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.     

Keratin filament suspensions show unique micromechanical properties.

All epithelial cells feature a prominent keratin intermediate filament (IF) network in their cytoplasm. Studies in transgenic mice and in patients with inherited epithelial fragility syndromes showed that a major function of keratin IFs is to provide mechanical support to epithelial cell sheets. Yet the micromechanical properties of keratin IFs themselves remain unknown. We used rheological methods to assess the properties of suspensions of epidermal type I and type II keratin IFs and of vimentin, a type III IF polymer. We find that both types of IFs form gels with properties akin to visco-elastic solids. With increasing deformation they display strain hardening and yield relatively rapidly. Remarkably, both types of gels recover their preshear properties upon cessation of the deformation. Repeated imposition of small deformations gives rise to a progressively stiffer gel for keratin but not vimentin IFs. The visco-elastic moduli of both gels show a weak dependence upon the frequency of the input shear stress and the concentration of the polymer, suggesting that both steric and nonsteric interactions between individual polymers contribute to the observed mechanical properties. In support of this, the length of individual polymers contributes only modestly to the properties of IF gels. Collectively these properties render IFs unique among cytoskeletal polymers and have strong implications for their function in vivo.     

The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions

Thread cell differentiation in the slime gland of the Pacific hagfish Eptatretus stouti has been studied using light microscopy and scanning and transmission electron microscopy. Thread cell differentiation is remarkable in that the life history of the cell is largely dedicated to the production of a single, tapered, cylindrical, highly coiled, and precisely packaged cytoplasmic thread that may attain lengths of 60 cm and diameters approaching 1.5 micron. Each tapered thread, in turn, is comprised almost entirely of large numbers of intermediate filaments (IFs) bundled in parallel. During differentiation of the thread, the IFs become progressively more tightly packed. Various numbers of microtubules (MTs) are found among the bundled IFs during differentiation of the thread but disappear during the latter stages of thread differentiation. Observations of regularly spaced dots in longitudinal bisections of developing threads, diagonal striations in tangential sections of developing threads, and circumferentially oriented, filament-like structures observed at the periphery of developing threads cut in cross section have led us to postulate a helically oriented component(s) wrapped around the periphery of the developing thread. The enormous size of the fully differentiated thread cell, its apparent singular dedication to the production of IFs, the ease of isolating and purifying the threads and IF subunits (see accompanying paper), and the unique position of the hagfish in the phylogenetic scheme of vertebrate evolution all contribute to the attractiveness of the hagfish slime gland thread cell as a potential model system for studying IF subunit synthesis, IF formation from IF subunits, aggregation of IFs into IF bundles and the interaction(s) of IFs and MTs.     

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.     

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.     

Structural changes in trichocyte keratin intermediate filaments during keratinization

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.     

Mathematical Modeling of the Impact of Actin and Keratin Filaments on?Keratinocyte Cell Spreading

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.     

Ubiquitin-proteasome-mediated degradation of keratin intermediate filaments in mechanically stimulated A549 cells

We previously reported that shear stress induces phosphorylation and disassembly of keratin intermediate filaments (IFs). Shear stress also induces a time- and strain-dependent degradation of keratin IFs, and the current study examines the mechanisms involved in degradation of keratin proteins in human A549 cells exposed to 0-24 h of shear stress (7.5-30 dynes/cm(2)). Ubiquitin was found to be covalently associated with keratin proteins immunoprecipitated from shear-stressed cells, and pretreatment with the proteasomal inhibitor MG132 prevented the degradation of the keratin IF network. Importantly, phosphorylation of K8 Ser-73 is required for the shear stress-mediated ubiquitination, disassembly, and degradation of the keratin IF network. Immunofluorescence microscopy revealed that shear stress caused the thin array of keratin fibrils observed in control cells to be reorganized into a perinuclear aggregate, known as an aggresome, and that ubiquitin was also associated with this structure. Finally, the E2 enzymes, UbcH5b, -c, and Ubc3, but not E2-25K are required for the shear stress-mediated ubiquitin-proteasomal degradation of keratin proteins. These data suggest that shear stress promotes the disassembly and degradation of the keratin IF network via phosphorylation and the ubiquitin-proteasome pathway.     

Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease.

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.     

Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.     

