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.     

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.     

Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with alpha-actinin may anchor synemin-containing heterofilaments.

Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with alpha-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and alpha-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim- cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity.     

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.     

Functions of intermediate filaments in neuronal development and disease

Five major types of intermediate filament (IF) proteins are expressed in mature neurons: the three neurofilament proteins (NF-L, NF-M, and NF-H), alpha-internexin, and peripherin. While the differential expression of IF genes during embryonic development suggests potential functions of these proteins in axogenesis, none of the IF gene knockout experiments in mice caused gross developmental defects of the nervous system. Yet, deficiencies in neuronal IF proteins are not completely innocuous. Substantial developmental loss of motor axons was detected in mice lacking NF-L and in double knockout NF-M;NF-H mice, supporting the view of a role for IFs in axon stabilization. Moreover, the absence of peripherin resulted in approximately 30% loss of small sensory axons. Mice lacking NF-L had a scarcity of IF structures and exhibited a severe axonal hypotrophy, causing up to 50% reduction in conduction velocity, a feature that would be very detrimental for large animal species. Unexpectedly, the NF-M rather than NF-H protein turned out to be required for proper radial growth of large myelinated axons. Studies with transgenic mice suggest that some types of IF accumulations, reminiscent of those found in amyotrophic lateral sclerosis (ALS), can have deleterious effects and even cause neurodegeneration. Additional evidence for the involvement of IFs in pathogenesis came from the recent discovery of neurofilament gene mutations linked to ALS and Charcot-Marie-Tooth disease (CMT2E). Conversely, we discuss how certain types of perikaryal neurofilament aggregates might confer protection in motor neuron disease.     

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.     

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.     

Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells

Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.     

Molecular mechanism of intermediate filament recognition by plakin proteins

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.     

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.     

Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds

The obligate intracellular bacterial pathogen Chlamydia trachomatis replicates within a large vacuole or "inclusion" that expands as bacteria multiply but is maintained as an intact organelle. Here, we report that the inclusion is encased in a scaffold of host cytoskeletal structures made up of a network of F-actin and intermediate filaments (IF) that act cooperatively to stabilize the pathogen-containing vacuole. Formation of F-actin at the inclusion was dependent on RhoA, and its disruption led to the disassembly of IFs, loss of inclusion integrity, and leakage of inclusion contents into the host cytoplasm. In addition, IF proteins were processed by the secreted chlamydial protease CPAF to form filamentous structures at the inclusion surface with altered structural properties. We propose that Chlamydia has co-opted the function of F-actin and IFs to stabilize the inclusion with a dynamic, structural scaffold while minimizing the exposure of inclusion contents to cytoplasmic innate immune-surveillance pathways.     

Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.     

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.     

Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin

As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.     

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.     

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.     

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.     

The 'ins' and 'outs' of intermediate filament organization

A major function shared by several types of cytoplasmic intermediate filaments (IFs) is to stabilize cellular architecture against the mechanical forces it is subjected to. As for other fibrous cytoskeletal arrays, a crucial determinant of this function is the spatial organization of IFs in the cytoplasm. However, very few crossbridging proteins are specific for IFs - most IF-associated proteins known to exert a structural role act to tether IFs to other major cytoskeletal elements, such as F-actin, microtubules or adhesion complexes. In addition, IFs are endowed with the ability to participate in their own organization. This intriguing property is probably connected to the unusual degree of sequence diversity and sequence-specific regulation that characterize IF genes and their proteins. This dependence upon a combination of extrinsic and intrinsic determinants contributes to distinguish IFs from other fibrous cytoskeletal polymers and is key to their function.     

An epithelium-type cytoskeleton in a glial cell: astrocytes of amphibian optic nerves contain cytokeratin filaments and are connected by desmosomes

In higher vertebrates the cytoskeleton of glial cells, notably astrocytes, is characterized (a) by masses of intermediate filaments (IFs) that contain the hallmark protein of glial differentiation, the glial filament protein (GFP); and (b) by the absence of cytokeratin IFs and IF-anchoring membrane domains of the desmosome type. Here we report that in certain amphibian species (Xenopus laevis, Rana ridibunda, and Pleurodeles waltlii) the astrocytes of the optic nerve contain a completely different type of cytoskeleton. In immunofluorescence microscopy using antibodies specific for different IF and desmosomal proteins, the astrocytes of this nerve are positive for cytokeratins and desmoplakins; by electron microscopy these reactions could be correlated to IF bundles and desmosomes. By gel electrophoresis of cytoskeletal proteins, combined with immunoblotting, we demonstrate the cytokeratinous nature of the major IF proteins of these astroglial cells, comprising at least three major cytokeratins. In this tissue we have not detected a major IF protein that could correspond to GFP. In contrast, cytokeratin IFs and desmosomes have not been detected in the glial cells of brain and spinal cord or in certain peripheral nerves, such as the sciatic nerve. These results provide an example of the formation of a cytokeratin cytoskeleton in the context of a nonepithelial differentiation program. They further show that glial differentiation and functions, commonly correlated with the formation of GFP filaments, are not necessarily dependent on GFP but can also be achieved with structures typical of epithelial differentiation; i.e., cytokeratin IFs and desmosomes. We discuss the cytoskeletal differences of glial cells in different kinds of nerves in the same animal, with special emphasis on the optic nerve of lower vertebrates as a widely studied model system of glial development and nerve regeneration.     

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.