Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex.

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

N-acetylglucosamine-bearing polymers mimicking O-GlcNAc-modified proteins elicit anti-fibrotic activities in myofibroblasts and activated stellate cells.

O-linked ß-N-acetylglucosamine (O-GlcNAc)-modified proteins are post-translationally modified with GlcNAc conjugated to serine and threonine residues. This modification is associated with various physiological functions such as serine and threonine phosphorylation and Notch signaling. Here, we demonstrated that O-GlcNAc-modified proteins leaked from dead cells and GlcNAc-bearing polymers mimicking the multivalent GlcNAc moiety of these proteins induced anti-fibrotic activities, such as the suppression of α-smooth muscle actin and collagen and the induction of matrix metalloprotease 1 in myofibroblasts. We have previously reported that O-GlcNAc-modified proteins and GlcNAc-bearing polymers could interact with cell surface vimentin and desmin. In the current study, it was demonstrated that a multivalent GlcNAc moiety structure of these molecules activated PI3K/Akt and p38MAPK pathway and elicited these anti-fibrotic activities in myofibroblasts by interacting with cell surface vimentin. Since the interaction of O-GlcNAc-modified proteins with desmin was observed in the fibrotic liver of carbon tetrachloride-treated mice via an in situ proximity ligation assay, it was assumed that the activated stellate cells could bind to the O-GlcNAc-modified proteins from the damaged hepatocytes. In addition, the administration of anti-O-GlcNAc antibody to inhibit the interaction exacerbated liver fibrosis in the mice. Moreover, administration of the GlcNAc-bearing polymers into carbon tetrachloride-treated mice could ameliorate liver fibrosis. Thus, O-GlcNAc-modified proteins leaked from dead cells can interact with myofibroblasts and activated stellate cells and function as fibrosis suppressors. Moreover, we anticipate that GlcNAc-bearing polymers mimicking O-GlcNAc-modified proteins will be applied as novel therapeutic tools for fibrosis.

The vimentin cytoskeleton: when polymer physics meets cell biology.

The proper functions of tissues depend on the ability of cells to withstand stress and maintain shape. Central to this process is the cytoskeleton, comprised of three polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). IF proteins are among the most abundant cytoskeletal proteins in cells; yet they remain some of the least understood. Their structure and function deviate from those of their cytoskeletal partners, F-actin and microtubules. IF networks show a unique combination of extensibility, flexibility and toughness that confers mechanical resilience to the cell. Vimentin is an IF protein expressed in mesenchymal cells. This review highlights exciting new results on the physical biology of vimentin intermediate filaments and their role in allowing whole cells and tissues to cope with stress.

Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids.

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

The function of intermediate filaments in cell shape and cytoskeletal integrity.

This study describes the development and use of a specific method for disassembling intermediate filament (IF) networks in living cells. It takes advantage of the disruptive effects of mimetic peptides derived from the amino acid sequence of the helix initiation 1A domain of IF protein chains. The results demonstrate that at 1:1 molar ratios, these peptides disassemble vimentin IF into small oligomeric complexes and monomers within 30 min at room temperature in vitro. Upon microinjection into cultured fibroblasts, these same peptides induce the rapid disassembly of IF networks. The disassembly process is accompanied by a dramatic alteration in cell shape and the destabilization of microtubule and actin-stress fiber networks. These changes in cell shape and IF assembly states are reversible. The results are discussed with respect to the roles of IF in cell shape and the maintenance of the integrity and mechanical properties of the cytoplasm, as well as the stability of the other major cytoskeletal systems.

Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.

Unbiased pattern analysis reveals highly diverse responses of cytoskeletal systems to cyclic straining.

