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.     

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.     

Plasticity of Intermediate Filament Subunits

Intermediate filaments (IFs) assembled in vitro from recombinantly expressed proteins have a diameter of 8–12 nm and can reach several micrometers in length. IFs assemble from a soluble pool of subunits, tetramers in the case of vimentin. Upon salt addition, the subunits form first unit length filaments (ULFs) within seconds and then assembly proceeds further by end-to-end fusion of ULFs and short filaments. So far, IF subunits have mainly been observed by electron microscopy of glycerol sprayed and rotary metal shadowed specimens. Due to the shear forces during spraying the IF subunits appear generally as straight thin rods. In this study, we used atomic force microscopy (AFM), cryo-electron microscopy (cryo-EM) combined with molecular modeling to investigate the conformation of the subunits of vimentin, desmin and keratin K5/K14 IFs in various conditions. Due to their anisotropic shape the subunits are difficult to image at high resolution by cryo-EM. In order to enhance contrast we used a cryo-negative staining approach. The subunits were clearly identified as thin, slightly curved rods. However the staining agent also forced the subunits to aggregate into two-dimensional networks of dot-like structures. To test this conformational change further, we imaged dried unfixed subunits on mica by AFM revealing a mixture of extended and dot-like conformations. The use of divalent ions such as calcium and magnesium, as well as glutaraldehyde exposure favored compact conformations over elongated ones. These experimental results as well as coarse-grained molecular dynamics simulations of a vimentin tetramer highlight the plasticity of IF subunits.     

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.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

Characterization of the in vitro co-assembly process of the intermediate filament proteins vimentin and desmin: mixed polymers at all stages of assembly

We have investigated the co-assembly properties of the intermediate filament (IF) proteins vimentin and desmin. First, the soluble complexes formed by both proteins separately in 5 mM Tris-HCl, pH 8.4, were characterized by analytical ultracentrifugation. In both cases, s-values of around 5 S were obtained corresponding to the formation of tetramers. However, at pH 7.5 and in the presence of 1 mM EDTA, both proteins behaved quite differently; whereas vimentin sedimented at 7.2 S, desmin assembled into much larger complexes of about 13 S. A mixture of equimolar amounts of vimentin and desmin in 8 M urea yielded, after reconstitution into 5 mM Tris-HCl, pH 7.5, and 1 mM EDTA, complexes exhibiting a sharp peak at 10.9 S. This intermediate s-value indicated that co-assembly into a distinct new set of complexes had occurred. As judged by electron microscopy and viscometry, these mixtures assembled into IFs with characteristics similar to those of pure vimentin and desmin. Furthermore, when vimentin and desmin tetramers were mixed in 5 mM Tris-HCl, pH 8.4, and subsequently subjected to IF assembly conditions, again "hybrid" filaments were obtained. Most interestingly, after 10 min of assembly, mass-per-length (MPL) measurements by scanning transmission electron microscopy yielded IFs with an MPL-peak value of 36 +/- 5 kDa/nm, hence closer to that of vimentin IFs (33 +/- 4 kDa/nm) than to that of desmin IFs (48 +/- 8 kDa/nm). Finally, when unit length-filaments (ULF) of vimentin and desmin were mixed and assembled further, the diameters of individual mature IFs formed exhibited a significantly higher degree of width inhomogeneity along their length than vimentin and desmin IFs as might be expected for a modular mode of assembly. Last but not least, atomic force microscopy provided further direct evidence that desmin IFs are able to fuse end-to-end with vimentin IFs. In summary, we have shown that vimentin and desmin are able to co-assemble at the dimer, tetramer, ULF and even the mature IF level.     

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.     

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.     

Deconstructing the late phase of vimentin assembly by total internal reflection fluorescence microscopy (TIRFM)

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several μm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 μm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The 'patchy' structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments.     

Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens- epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host- specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.     

Direct Observation of Subunit Exchange along Mature Vimentin Intermediate Filaments

Actin filaments, microtubules, and intermediate filaments (IFs) are central elements of the metazoan cytoskeleton. At the molecular level, the assembly mechanism for actin filaments and microtubules is fundamentally different from that of IFs. The former two types of filaments assemble from globular proteins. By contrast, IFs assemble from tetrameric complexes of extended, half-staggered, and antiparallel oriented coiled-coils. These tetramers laterally associate into unit-length filaments; subsequent longitudinal annealing of unit-length filaments yields mature IFs. In vitro, IFs form open structures without a fixed number of tetramers per cross-section along the filament. Therefore, a central question for the structural biology of IFs is whether individual subunits can dissociate from assembled filaments and rebind at other sites. Using the fluorescently labeled IF-protein vimentin for assembly, we directly observe and quantitatively determine subunit exchange events between filaments as well as with soluble vimentin pools. Thereby we demonstrate that the cross-sectional polymorphism of donor and acceptor filaments plays an important role. We propose that in segments of donor filaments with more than the standard 32 molecules per cross-section, subunits are not as tightly bound and are predisposed to be released from the filament.     