Contribution of intermediate filaments to cell stiffness, stiffening, and growth

It has been shown previously thatintermediate filament (IF) gels in vitro exhibit stiffening athigh-applied stress, and it was suggested that this stiffening propertyof IFs might be important for maintaining cell integrity at largedeformations (Janmey PA, Evtenever V, Traub P, and Schliwa M, JCell Biol 113: 155-160, 1991). In this study, the contribution ofIFs to cell mechanical behavior was investigated by measuring cellstiffness in response to applied stress in adherent wild-type andvimentin-deficient fibroblasts using magnetic twisting cytometry. Itwas found that vimentin-deficient cells were less stiff andexhibited less stiffening than wild-type cells, except at the lowestapplied stress (10 dyn/cm²) where the difference in thestiffness was not significant. Similar results were obtained frommeasurements on wild-type fibroblasts and endothelial cells aftervimentin IFs were disrupted by acrylamide. If, however, cells wereplated over an extended period of time (16 h), they exhibited asignificantly greater stiffness before than after acrylamide, even atthe lowest applied stress. A possible reason could be that theinitially slack IFs became fully extended due to a high degree of cellspreading and thus contributed to the transmission of mechanical stressacross the cell. Taken together, these findings were consistent withthe notion that IFs play important roles in the mechanical propertiesof the cell during large deformation. The experimental data also showedthat depleting or disrupting IFs reduced, but did not entirely abolish,cell stiffening. This residual stiffening might be attributed to theeffect of geometrical realignment of cytoskeletal filaments in thedirection of applied load. It was also found that vimentin-deficientcells exhibited a slower rate of proliferation and DNA synthesis thanwild-type cells. This could be a direct consequence of the absence ofthe intracellular IFs that may be necessary for efficient mediation ofmechanical signals within the cell. Taken together, results of thisstudy suggest that IFs play important roles in the mechanical properties of cells and in cell growth.

     

Intermediate filaments: from cell architecture to nanomechanics

Intermediate filaments (IFs) constitute a major structural element of animal cells. They build two distinct systems, one in the nucleus and one in the cytoplasm. In both cases, their major function is assumed to be that of a mechanical stress absorber and an integrating device for the entire cytoskeleton. In line with this, recent disease mutations in human IF proteins indicate that the nanomechanical properties of cell-type-specific IFs are central to the pathogenesis of diseases as diverse as muscular dystrophy and premature ageing. However, the analysis of these various diseases suggests that IFs also have an important role in cell-type-specific physiological functions.     

Three-dimensional architecture of macrofibrils in the human scalp hair cortex

Human scalp hairs are comprised of a central cortex enveloped by plate-like cuticle cells. The elongate cortex cells of mature fibres are composed primarily of macrofibrils-bundles of hard-keratin intermediate filaments (IFs) chemically cross-linked within a globular protein matrix. In wool, three cell types (ortho-, meso- and paracortex) contain macrofibrils with distinctly different filament arrangements and matrix fractions, but in human hair macrofibril-cell type relationships are less clear. Here we show that hair macrofibrils all have a similar matrix fraction (∼0.4) and are typically composed of a double-twist architecture in which a central IF is surrounded by concentric rings of tangentially-angled IFs. The defining parameter is the incremental angle increase (IF-increment) between IFs of successive rings. Unlike the wool orthocortex, hair double-twist macrofibrils have considerable inter-macrofibril variation in IF increment (0.05–0.35°/nm), and macrofibril size and IF increment are negatively correlated. Correspondingly, angular difference between central and outer-most IFs is up to 40° in small macrofibrils, but only 5–10° in large macrofibrils. Single cells were observed containing mixtures of macrofibrils with different diameters. These new observations advance our understanding of the nano-level and cell-level organisation of human hair, with implications for interpretation of structure with respect the potential roles of cortex cell types in defining the mechanical properties of hair.     

Softness, strength and self-repair in intermediate filament networks

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.     

Hierarchical Structure Controls Nanomechanical Properties of Vimentin Intermediate Filaments

Intermediate filaments (IFs), in addition to microtubules and microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells, playing a vital role in mechanotransduction and in providing mechanical stability to cells. Despite the importance of IF mechanics for cell biology and cell mechanics, the structural basis for their mechanical properties remains unknown. Specifically, our understanding of fundamental filament properties, such as the basis for their great extensibility, stiffening properties, and their exceptional mechanical resilience remains limited. This has prevented us from answering fundamental structure-function relationship questions related to the biomechanical role of intermediate filaments, which is crucial to link structure and function in the protein material''s biological context. Here we utilize an atomistic-level model of the human vimentin dimer and tetramer to study their response to mechanical tensile stress, and describe a detailed analysis of the mechanical properties and associated deformation mechanisms. We observe a transition from alpha-helices to beta-sheets with subsequent interdimer sliding under mechanical deformation, which has been inferred previously from experimental results. By upscaling our results we report, for the first time, a quantitative comparison to experimental results of IF nanomechanics, showing good agreement. Through the identification of links between structures and deformation mechanisms at distinct hierarchical levels, we show that the multi-scale structure of IFs is crucial for their characteristic mechanical properties, in particular their ability to undergo severe deformation of ≈300% strain without breaking, facilitated by a cascaded activation of a distinct deformation mechanisms operating at different levels. This process enables IFs to combine disparate properties such as mechanosensitivity, strength and deformability. Our results enable a new paradigm in studying biological and mechanical properties of IFs from an atomistic perspective, and lay the foundation to understanding how properties of individual protein molecules can have profound effects at larger length-scales.     

