Classes Of Intermediate Filaments Research Articles

Intermediate filaments at a glance.

Intermediate filaments (IFs) comprise a large family of versatile cytoskeletal proteins, divided into six subtypes with tissue-specific expression patterns. IFs have a wide repertoire of cellular functions, including providing structural support to cells, as well as active roles in mechanical support and signaling pathways. Consequently, defects in IFs are associated with more than 100 diseases. In this Cell Science at a Glance article, we discuss the established classes of IFs and their general features, their functions beyond structural support, and recent advances in the field. We also highlight their involvement in disease and potential use as clinical markers of pathological conditions. Finally, we provide our view on current knowledge gaps and the future directions of the IF field.

Structural Transition of Trichocyte Keratin Intermediate Filaments During Development in the Hair Follicle.

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.

Specialized roles of neurofilament proteins in synapses: Relevance to neuropsychiatric disorders.

Neurofilaments are uniquely complex among classes of intermediate filaments in being composed of four subunits (NFL, NFM, NFH and alpha-internexin in the CNS) that differ in structure, regulation, and function. Although neurofilaments have been traditionally viewed as axonal structural components, recent evidence has revealed that distinctive assemblies of neurofilament subunits are integral components of synapses, especially at postsynaptic sites. Within the synaptic compartment, the individual subunits differentially modulate neurotransmission and behavior through interactions with specific neurotransmitter receptors. These newly uncovered functions suggest that alterations of neurofilament proteins not only underlie axonopathy in various neurological disorders but also may play vital roles in cognition and neuropsychiatric diseases. Here, we review evidence that synaptic neurofilament proteins are a sizable population in the CNS and we advance the concept that changes in the levels or post-translational modification of individual NF subunits contribute to synaptic and behavioral dysfunction in certain neuropsychiatric conditions.

Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy.

Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients.

The soft framework of the cellular machine

The mechanical properties of cells are essential in determining a myriad of functions, from mitosis to locomotion. The functional rigidity of a cell is usually thought to result from three interpenetrating networks of filamentous biopolymers: actin microfilaments, microtubules, and intermediate filaments (IF). The mechanical properties of both filamentous actin (F-actin) and microtubule networks have been extensively studied, both directly in cells and in model in vitro systems, consisting of reconstituted networks of purified proteins. The assembly and structures of actin and tubulin polymers, coupled to ATP and GTP hydrolysis, respectively, give rise to fascinating dynamics that have attracted experimentalists, theorists, and modelers for decades, trying to understand their properties and functions (1, 2). In addition, the diverse molecular motors that run along these tracks are central to much of cell dynamics and vesicle transport. By contrast, intermediate filaments do not hydrolyze nucleotides, do not exhibit structural polarity, and have no motors that run along them. Also, unlike actin and tubulin, which exist in very similar forms in nearly all eukaryotic cells, IF proteins appeared later in evolution and mutated rapidly to form distinct molecular species in different cell types (3). Some classes of IFs can be genetically ablated in mice without the mice necessarily losing viability and resulting in some cases in a barely discernable phenotype (4). However, the IFs expressed in epithelial cells, keratin IFs (or KIFs), are required for normal epithelial function, and mutations in these proteins can cause devastating human diseases (5–7). In an article in a recent issue of PNAS, Sivaramakrishnan et al. (8) reported on the results of a remarkable study of the micromechanical properties of IF networks that should begin to redress our imbalance in understanding of the mechanics of the different biopolymer networks. These authors show that the KIF networks are essential for the mechanical integrity of the cell, and without them, cells such as alveolar epithelial cells would be helpless to withstand the forces they experience as the lung inflates and stretches them.

The membrane skeleton of Tetrahymena contains immunoanalogs of GFAP, the intermediate filament protein expressed in astrocytes and cells of glial origin

Summary The membrane skeleton of ciliated protozoa, called the epiplasm, is well developed and serves to anchor kinetosomes and other structures to the cell surface. Like intermediate filaments (IFs) in metazoa, epiplasmic proteins characterized so far in ciliates exhibit strong heterogeneity in number and apparent molecular weight. Only a few studies have examined the relationship between the epiplasm and IFs. We used a monoclonal antibody raised against “band C”, a major epiplasmic component in Tetrahymena pyriformis , to identify antigenically related proteins among members of the main classes of IFs in mammalian cell lines and tissues. The antibody cross-reacts with a 51 kDa protein occurring in brain, cerebellar, spinal cord and in C6 rat neuroglioma cell line. Using western blot and immunolocalization experiments, we demonstrate that the 51 kDa antigen is GFAP, the IF protein expressed in astrocytes and cells of glial origin. Immunolabelling of recombinantly-expressed, truncated GFAP constructs suggests that the cognate epitope may be localized to the central rod domain, which is highly conserved between different classes of IF proteins.

