Intermediate filaments in muscle and epithelial cells of nematodes

Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.     

Novel filamentous bundles in the cytoplasm of a unicellular eukaryote,Crithidia fasciculata

Summary Trypanosomes, an evolutionarily ancient group of unicellular eukaryotic parasites, appear to lack both microfilaments (actin) and intermediate filaments (IFs): the major cytoskeletal component common to all trypanosomes consists of a stable microtubular array intimately associated with the plasma membrane. We present here evidence of bundles of trans-cytoplasmic filaments ca. 10 nm in diameter, seen by transmission electron microscopy, that are formed in stationary cultures of an insect trypanosome,Crithidia fasciculata. Immunofluorescent labelling with an antibody raised against plant fibrillar bundles (AFB) and Western blotting with an antibody that cross-reacts with a broad range of IFs (anti-IFA) as well as with fibrillar bundles, indicates that these filaments appear to share antigenic determinants common to animal IFs and to fibrillar bundles of plant origin.Abbreviations AFB anti-fibrillar bundle antibody - anti-IFA anti-intermediate filament antibody - IF intermediate filament - SEM scanning electron microscope - TEM transmission electron microscope - YOL 1/34 anti--tubulin antibody     

Modeling the self-organization property of keratin intermediate filaments

Keratin intermediate filaments (IFs) fulfill an important function of structural support in epithelial cells. The necessary mechanical attributes require that IFs be organized into a crosslinked network and accordingly, keratin IFs are typically organized into large bundles in surface epithelia. For IFs comprised of keratins 5 and 14 (K5, K14), found in basal keratinocytes of epidermis, bundling can be self-driven through interactions between K14's carboxy-terminal tail domain and two regions in the central α-helical rod domain of K5. Here, we exploit theoretical principles and computational modeling to investigate how such cis-acting determinants best promote IF crosslinking. We develop a simple model where keratin IFs are treated as rigid rods to apply Brownian dynamics simulation. Our findings suggest that long-range interactions between IFs are required to initiate the formation of bundlelike configurations, while tail domain-mediated binding events act to stabilize them. Our model explains the differences observed in the mechanical properties of wild-type versus disease-causing, defective IF networks. This effort extends the notion that the structural support function of keratin IFs necessitates a combination of intrinsic and extrinsic determinants, and makes specific predictions about the mechanisms involved in the formation of crosslinked keratin networks in vivo.     

Biomechanical properties of intermediate filaments: from tissues to single filaments and back

The animal cell cytoskeleton consists of three interconnected filament systems: actin-containing microfilaments (MFs), microtubules (MTs), and the lesser known intermediate filaments (IFs). All IF proteins share a common tripartite domain structure and the ability to assemble into 8-12 nm wide filaments. Electron microscopy data suggest that IFs are built according to a completely different plan from that of MFs and MTs. IFs are known to impart mechanical stability to cells and tissues but, until recently, the biomechanical properties of single IFs were unknown. However, with the discovery of naturally occurring micrometer-wide IF bundles and the development of new methodologies to mechanically probe single filaments, it is now possible to propose a more unified view of IF biomechanics. Unlike MFs and MTs, single IFs can now be described as flexible, extensible and tough, which has important implications for our understanding of cell and tissue mechanics. Furthermore, the molecular mechanisms at play when IFs are deformed point toward a pivotal role for them in mechanotransduction.     

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.     

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.     

Assembly and structure of calcium-induced thick vimentin filaments.

Using a viscometric assay and various electron microscopic procedures (negative staining, rotary shadowing, ultrathin sectioning) we have determined the influences of different kinds of ions and of ionic strength on the structures formed by assembly of soluble subunits of vimentin from bovine lens tissue or from Escherichia coli transformed with Xenopus vimentin cDNA. In contrast to the assembly of typical, i.e., 8 to 14-nm, intermediate-sized filaments (IFs) at elevated (e.g., 160 mM) concentrations of monovalent cations and at millimolar Mg2+ concentrations, filaments formed in the presence of Ca2+ ions (e.g., 5 mM) appeared at a lower rate, attained lower viscosity and were considerably thicker and shorter. The largest diameter measured was that for the recombinant amphibian protein: 24.2 +/- 8.5 nm in negative staining, 28.7 +/- 5.6 nm in sections. These thick Ca(2+)-induced filaments, however, revealed the same approximately 2 nm protofilament composition and approximately 20 nm cross-striation pattern as typical IFs, indicative of a similar molecular arrangement. The significance of this unusual structural IF protein assembly is discussed.     

Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics

The assembly of intermediate filament (IF) arrays involves the recruitment of a complex set of cell-type-specific IF-associated proteins. Some of them are integral membrane proteins, others act as crosslinking proteins with vectorial binding activities, and yet others comprise motor proteins. In vivo IFs appear to be predominantly heteropolymers, although in vitro several IF proteins (e.g. vimentin, desmin, neurofilament (NF)-L and the nuclear lamins) do self-assemble into IF-like polymers. In contrast, NF-M, NF-H, nestin, synemin and paranemin, all bona fide IF proteins, are unable to self-assemble into IFs either in vitro or in vivo. The individual IF proteins of this large multigene family are chemically heterogeneous, exhibiting different assembly kinetics and yielding discrete types of filaments. The unique physical properties and interaction capabilities of these distinct IF molecular building blocks, in combination with accessory proteins, mediate the generation of a highly dynamic and interconnected, cell-type-specific cytoarchitecture.     

Purification of the 300K intermediate filament-associated protein and its in vitro recombination with intermediate filaments

IFAP-300K is a 300,000-mol-wt intermediate filament-associated protein previously identified in the baby hamster kidney fibroblastic cell line (BHK-21) by a monoclonal antibody (Yang H.-Y., N. Lieska, A. E. Goldman, and R. D. Goldman, 1985, J. Cell Biol., 100: 620-631). In the present study, this molecule was purified from the high salt/detergent-insoluble cytoskeletal preparation of these cells. Gel filtration on Sephacryl S-400 in the presence of 7.2 M urea allowed separation of the high molecular weight fraction from the structural intermediate filament (IF) subunits desmin and vimentin, designated 54K and 55K, respectively, and other low molecular weight polypeptides. DE-52 cellulose chromatography of the high molecular weight fraction using a linear NaCl gradient in 8 M urea yielded a pure 300,000-mol-wt species which was confirmed to be IFAP-300K by immunological and peptide mapping criteria. Two-dimensional PAGE of native BHK IF preparations followed by immunoblot analysis demonstrated the inability of the IFAP-300K-immunoreactive material to enter the first dimensional gel except as a 200,000-mol-wt doublet which presumably represented a major proteolytic derivative of IFAP-300K. The molecule's pl of 5.35, as determined by chromatofocusing, and its amino acid composition were extremely similar to those of BHK cell vimentin/desmin despite their non-identity. Ultrastructurally, IFAP-300K preparations in low salt buffers existed as particles composed of one or two elliptical units measuring 16 X 20 nm. In physiological salt buffers, the predominant entities were large, elongated aggregates of the elliptical units, which were able to be decorated by using the immunogold technique with monoclonal anti-IFAP-300K. Compared with the morphology of homopolymer vimentin IF, in vitro recombination studies using column-purified vimentin and IFAP-300K demonstrated the additional presence of aggregates similar in appearance to IFAP-300K at points of contact between IFs. Antibody decoration and immunogold labeling of these recombined preparations using rabbit antidesmin/vimentin and monoclonal anti-IFAP-300K confirmed the identity of the inter-filament, amorphous material as IFAP-300K. The presence of IFAP-300K at many points of intersection and lateral contact between IFs, as well as at apparent inter-filament "bridges," in these recombined specimens was identical to that seen both in situ and in native IF preparations. No such co-sedimentation was found in vitro between actin and IFAP-300K. No effects of IFAP-300K upon the kinetics of IF polymerization were detected by turbidimetric measurements.     

Molecular architecture of intermediate filaments

Together with microtubules and actin microfilaments, approximately 11 nm wide intermediate filaments (IFs) constitute the integrated, dynamic filament network present in the cytoplasm of metazoan cells. This network is critically involved in division, motility and other cellular processes. While the structures of microtubules and microfilaments are known in atomic detail, IF architecture is presently much less understood. The elementary 'building block' of IFs is a highly elongated, rod-like dimer based on an alpha-helical coiled-coil structure. Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs). Subsequently ULFs start to anneal longitudinally, ultimately yielding mature IFs after a compaction step. For nuclear lamins, however, assembly starts with a head-to-tail association of dimers. Recently, X-ray crystallographic data were obtained for several fragments of the vimentin dimer. Based on the dimer structure, molecular models of the tetramer and the entire filament are now a possibility.     