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.     

Immunolocalization of alpha-keratins and associated beta-proteins in lizard epidermis shows that acidic keratins mix with basic keratin-associated beta-proteins

The differentiation of the corneous layers of lizard epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against alpha-keratins and keratin associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins). Both beta-cells and alpha-cells of the corneous layer derive from the same germinal layer. An acidic type I alpha-keratin is present in basal and suprabasal layers, early differentiating clear, oberhautchen, and beta-cells. Type I keratin apparently disappears in differentiated beta- and alpha-layers of the mature corneous layers. Conversely, a basic type II alpha-keratin rich in glycine is absent or very scarce in basal and suprabasal layers and this keratin likely does not pair with type I keratin to form intermediate filaments but is weakly detected in the pre-corneous and corneous alpha-layer. Single and double labeling experiments show that in differentiating beta-cells, basic KAbetaPs are added and replace type-I keratin to form the hard beta-layer. Epidermal alpha-keratins contain scarce cysteine (0.2–1.4 %) that instead represents 4–19 % of amino acids present in KAbetaPs. Possible chemical bonds formed between alpha-keratins and KAbetaPs may derive from electrostatic interactions in addition to cross-linking through disulphide bonds. Both the high content in glycine of keratins and KAbetaPs may also contribute to increase the hydrophobicy of the beta- and alpha-layers and the resistance of the corneous layer. The increase of gly-rich KAbetaPs amount and the bonds to the framework of alpha-keratins give rise to the inflexible beta-layer while the cys-rich KAbetaPs produce a pliable alpha-layer.     

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.     

Highly extensible, tough, and elastomeric nanocomposite hydrogels from poly(ethylene glycol) and hydroxyapatite nanoparticles

Unique combinations of hard and soft components found in biological tissues have inspired researchers to design and develop synthetic nanocomposite gels and hydrogels with elastomeric properties. These elastic materials can potentially be used as synthetic mimics for diverse tissue engineering applications. Here we present a set of elastomeric nanocomposite hydrogels made from poly(ethylene glycol) (PEG) and hydroxyapatite nanoparticles (nHAp). The aqueous nanocomposite PEG-nHAp precursor solutions can be injected and then covalently cross-linked via photopolymerization. The resulting PEG-nHAp hydrogels have interconnected pore sizes ranging from 100 to 300 nm. They have higher extensibilities, fracture stresses, compressive strengths, and toughness when compared with conventional PEO hydrogels. The enhanced mechanical properties are a result of polymer nanoparticle interactions that interfere with the permanent cross-linking of PEG during photopolymerization. The effect of nHAp concentration and temperature on hydrogel swelling kinetics was evaluated under physiological conditions. An increase in nHAp concentration decreased the hydrogel saturated swelling degree. The combination of PEG and nHAp nanoparticles significantly improved the physical and chemical hydrogel properties as well as some biological characteristics such as osteoblast cell adhesion. Further development of these elastomeric materials can potentially lead to use as a matrix for drug delivery and tissue repair especially for orthopedic applications.     

Keratin function in skin epithelia: a broadening palette with surprising shades

Keratins make up the largest subgroup of intermediate filament (IF) proteins and form a dynamic network of 10-12 nm filaments, built from type I/type II heterodimers, in the cytoplasm of epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical and non-mechanical stresses that cause cell rupture and death. Interference with this role is the root cause of a large number of inherited epithelial fragility conditions. Additional functions, non-mechanical in nature, are manifested in a way that depends on the specific keratin and on the epithelial context. The recent discovery of unusual mutations affecting keratin proteins has uncovered a novel dimension of their mechanical support function, and has synergized with mouse genetics to reveal a role in skin pigmentation. Other studies extended the role of keratin proteins in regulating the response to pro-apoptotic signals, and revealed their ability to modulate protein synthesis and cell size in epithelial cells challenged to grow.