To bead or not to bead? Lens-specific intermediate filaments revisited.

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to 'mainstream' IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

Intermediate filament structure: hard α-keratin

Structurally there are four classes of intermediate filaments (IF) with distinct but closely related axial organisations. One of these, hard α-keratin IF, has been studied to clarify several apparently exceptional features which include the number of molecules in the IF cross-section and the mode by which the axial organisation of its constituent molecules is stabilised. Using the dark-field mode of the STEM at the Brookhaven National Laboratory (USA) mass measurements were obtained from unstained IF isolated from hair keratin. The data thus obtained show that the number of chains in cross-section is about 30 (±3: standard deviation) and is very similar to the numbers determined in previous STEM experiments for the dominant filament type in other classes of IF (about 32). Furthermore, re-analysis of the low-angle equatorial X-ray diffraction pattern reveals, in contrast to earlier work, solutions that are compatible with the number of chains in cross-section indicated by the STEM data. The absence of the head-to-tail overlap between parallel molecules characteristic of most IF may be compensated in hard α-keratin by a network of intermolecular disulfide bonds. It is concluded that native IF of hard α-keratin and desmin/vimentin —and probably many other kinds of IF as well— contain about 32 chains in cross-section, and that the axial structures of these various kinds of IF differ in small but significant ways, while generally observing the same basic modes of aggregation.

HTLV-I Tax and Cytokeratin: Tax-Expressing Cells Show Morphological Changes in Keratin-Containing Cytoskeletal Networks.

Human T cell leukemia virus type I (HTLV-I) has been linked to the development of an aggressive lymphoproliferative disorder (adult T cell leukemia), a chronic neurodegenerative presentation (HTLV-I-associated myelopathy/tropical spastic paraparesis) and numerous less well-defined inflammatory conditions. The viral regulatory protein Tax has been implicated in cellular transformation events leading to the onset of adult T cell leukemia. Details on the stepwise processes through which Tax induces morphological changes in cells are poorly understood. We show here that Tax can bind to a class of intermediate filaments, the cytokeratins (Ker). Tax interacts with the 1B helical coil of keratin 8, a domain critical for higher-order intermediate filament matrix formation. Expression of Tax in epithelial cells visibly altered the structural pattern of the Ker network. In a T lymphocyte cell line, induction of Tax expression resulted in increased cellular adherence/invasion of Matrigel filters. We propose that one aspect of Tax function is the induction of morphological changes in cellular cytoskeletal structures. This finding for Tax-expressing cells might be one factor contributing directly to the pathogenesis of HTLV-I disease(s). Copyright 1997 S. Karger AG, Basel

Presence of a 300-kDa Intermediate-Filament-Associated Protein (IFAP-300kDa) in Bovine Chromaffin Cells

Intermediate filaments (IFs) are cell-type-specific filaments that constitute a major part of the cellular cytoskeleton. Neurofilaments (NFs) are representative of a class of IFs which are excellent markers for neurons. NFs are also present in some cells of neural crest origin. A number of proteins have now been identified as being associated with IFs. Previously, a 300-kDa intermediate-filament-associated protein (IFAP-300kDa) was identified in baby hamster kidney cells (BHK-21). This IFAP is developmentally regulated and is not found in the adult CNS. To learn more about the expression of IFAP-300kDa, this study investigated the expression of IFAP-300kDa in neural-crest-derived chromaffin cells, bothin situandin vitro.Immunofluorescence localization of IFAP-300kDa in cryosections of bovine adrenal gland demonstrated IFAP-300kDa immunoreactivity in the dopamine-beta-hydroxylase-positive chromaffin cells of the adrenal medulla. When rounded chromaffin cells in culture were examined, double-label immunofluorescence microscopy revealed an IFAP-300kDa/NF-L-positive juxtanuclear aggregate. The plasma membrane was also IFAP-300kDa positive, but NF-L immunoreactivity was lacking. In cells which have spread under the influence of NGF, slender IFAP-300kDa-positive immunofluorescent strands were frequently seen radiating from a juxtanuclear area of immunoreactivity. Double-labeling revealed these filaments and juxtanuclear area to also be positive for NF-L immunoreactivity. The presence of IFAP-300kDain situandin vitrowas further confirmed by immunoblot analysis. This study is the first demonstration of an IFAP in neuron-like cells located outside the central nervous system. In addition, these data indicate that the developmentally regulated IFAP-300kDa may persist in some cells of neural crest origin.