In mammalian cells, actin, microtubules, and various types of cytoplasmic intermediate filaments respond to external stretching. Here, we investigated the underlying processes in endothelial cells plated on soft substrates from silicone elastomer. After cyclic stretch (0.13 Hz, 14% strain amplitude) for periods ranging from 5 min to 8 h, cells were fixed and double-stained for microtubules and either actin or vimentin. Cell images were analyzed by a two-step routine. In the first step, micrographs were segmented for potential fibrous structures. In the second step, the resulting binary masks were auto- or cross-correlated. Autocorrelation of segmented images provided a sensitive and objective measure of orientational and translational order of the different cytoskeletal systems. Aligning of correlograms from individual cells removed the influence of only partial alignment between cells and enabled determination of intrinsic cytoskeletal order. We found that cyclic stretching affected the actin cytoskeleton most, microtubules less, and vimentin mostly only via reorientation of the whole cell. Pharmacological disruption of microtubules had barely any influence on actin ordering. The similarity, i.e., cross-correlation, between vimentin and microtubules was much higher than the one between actin and microtubules. Moreover, prolonged cyclic stretching slightly decoupled the cytoskeletal systems as it reduced the cross-correlations in both cases. Finally, actin and microtubules were more correlated at peripheral regions of cells whereas vimentin and microtubules correlated more in central regions.

Separation and characterization of homo and hetero-oligomers of the intermediate filament proteins desmin and vimentin.

Affinity chromatography on single-stranded (ss)DNA-cellulose in conjunction with gel permeation chromatography in the presence of urea was employed to separate the intermediate filament (IF) protein complement of catalytically oxidized BHK-21 cell Triton cytoskeletons into disulfide-cross-linked homo- and heterodimers of desmin and vimentin and uncross-linked homodimers. The same separation was performed on a desmin-vimentin mixture under autoxidizing conditions in 6 M-urea to obtain the respective cross-linked collision complexes of both proteins. In 5 M-urea, the oxidation products were identified as dimers that were physically indistinguishable from uncross-linked homodimers, suggesting that they were in the form of partially denatured face-to face pairs. Heterodimers derived from intact IFs were identical to those derived from collision complexes. In the presence of 2-mercaptoethanol, heterodimers were unstable and transformed spontaneously into homodimers. After removal of urea, all cross-linked dimers were totally unable to polymerize into filaments; however, in the presence of 2-mercaptoethanol they showed a normal assembly competence. This inability of oxidized homo- and heterodimers to polymerize, together with the relatively low yield of cross-linked dimers obtained from cytoskeletons, is probably due to the introduction of steric strain into the dimers by disulfide bond formation. Substantial amounts of cross-linked heterodimers could also be isolated from IFs reconstituted from mixtures of desmin and vimentin in their homodimeric or tetrameric forms. Taken together, these results suggest that the cross-linked dimers isolated from cytoskeletons arise from a reaction between subfilament strands of IFs rather than from disulfide bond formation within pre-existing dimers and that the heterotypic IFs of BHK-21 cells are largely formed from homodimers and tetramers, respectively, rather than from heterodimers. The differential capacity of desmin and vimentin to interact with ssDNA has also been exploited to distinguish between homotypic and heterotypic protofilaments, the latter consisting of one homodimer of each protein species. This distinction could be made on the basis of characteristic differences in the sedimentation behavior of the respective protein-DNA complexes.

Dynamics of counterion-induced attraction between vimentin filaments followed in microfluidic drops.

Intermediate filaments (IFs) are fiber-forming proteins and part of the cytoskeleton of eukaryotes. In vitro the network formation of purified IF systems is mediated, for example, by the interaction with multivalent ions. The understanding of these interaction mechanisms increases the knowledge of the cytoskeleton on a fundamental level. Here, we employ time-lapse fluorescence microscopy to directly image the evolution of network formation of vimentin IFs upon addition of divalent ions. We are thus able to follow the process starting a few seconds after the first encounter of free filaments and ions up to several minutes when the networks are in equilibrium. The local protein density in the compacted networks can reach a factor of 45 higher than the original solution concentration. The competition between mono- and divalent ion condensation onto the protein explains our observations and reveals the polyelectrolyte nature of vimentin as a reason for the protein attraction in the presence of small cations. The method for time-lapse studies in microfluidic drops presented here can be generalized to other dynamic systems.