Morphological analysis of glutaraldehyde-fixed vimentin intermediate filaments and assembly-intermediates by atomic force microscopy

Atomic force microscopy (AFM) was used to study the morphology of vimentin intermediate filaments (IFs) and their assembly intermediates. At each time after initiation of IF assembly in vitro of recombinant mouse vimentin, the sample was fixed with 0.1% glutaraldehyde and then applied to AFM analysis. When mature vimentin IFs were imaged in air on mica, they appeared to have a width of approximately 28 nm, a height of approximately 4 nm and a length of several micrometers. Taking into account the probe tip's distortion effect, the exact width was evaluated to be approximately 25 nm, suggesting that the filaments flatten on the substrate rather than be cylindrical with a diameter of approximately 10 nm. Vimentin IFs in air clearly demonstrated approximately 21-nm repeating patterns along the filament axis. The three-dimensional profiles of vimentin IFs indicated that the characteristic patterns were presented by repeating segments with a convex surface. The repeating patterns close to 21 nm were also observed by AFM analysis in a physiological solution condition, suggesting that the segments along the filaments are an intrinsic substructure of vimentin IFs. In the course of IF assembly, assembly intermediates were analyzed in air. Many short filaments with a full-width and an apparent length of approximately 78 nm (evaluated length approximately 69 nm) were observed immediately after initiation of the assembly reaction. Interestingly, the short full-width filaments appeared to be composed of the four segments. Further incubation enabled the short full-width filaments to anneal longitudinally into longer filaments with a distinct elongation step of approximately 40 nm, which corresponds to the length of the two segments. To explain these observations, we propose a vimentin IF formation model in which vimentin dimers are supercoiling around the filament axis.     

The mechanical properties of hydrated intermediate filaments: insights from hagfish slime threads

Intermediate filaments (IFs) impart mechanical integrity to cells, yet IF mechanics are poorly understood. It is assumed that IFs in cells are as stiff as hard alpha-keratin, F-actin, and microtubules, but the high bending flexibility of IFs and the low stiffness of soft alpha-keratins suggest that hydrated IFs may be quite soft. To test this hypothesis, we measured the tensile mechanics of the keratin-like threads from hagfish slime, which are an ideal model for exploring the mechanics of IF bundles and IFs because they consist of tightly packed and aligned IFs. Tensile tests suggest that hydrated IF bundles possess low initial stiffness (E(i) = 6.4 MPa) and remarkable elasticity (up to strains of 0.34), which we attribute to soft elastomeric IF protein terminal domains in series with stiffer coiled coils. The high tensile strength (180 MPa) and toughness (130 MJ/m(3)) of IF bundles support the notion that IFs lend mechanical integrity to cells. Their long-range elasticity suggests that IFs may also allow cells to recover from large deformations. X-ray diffraction and congo-red staining indicate that post-yield deformation leads to an irreversible alpha-->beta conformational transition in IFs, which leads to plastic deformation, and may be used by cells as a mechanosensory cue.     

Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium.

Intermediate filaments (IF) isolated from the oesophagus epithelium of the snail Helix pomatia contain two polypeptides of mol. wt 66,000 (A) and 52,000 (B), which we have now characterized by in vitro self-assembly studies and by protein sequences. A and B can each form morphologically normal IF and share extended regions of sequence identity. All A-specific sequences seem to locate to an extension of the carboxyl-terminal domain. Although the Helix protein(s) reveal the IF-consensus sequences at the ends of the coiled-coil, the remainder of the rod domain shows conservation of sequence principles rather than extended homology, when compared with any subtype of vertebrate IF proteins. Interestingly, the Helix proteins have the longer coil 1b domain found in nuclear lamins and not in cytoplasmic IF proteins of vertebrates. They lack, however, the karyophilic signal sequence typical for lamins. Obvious implications for IF evolution and structure are discussed.     

Assessing the flexibility of intermediate filaments by atomic force microscopy

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.     