Continued expression of neuronal traits in chromaffin cells of the adult bovine adrenal gland

Intermediate filaments (IF) are a major constituent of the cytoskeleton of most eukaryotic cells.While there are five major classes of IF, amino acid sequence and secondary structure homologies suggest that all IFs evolved from a single precursor gene early in the history of life. Characteristically, IF are 10 nm filaments whose distribution, in most cases, is tissue-type specific. For example, the IFs of muscle cells contain the muscle-specific IF protein desmin, and those of epithelial cells contain subunits of the keratin family. Neurofilaments (NF) are a class of IFs expressed in most neuronal cells and are composed of three polypeptide subunits with apparent molecular weights of 68,000 (NF-L), 150,000 (NF-M) and 200,000 (NF-H). Interestingly, NFs are present in chromaffin cells cultured from bovine adrenal medulla. Chromaffin cells are derived from the neural crest, and the presence of NFs confirms their relatedness to neurons. However, the presence of NFs in vivo has been the subject of debate.

Neurofilament-like immunoreactivity in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa).

The neuronal cytoskeleton contains neurofilament proteins that serve as markers for nervous tissue in many species across phyla. Antiserum generated to mammalian neurofilaments was used for immunocytochemical staining of tissues in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa). Specific staining, visible at the light and electron microscope levels, was found in the tissues of the tentacle. Proteins were extracted from the tissues and solubilized. SDS-polyacrylamide gel electrophoresis and Western blotting revealed two bands of MWr 156 kD and 74 kD that reacted with antiserum generated to neurofilaments. The protein bands also bound a monoclonal antibody shown to react with a highly conserved epitope in many classes of intermediate filaments. These data suggest that the neurons of this anthozoan contain neurofilament-like proteins with molecular properties similar to those of mammalian neurons.

Simultaneous labelling of microtubules and fibrillar bundles in tobacco BY-2 cells by the anti-intermediate filament antibody, ME 101

In previous studies on plant cells, antibodies directed against intermediate filaments (IFs) have shown that IF antigens are distributed in one of two quite distinct forms. The first co-distributes with each of the four microtubule arrays (cortical, preprophase band, spindle and phragmoplast), while the second form is associated with cytoplasmic paracrystalline fibrillar bundles (FBs) of 10 nm filaments. Conditions allowing one form to be labelled with antibody have generally proved unsuitable for labelling of the other; this has prevented the simultaneous visualization of the two forms of IF antigen in plants and the study of any possible physical relationships between them at the electron microscopic level. In this paper, we show that ME 101, which recognizes an epitope in the N-terminal portion of all classes of intermediate filaments, stains both forms of plant IF antigen simultaneously in tobacco suspension cells using immunofluorescence or immunogold labelling techniques. These cells contain in their cortex short (ca. l μm) fibrillar bundles which stain with ME 101. These bundles appear to be independent of the microtubule-associated epitope which stains in a continuous linear manner with ME 101. When protoplasts are either cleaved open on grids or sequentially extracted with detergents prior to critical point drying, the short fibrillar bundles are specifically labelled by ME 101 tagged with colloidal gold. ME 101 also co-distributed with underlying linear filaments, which appeared to be microtubules. In addition to these structures, the cortex also contains a meshwork of variably-sized fine filaments but these are not labelled with ME 101 nor with an antibody raised against the plant cytoskeleton, which recognizes cytokeratin 8. These results confirm that the fibrillar bundles and the microtubule-associated form of plant IF antigens are present simultaneously rather than experimentally-interconvertible, and that they appear to be physically unconnected.