Gelatin embedding for the preparation of thermoreversible or delicate scaffolds for histological analysis.

Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples.

Structure and dynamics of human vimentin intermediate filament dimer and tetramer in explicit and implicit solvent models.

Intermediate filaments, in addition to microtubules and microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells, and play an important role in mechanotransduction as well as in providing mechanical stability to cells at large stretch. The molecular structures, mechanical and dynamical properties of the intermediate filament basic building blocks, the dimer and the tetramer, however, have remained elusive due to persistent experimental challenges owing to the large size and fibrillar geometry of this protein. We have recently reported an atomistic-level model of the human vimentin dimer and tetramer, obtained through a bottom-up approach based on structural optimization via molecular simulation based on an implicit solvent model (Qin et al. in PLoS ONE 2009 4(10):e7294, 9). Here we present extensive simulations and structural analyses of the model based on ultra large-scale atomistic-level simulations in an explicit solvent model, with system sizes exceeding 500,000 atoms and simulations carried out at 20 ns time-scales. We report a detailed comparison of the structural and dynamical behavior of this large biomolecular model with implicit and explicit solvent models. Our simulations confirm the stability of the molecular model and provide insight into the dynamical properties of the dimer and tetramer. Specifically, our simulations reveal a heterogeneous distribution of the bending stiffness along the molecular axis with the formation of rather soft and highly flexible hinge-like regions defined by non-alpha-helical linker domains. We report a comparison of Ramachandran maps and the solvent accessible surface area between implicit and explicit solvent models, and compute the persistence length of the dimer and tetramer structure of vimentin intermediate filaments for various subdomains of the protein. Our simulations provide detailed insight into the dynamical properties of the vimentin dimer and tetramer intermediate filament building blocks, which may guide the development of novel coarse-grained models of intermediate filaments, and could also help in understanding assembly mechanisms.

Filamentous biopolymers on surfaces: atomic force microscopy images compared with Brownian dynamics simulation of filament deposition.

Nanomechanical properties of filamentous biopolymers, such as the persistence length, may be determined from two-dimensional images of molecules immobilized on surfaces. For a single filament in solution, two principal adsorption scenarios are possible. Both scenarios depend primarily on the interaction strength between the filament and the support: i) For interactions in the range of the thermal energy, the filament can freely equilibrate on the surface during adsorption; ii) For interactions much stronger than the thermal energy, the filament will be captured by the surface without having equilibrated. Such a 'trapping' mechanism leads to more condensed filament images and hence to a smaller value for the apparent persistence length. To understand the capture mechanism in more detail we have performed Brownian dynamics simulations of relatively short filaments by taking the two extreme scenarios into account. We then compared these 'ideal' adsorption scenarios with observed images of immobilized vimentin intermediate filaments on different surfaces. We found a good agreement between the contours of the deposited vimentin filaments on mica ('ideal' trapping) and on glass ('ideal' equilibrated) with our simulations. Based on these data, we have developed a strategy to reliably extract the persistence length of short worm-like chain fragments or network forming filaments with unknown polymer-surface interactions.

Investigation of the morphology of intermediate filaments adsorbed to different solid supports.

Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with a width of 8-12 nm when observed by electron microscopy. Sometimes, the filaments are even unraveled on the carbon-coated grid and reveal a protofilamentous architecture. In this study, we have used atomic force microscopy to further investigate the morphology of IFs in a more physiological environment. First, we have imaged hydrated glutaraldehyde-fixed IFs adsorbed to a graphite support. In such conditions, human vimentin and desmin IFs appeared compact with a height of 5-8 nm and revealed either a beading repeat or a helical morphology. Second, we have analyzed the architecture of hydrated vimentin, desmin, and neurofilament IFs adsorbed to mica, graphite, and hydrophilic glass without the presence of fixative. On mica, vimentin IFs had a height of only 3-5 nm, whereas desmin IFs appeared as 8-10 nm height filaments with a helical twist. Neurofilaments were 10-12 nm in height with a pronounced 30-50 nm beading along their length. On graphite, the different IFs were either not adsorbing properly or their architecture was modified yielding, for example, broad, flattened filaments. Finally, hydrophilic glass was the surface which seemed to best preserve the architecture of the three IFs, even if, in some cases, unraveled vimentin filaments were observed on this support. These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished from each others by their physicochemical properties.

