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.     

Isomin: a novel cytoplasmic intermediate filament protein from an arthropod species

Background  

The expression of intermediate filaments (IFs) is a hallmark feature of metazoan cells. IFs play a central role in cell organization and function, acting mainly as structural stress-absorbing elements. There is growing evidence to suggest that these cytoskeletal elements are also involved in the integration of signalling networks. According to their fundamental functions, IFs show a widespread phylogenetic expression, from simple diblastic animals up to mammals, and their constituent proteins share the same molecular organization in all species so far analysed. Arthropods represent a major exception in this scenario. Only lamins, the nuclear IF proteins, have so far been identified in the model organisms analysed; on this basis, it has been considered that arthropods do not express cytoplasmic IFs.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks.     

Absence of intermediate filaments in a human adrenal cortex carcinoma-derived cell line

Subclones of a human adrenal cortex carcinoma-derived cell line (SW13) are described which by immunofluorescence lack detectable expression of any of the five known classes of intermediate filament (IF) proteins. Further investigation for vimentin and keratins in these subclones by two-dimensional gel analysis and by immunoblotting gave results consistent with the immunofluorescence results. Despite the apparent absence of IFs, SW13 subclones have organized actin and microtubule cytoskeletal networks, maintain an epithelial shape and colony pattern, and grow well in culture. Although a rat hepatoma cell line which similarly appears to have ceased IF expression has been reported, this is the first such report of a human cell line. Although rare, these cases provide evidence that IFs in general are not essential to growth in culture, nor are the keratin-containing IFs in particular necessarily responsible for the 'cobblestone' morphology or colony-type growth pattern characteristic of cultured epithelial cells.     

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.     

中间纤维与细胞信号通路的研究进展

中间纤维家族由约70个中间纤维蛋白组成,在真核细胞内组成横跨核膜和胞质的网状骨架.中间纤维最初仅仅被当做是细胞骨架的一种,主要起机械支撑作用.这个观点正发生快速的改变,因为越来越多的研究发现中间纤维蛋白参与各种主要的细胞信号通路,如细胞应激、细胞凋亡和14-3-3信号通路等.     

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.     

Deletion mutagenesis of the amino-terminal head domain of vimentin reveals dispensability of large internal regions for intermediate filament assembly and stability

Previous studies have shown that the non-alpha-helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino-terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25-38, 44-95, or 40-95 polymerized into wild-type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44-69 or 25-63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25-68, 44-103, or 88-103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non-alpha-helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non-alpha-helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non-alpha-helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C-terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as-yet-unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild-type vimentin.     

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.     

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.     

Cellular integrity plus: organelle-related and protein-targeting functions of intermediate filaments

Intermediate filament proteins (IFs) maintain cell and tissue integrity, based on evidence of their polymerization and mechanical properties, abundance and disease-associated phenotypes. This 'traditional' function is now augmented by organelle-related and protein-targeting roles. Mitochondrial location and function depend on intact IFs, as demonstrated for desmin, keratins and neurofilaments. Golgi positioning is regulated by several IFs, and endosomal/lysosomal protein distribution by vimentin. IFs dramatically affect nuclear function and shape and play a role in subcellular and membrane targeting of proteins. These functions have been noted in tissues but in some cases only in cell culture. The IF-related organelle-specific and protein-targeting roles, which are likely interrelated, provide functions beyond cell scaffolding and integrity and contribute to the cytoprotective and tissue-specific functions of IF proteins.     

"Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights

Intermediate filaments (IFs) are major components of the mammalian cytoskeleton. They are among the most abundant cellular phosphoproteins; their phosphorylation typically involves multiple sites at repeat or unique motifs, preferentially within the "head" or "tail" domains. Phosphorylation and dephosphorylation are essential for the regulation of IF dynamics by modulating the intrinsic properties of IFs: solubility, conformation and filament organization, and, in addition, for the regulation of other IF post-translational modifications. These phosphorylation-regulated properties dictate generalized and context-dependent IF functions that reflect their tissue-specific expression. Most important among IF phosphorylation-mediated functions are the regulation of IF cellular or subcellular compartmentalization, levels and turnover, binding with associated proteins, susceptibility to cell stresses (including apoptosis), tissue-specific functions and IF-associated disease pathogenesis (where IF hyperphosphorylation also serves as a tissue-injury marker).     