The tektin family of microtubule-stabilizing proteins

Tektins are insoluble alpha-helical proteins essential for the construction of cilia and flagella and are found throughout the eukaryotes apart from higher plants. Being almost universal but still fairly free to mutate, their coding sequences have proved useful for estimating the evolutionary relationships between closely related species. Their protein molecular structure, typically consisting of four coiled-coil rod segments connected by linkers, resembles that of intermediate filament (IF) proteins and lamins. Tektins assemble into continuous rods 2 nm in diameter that are probably equivalent to subfilaments of the 10 nm diameter IFs. Tektin and IF rod sequences both have a repeating pattern of charged amino acids superimposed on the seven-amino-acid hydrophobic pattern of coiled-coil proteins. The length of the repeat segment matches that of tubulin subunits, suggesting that tektins and tubulins may have coevolved, and that lamins and IFs may have emerged later as modified forms of tektin. Unlike IFs, tektin sequences include one copy of a conserved peptide of nine amino acids that may bind tubulin. The 2 nm filaments associate closely with tubulin in doublet and triplet microtubules of axonemes and centrioles, respectively, and help to stabilize these structures. Their supply restricts the assembled lengths of cilia and flagella. In doublet microtubules, the 2 nm filaments may also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes.     

Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly

Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF 'building block' is an elongated coiled-coil dimer consisting of four consecutive alpha-helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4-2.3 A resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic alpha-helix, which is compatible with a coiled-coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer-dimer interactions during IF assembly. The 2B segment reveals a double-stranded coiled coil, which unwinds near residue Phe351 to accommodate a 'stutter'. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level.     

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.     

Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.     

Recent insight into intermediate filament structure

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal ‘head’ and C-terminal ‘tail’ domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans.     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

Characterization of early assembly intermediates of recombinant human keratins

The intermediate filaments (IFs) form major structural elements of the cytoskeleton. In vitro analyses of these fibrous proteins reveal very different assembly properties for the nuclear and cytoplasmic IF proteins. However, keratins in particular, the largest and most heterogenous group of cytoplasmic IF proteins, have been difficult to analyze due to their rapid assembly dynamics under the near-physiological conditions used for other IF proteins. We show here that keratins, like other cytoplasmic IF proteins, go through a stage of assembling into full-width soluble complexes, i.e., "unit-length filaments" (ULFs). In contrast to other IF proteins, however, longitudinal annealing of keratin ULFs into long filaments quasi-coincides with their formation. In vitro assembly of IF proteins into filaments can be initiated by an increase of the ionic strength and/or lowering of the pH of the assembly buffer. We now document that 23-mer peptides from the head domains of various IF proteins can induce filament formation even under conditions of low salt and high pH. This suggests that the "heads" are involved in the formation and longitudinal association of the ULFs. Using a Tris-buffering protocol that causes formation of soluble oligomers at pH 9, the epidermal keratins K5/14 form less regular filaments and less efficiently than the simple epithelial keratins K8/18. In sodium phosphate buffers (pH 7.5), however, K5/14 were able to form long partially unraveled filaments which compacted into extended, regular filaments upon addition of 20 mM KCl. Applying the same assembly regimen to mutant K14 R125H demonstrated that mutations causing a severe disease phenotype and morphological filament abnormalities can form long, regular filaments with surprising efficiency in vitro.     

Probing of the structural stability of vimentin and desmin-type intermediate filaments with Ca2+-activated proteinase, thrombin and lysine-specific endoproteinase Lys-C

Intermediate filaments (IFs) reconstituted from purified, delipidated vimentin and desmin as well as respective protofilaments were subjected to degradation by Ca2+-activated neutral thiol proteinase, thrombin and lysine-specific endoproteinase Lys-C, respectively. The breakdown products were analyzed by SDS-polyacrylamide gel electrophoresis and negative stain electron microscopy. While Ca2+-activated proteinase and thrombin caused rapid and complete degradation of IFs with kinetics not significantly different from those of the degradation of protofilaments, lysine-specific endoproteinase did not exert any electron microscopically detectable effect on filament structure. Although both types of subunit proteins were truncated at their non-alpha-helical, C-terminal polypeptides by this proteinase, they were still able to assemble into 10 nm filaments. Closer electron microscopic inspection of IFs treated with Ca2+-activated proteinase revealed numerous ruptures along the filaments already at very early stages of digestion. SDS-polyacrylamide gel electrophoresis of the processed filaments in conjunction with previous biochemical characterizations of the breakdown of protofilaments by Ca2+-activated proteinase showed that these inhomogeneities primarily arose from degradation of the arginine-rich, non-alpha-helical N-termini of the filament proteins. These findings demonstrate that, although the N-terminus of vimentin and desmin is essential for filament stability, it is still highly susceptible to proteolytic attack in particular and very likely to posttranslational modification in general. Such structural modifications of the N-termini of IF proteins might exert great influences on the intracellular distribution and molecular organization of IFs in various physiological and pathological conditions.     

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.