Molecular modeling of vimentin filament assembly.

Intermediate-filament forming proteins are known to form rod-shaped dimers that are calculated to be 45 nm in length. Molecular modeling indicates that the dimerization is promoted by interchain hydrophobic interactions between sections of alpha helix and beta helix. Further aggregation involves the formation of tetramers in which two dimers are anti-parallel and staggered to two characteristic degrees of overlap. Modeling indicated that the degrees of stagger are dictated by the association of sections of alpha helix in 4-chain bundles, in which hydrophobic side chains are sequestered from contact with water. The staggered arrangement of two dimers produces a tetramer having sections of 2-chain rod in which hydrophobic side chains are exposed to water. Extension of the tetramer to form protofilaments may be driven by associations with the 2-chain regions that reduce aqueous exposure of the hydrophobic side chains. Exposure of hydrophobic groups may be reduced by the 2-chain regions folding back upon themselves so that the entire tetramer becomes a 4-chain conformation. This prediction is in line with electron microscope data showing that mixtures of the lower oligomers contain rods of uniform thickness ranging upwards from 45 nm in a series having incremental increases in length. Data from previous chemical crosslinking studies support this model and also the idea that the completed intermediate filaments each consist of seven 4-chain protofilaments.

Transient electric birefringence study of intermediate filament formation from vimentin and glial fibrillary acidic protein.

Mg2+-induced polymerization of type III intermediate filament proteins vimentin and glial fibrillary acidic protein was studied by transient electric birefringence. In the absence of MgCl2 we found a net permanent dipole moment, approximately 45-nm-long dimers for vimentin, approximately 65-nm-long tetramers, hexamers, and possibly octamers for both proteins, and 100-nm aggregates for glial fibrillary acidic protein. Controlled oligomerization occurred after the addition of MgCl2. Although the solutions contained (small) aggregates of different sizes, more or less discrete steps in polymer formation were observed, and it was possible to discriminate between an increase in width and length. At the first stage of polymerization (in 0.3 mM MgCl2 for vimentin and 0.2 mM MgCl2 for glial fibrillary acidic protein), the permanent dipole moment disappeared without a change in length of the particles. At higher MgCl2 concentrations, structures of approximately 100 nm were formed, which strongly tended to laterally assemble into full-width intermediate filament structures consisting of about 32 monomers. This contrasts with previous models where first full-width (approximately 10-nm) aggregates are formed, which then increase in length. Subsequently, two discrete elongation steps of 35 nm are observed that increase the length to 135 and 170 nm, respectively. Possible structural models are suggested for the polymerization.

Diversity of intermediate filament structure. Evidence that the alignment of coiled-coil molecules in vimentin is different from that in keratin intermediate filaments.