Chasing Coiled Coils: Intermediate Filaments in Plants

The cytoskeleton's structurally most resilient components, the intermediate filaments (IFs), have attracted the interest of cell biologists for more than two decades. IFs form extensive networks in many animal cells, and are thought to provide considerable tensile strength to the cells and tissues. In fact, the term “cytoskeleton” has originally been coined for the insoluble fibrous remains of detergent extracted animal cells. Nevertheless, cells can survive quite well without an IF network, and even without the subunit proteins that build the 10 nm wide polymeric filaments. Hence, the vital function of these cytoskeletal components is still hotly debated. Against this background, it may be premature to start suggesting functions for IFs in plants. Yet this is exactly what quite a number of researchers have begun to do. Because much recent evidence supports the idea of a plant IF cytoskeleton, it seems timely to examine this evidence and discuss its impact on our current understanding of IF function.     

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.     

Identification of a repeated domain within mammalian alpha-synemin that interacts directly with talin

The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, alpha-actinin, and talin. We report herein a direct interaction of human alpha-synemin with the cytoskeletal protein talin by protein-protein interaction assays. The 312 amino acid insert (SNTIII) present only within alpha-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human alpha- and beta-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human alpha-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.     

Microsporidia Can Acquire Lamin‐like Intermediate Filaments and Cell Adhesion Catenin‐cadherin Complexes from the Host (?)

On their spore surfaces, Microsporidia often develop a canopy of filaments with characteristics of intermediate filaments (IF), as we demonstrated in previous studies on Thelohania sp., Ameson michaelis, and Spraguea lophii. Genomic studies indicate that among invertebrates, lamins that may localize in the cytoplasm or nucleus, are the only known IF type. These IFs can bind to the substrate containing cell adhesion molecules (CAMs) cadherins, associated with β and γ catenins. The objects of this study were to determine whether microsporidia have CAMs with the attached IFs on their envelopes and to find out if these proteins are provided by the host. An examination was made for localization of lamins and CAMs on the spores of the mentioned above species and Anncaliia algerae, plus in the host animals. Then, we determined whether the spores of A. michaelis and A. algerae could bind vertebrate nuclear lamin onto the spore surface. We also tested transgenic Drosophila melanogaster stocks bearing cadherin‐GFP to see whether developing A. algerae parasites in these hosts could acquire host CAMs. The tests were positive for all these experiments. We hypothesize that microsporidia are able to acquire host lamin IFs and cell adhesion catenin–cadherin complexes from the host.     

Small heat shock proteins and the cytoskeleton: An essential interplay for cell integrity?

The cytoskeleton is a highly complex network of three major intracellular filaments, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs). This network plays a key role in the control of cell shape, division, functions and interactions in animal organs and tissues. Dysregulation of the network can contribute to numerous human diseases. Although small HSPs (sHSPs) and in particular HSP27 (HSPB1) or αB-crystallin (HSPB5) display a wide range of cellular properties, they are mostly known for their ability to protect cells under stress conditions. Mutations in some sHSPs have been found to affect their ability to interact with cytoskeleton proteins, leading to IF aggregation phenotypes that mimick diseases related to disorders in IF proteins (i.e. desmin, vimentin and neuro-filaments). The aim of this review is to discuss new findings that point towards the possible involvement of IFs in the cytoprotective functions of sHSPs, both in physiological and pathological settings, including the likelihood that sHSPs such as HSPB1 may play a role during epithelial-to-mesenchymal transition (EMT) during fibrosis or cancer progression. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

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 in cardiomyopathy

Intermediate filament (IF) proteins are critical regulators in health and disease. The discovery of hundreds of mutations in IF genes and posttranslational modifications has been linked to a plethora of human diseases, including, among others, cardiomyopathies, muscular dystrophies, progeria, blistering diseases of the epidermis, and neurodegenerative diseases. The major IF proteins that have been linked to cardiomyopathies and heart failure are the muscle-specific cytoskeletal IF protein desmin and the nuclear IF protein lamin, as a subgroup of the known desminopathies and laminopathies, respectively. The studies so far, both with healthy and diseased heart, have demonstrated the importance of these IF protein networks in intracellular and intercellular integration of structure and function, mechanotransduction and gene activation, cardiomyocyte differentiation and survival, mitochondrial homeostasis, and regulation of metabolism. The high coordination of all these processes is obviously of great importance for the maintenance of proper, life-lasting, and continuous contraction of this highly organized cardiac striated muscle and consequently a healthy heart. In this review, we will cover most known information on the role of IFs in the above processes and how their deficiency or disruption leads to cardiomyopathy and heart failure.