that antibody directed at something other than axial filaments causes immobilization of T. zuelzerae. The results do not exclude the possibility that the axial filament is the organelle of motility. Titration of the immobilizing activity of anticell sera by adding a constant amount of motile cells to serial dilutions of serum gave two zones of maximum immobilization, one with undiluted serum and one at higher dilutions. For a fixed amount of antiserum, increasing the numbers of cells in the titration almost, but not quite, abolished the zone phenomenon. This phenomenon appears to result from the presence of two kinds of immobilizing antibodies in anticell serum. One of these belongs to the IgG class of immunoglobulins. The other has not been identified but is present in a serum globulin fraction which contains IgM. At intermediate concentrations, the "IgM fraction" inhibits the immobilizing activity of IgG without itself causing immobilization.     

Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S.

The supramolocular structure of hemoglobin S has been studied by electron microscopy and computer-based image reconstruction. Negatively stained fibers prepared by the lysis of sickled cells or the stirring of hemoglobin S hemolysates have been observed to be almost exclusively of the 20-nm diameter form. These fibers have a periodic variation in diameter between the extremes of 18 nm and 23 nm. Computed Fourier transforms of the fibers show a, highly complex pattern of reciprocal space maxima, with 30 maxima on 20 layer-lines clearly resolved. The Bessel orders of the maxima were assigned with the aid of a newly developed technique, a combined real-space Fourier-space reconstruction method (REFORM). This method utilizes the filtered image produced by the inverse Fourier transform of the low-resolution maxima to calculate in real space the crosssection of a helical fiber. The REFORM analysis indicated that the fibers have an elliptical cross-section and are composed of 14 hexagonally packed filaments with 10 outer filaments surrounding four inner filaments. On the basis of this cross-section, the Bessel orders of all the maxima were assigned, permitting the calculation of three-dimensional reconstructions by Fourier Bessel synthesis. From these reconstructions details of the location of hemoglobin S molecules of each filament were obtained. Hemoglobin S molecules are staggered in adjacent filaments to produce a closely packed helical structure. Reconstructions of fibers at various stages of disassembly revealed a stable intermediate containing 10 filaments which could be characterized in terms of the loss of two pairs of specific outer filaments. A partially disassembled fiber with only six filaments at positions corresponding to three inner and three outer filaments of the parent structure was also identified. The six-filament structure appears to be produced from the 10-filament structure by the loss of two specific pairs of filaments. Thus pairs of filaments are evidently significant structural units in the stabilization of the complete fibers and the orientation of the molecules in these pairs may be related to the filament pairs known to occur in crystals of hemoglobin S.     

Isoforms of alpha-actinin from cardiac, smooth, and skeletal muscle form polar arrays of actin filaments

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by alpha-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using alpha-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4-0.7 microm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that alpha-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by alpha-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of alpha-actinin.     

Intermediate filament protein partnership in astrocytes.

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.