Although vimentin intermediate filaments (IF) are morphologically similar to all other IF types, cells have evolved different ways of manipulating vimentin and keratin IF. The structural basis for such differences is unknown. We have explored this by use of cross-linking experiments on vimentin oligomers, polymers, and intact IF to determine the axial length of vimentin molecules and the degrees to which neighboring molecules are aligned in IF. Our data reveal that the homodimer vimentin molecule (43.9 nm) is clearly shorter than a keratin heterodimer molecule (46.2 nm). Vimentin assemblies contain three modes of antiparallel molecular alignments: A11 and A22 in two-molecule or larger oligomeric assemblies, in which the two molecules are staggered so as to bring their 1B and 2B rod domain segments, respectively, into register; and A12 in higher order molecular assemblies in which the two neighboring molecules are largely overlapped. Since the repeat axial length of the vimentin assemblies (42.6 nm) is less than the molecular length, this means there is an overlap (designated as alignment ACN) of about 1 nm (5-10 residues) between the end of the 2B and beginning of the 1A rod domain segments of similarly directed molecules in the IF. Interestingly, these four modes of nearest neighbor molecular alignments also occur in keratin IF. However, the degree of stagger of alignments in the A11 and A22 modes is different (staggers of -19.5 for vimentin versus -16.6 nm for keratin, and 23.3 and 28.6 nm, respectively). Two-dimensional surface lattice maps of the two IF types are very similar, except for differences in molecule alignments and different axial repeats of 21.4 nm in vimentin and 22.6 nm in keratin IF. Although vimentin-keratin hybrid molecules can be induced to form in vitro, they do not assemble into higher order structures. The data suggest that vimentin and keratin are incapable of assembly into IF in vitro or in vivo simply because their molecules are of different axial lengths and because the exact axial alignments of neighboring molecules are different.

Association of the beta isoform of protein kinase C with vimentin filaments.

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform.

Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin.

A number of structural and functional subnuclear compartments have been described, including regions exclusive of chromosomes previously hypothesized to form a reactive nuclear space. We have now explored this accessible nuclear space and interchromosomal nucleoplasmic domains experimentally using Xenopus vimentin engineered to contain a nuclear localization signal (NLS-vimentin). In stably transfected human cells incubated at 37 degrees C, the NLS-vimentin formed a restricted number of intranuclear speckles. At 28 degrees C, the optimal temperature for assembly of the amphibian protein, NLS-vimentin progressively extended with time out from the speckles into strictly orientated intranuclear filamentous arrays. This enabled us to observe the development of a system of interconnecting channel-like areas. Quantitative analysis based on 3-D imaging microscopy revealed that these arrays were localized almost exclusively outside of chromosome territories. During mitosis the filaments disassembled and dispersed throughout the cytoplasm, while in anaphase-telophase the vimentin was recruited back into the nucleus and reassembled into filaments at the chromosome surfaces, in distributions virtually identical to those observed in the previous interphase. The filaments also colocalized with specific nuclear RNAs, coiled bodies and PML bodies, all situated outside of chromosome territories, thereby interlinking these structures. This strongly implies that these nuclear entities coexist in the same interconnected nuclear compartment. The assembling NLS-vimentin is restricted to and can be used to delineate, at least in part, the formerly proposed reticular interchromosomal domain compartment (ICD). The properties of NLS-vimentin make it an excellent tool for performing structural and functional studies on this compartment.

Molecular parameters of type IV alpha-internexin and type IV-type III alpha-internexin-vimentin copolymer intermediate filaments.

During neuronal development, a dynamic replacement mechanism occurs in which the type VI nestin and type III vimentin intermediate filament proteins are replaced by a series of type IV proteins beginning with alpha-internexin. We have explored molecular details of how the type III to type IV replacement process may occur. First, we have demonstrated by cross-linking experiments that bacterially expressed forms of alpha-internexin and vimentin form heterodimer molecules in vitro that assemble into copolymer intermediate filaments. We show using a urea disassembly assay that alpha-internexin molecules are likely to be more stable than those of vimentin. Second, by analyses of the induced cross-links, we have determined the axial lengths of alpha-internexin homodimer and alpha-internexin-vimentin heterodimer molecules and their modes of alignments in filaments. We report that these dimensions are the same as those reported earlier for vimentin homopolymer molecules and, by implication, are also the same for the other neuronal type IV proteins. These data suggest that during neuronal development, alpha-internexin molecules are readily assimilated onto the pre-existing vimentin cytoskeletal intermediate filament network because the axial lengths and axial alignments of their molecules are the same. Furthermore, the dynamic replacement process may be driven by a positive equilibrium due to the increased stability of the alpha-internexin network.