Arrangement of trichokeratin intermediate filaments and matrix in the cortex of Merino wool

Tomograms of transverse sections of Merino wool fibers obtained from fleeces differing in fiber curvature were reconstructed from image series collected using a 300 kV transmission electron microscope. Trichokeratin intermediate filaments (IFs) from the ortho-, para- and mesocortices were modeled from the tomograms. IFs were predominantly arranged in left-handed concentric helices with the relative angle of IFs increasing progressively from the center to the periphery of orthocortex macrofibrils. The median increase in IF angle between adjacent IFs between the center and periphery was 2.5°. The length of one turn of the helical path of an IF was calculated to be approximately 1 μm for an IF tilted at 30° and positioned 100 nm from the macrofibril center. With the exception of one paracortex macrofibril that weakly resembled an orthocortex macrofibril, all para- and mesocortex macrofibrils modeled had a parallel arrangement of the IFs, with a more ordered arrangement found in the mesocortex. Within the limited sample set, there appeared to be no significant relationship between IF angle and fiber curvature. We examined the matrix/IF ratio (in the form of proportion of matrix to one IF, calculated from IF center-to-center distance and IF diameter) for 28 macrofibrils used for modeling. The proportion of matrix was significantly different in the different cortex cell types, with paracortex having the most (0.61), orthocortex having the least (0.42), and mesocortex being intermediate (0.54). Fibers of different crimp type (high, medium or low crimp) were not significantly different from each other with respect to matrix proportion.     

Exploring the mechanical behavior of single intermediate filaments

Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs. Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers in vivo.     

Influence of phospholipids on the formation and stability of vimentin-type intermediate filaments

The interaction of vesicles produced from individual phospholipids and mixtures thereof with preformed vimentin filaments as well as the influence of these vesicles on filament assembly were investigated employing negative stain electron microscopy and sucrose density gradient equilibrium centrifugation. Liposomes with a phospholipid composition characteristic of Ehrlich ascites tumor cells were able to bind efficiently to vimentin filaments without significantly affecting their morphology at higher concentrations. However, in sucrose density gradient centrifugation partial disintegration of the filaments was observed. In addition, larger quantities of phospholipid mixture totally blocked intermediate filament (IF) formation. Using vesicles of individual phospholipids, these effects could be shown to be due to the presence of negatively charged lipid species in the phospholipid mixture. While these were highly active in preventing filament assembly and in dissociating preformed filaments, electrically uncharged phospholipids were virtually inactive. The highest efficiency was shown by phosphatidylinositol-4,5-diphosphate. These results demonstrate that a negative surface charge of liposomes is an essential prerequisite for their successful and tight association with vimentin filaments. However, the high susceptibility of these filaments to photoaffinity labeling with the membrane-penetrating reagent 1-azidopyrene in the presence of phospholipid vesicles, points to additional interactions between hydrophobic regions of both reactants. Finally, the data also suggest a direct relationship between IFs and the lipid bilayer as the active principle underlying the association of IFs with natural membranes as observed by electron and immunofluorescence microscopy.     

Dual roles of intermediate filaments in apoptosis

New roles have emerged recently for intermediate filaments (IFs), namely in modulating cell adhesion and growth, and providing resistance to various forms of stress and to apoptosis. In this context, we first summarize findings on the IF association with the cell response to mechanical stress and growth stimulation, in light of growth-related signaling events that are relevant to death-receptor engagement. We then address the molecular mechanisms by which IFs can provide cell resistance to apoptosis initiated by death-receptor stimulation and to necrosis triggered by excessive oxidative stress. In the same way, we examine IF involvement, along with cytolinker participation, in sequential caspase-mediated protein cleavages that are part of the overall cell death execution, particularly those that generate new functional IF protein fragments and uncover neoantigen markers. Finally, we report on the usefulness of these markers as diagnostic tools for disease-related aspects of apoptosis in humans. Clearly, the data accumulated in recent years provide new and significant insights into the multiple functions of IFs, particularly their dual roles in cell response to apoptotic insults.     

A multi-scale approach to understand the mechanobiology of intermediate filaments

The animal cell cytoskeleton consists of three interconnected filament systems: actin microfilaments, microtubules and the lesser known intermediate filaments (IFs). All mature IF proteins share a common tripartite domain structure and the ability to assemble into 8–12 nm wide filaments. At the time of their discovery in the 1980s, IFs were only considered as passive elements of the cytoskeleton mainly involved in maintaining the mechanical integrity of tissues. Since then, our knowledge of IFs structure, assembly plan and functions has improved dramatically. Especially, single IFs show a unique combination of extensibility, flexibility and toughness that is a direct consequence of their unique assembly plan. In this review we will first discuss the mechanical design of IFs by combining the experimental data with recent multi-scale modeling results. Then we will discuss how mechanical forces may interact with IFs in vivo both directly and through the activation of other proteins such as kinases.