Expression of the intermediate filament‐associated protein related to β‐amyloid precursor protein is developmentally regulated in cultured cells

It was previously reported that a monoclonal antibody to beta-amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament-associated protein (beta APP-IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60-67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord-dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. beta APP-IFAP was expressed in all non-neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), beta APP-IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that beta APP-IFAP may be the product of a member of the beta APP multigene family expressed developmentally in non-neuronal cells.

Primary Mesenchymal Tumors of the Urinary Bladder: A Histological and Immunohistochemical Study of 30 Cases

The light microscopic and immunohistochemical features of 30 primary mesenchymal neoplasms of the urinary bladder are reported. Half of the cases represented smooth and striated muscle tumors (five leiomyomas, seven leiomyosarcomas including epithelioid and myxoid subtypes, one rhabdomyoma, one embryonal rhabdomyosarcoma and one alveolar rhabdomyosarcoma). One third of the tumors were of fibrohistiocytic origin (one fibrous histiocytoma and eight malignant fibrous histiocytomas including fascicular and storiform, inflammatory and pleomorphic subtypes). In addition, a malignant epithelioid schwannoma, a round cell liposarcoma, two hemangiomas and two mixed mesodermal tumors were observed. The morphology of the vesical mesenchymal tumors was identical to that of their counterparts known to occur in other sites, particularly in the soft tissue. Muscle-specific actin, alpha-1-antichymotrypsin, S-100-protein and neuron-specific enolase proved to be useful and reliable immunomarkers for differential diagnosis of poorly differentiated leio- and rhabdomyosarcomas, malignant fibrous histiocytomas and malignant schwannomas. Since some tumors coexpressed several classes of intermediate filaments, diagnostic immunocytochemistry should only be used considering a larger panel of antibodies and in close correlation with the histological and cytological features of the neoplasms.

Intermediate filaments in oral neoplasia. I. Oral cancer and epithelial dysplasia.

The major value of intermediate filaments (IFs) in biological and applied research lies in their high order of cell and tissue specificity. This is particularly well illustrated in keratin (K) expression in various oral epithelia. Although the original class of IF is usually conserved in tissues after neoplastic transformation, epithelia show a tendency to shift their pattern of keratin expression in a manner which, while not predictable with precision, may sometimes be of diagnostic or prognostic significance. This review compares the keratins in normal oral epithelia, which show a mainly site-dependent expression, with those in squamous cell carcinoma. Key changes in the latter are the presence of simple epithelial keratins, K8 and K18 (occasional K7), reduced expression of differentiation-linked keratins (K1, K10, K4 and K13) and a tendency for down-regulation of primary keratins, K5 and K14. Moderate and severe dysplasias also tend to exhibit K8 and K18 with concomitant disordered expression of differentiation-linked keratins. There are reports of similar changes after neoplastic transformation in other mucosal sites and skin. Before this information can be applied diagnostically in immunocytochemical studies, the anti-keratin antibodies must be fully characterised and their interaction with the relevant tissue, both frozen and conventionally processed, should be evaluated.

Novel eosinophilic intracytoplasmic inclusions in a meningioma

Two forms of inclusion, extracellular and intracytoplasmic, have been reported in meningiomas. The authors recently encountered a case of meningothelial meningioma with unusual round eosinophilic intracytoplasmic inclusions. Ultrastructural and immunohistochemical studies were performed on the surgically removed tissue. Ultrastructurally, the inclusions were found in the cytoplasm and processes of the tumor cells. Each inclusion consisted of a dense central mass of filaments with a looser, less dense periphery of radiating filaments. At the periphery, crystalloid lamellar structures and granules were intermingled frequently. No limiting membrane was observed. Immunohistochemically, the inclusions were unreactive for five classes of intermediate filaments, actin, ubiquitin, and amyloid. The intracytoplasmic inclusions were ultrastructurally distinct from those reported previously. The molecular aspect of these novel eosinophilic intracytoplasmic inclusions remained uncertain.

A cell surface antigen expressed by astrocytes and their precursors

Increasing evidence suggests that astrocytes of the mammalian CNS are a heterogeneous population of cells that express a number of common characteristics. In most cases, astrocytes or their precursors contain a class of intermediate filaments, composed in large part of glial fibrillary acidic protein (GFAP). While the expression of GFAP immunoreactivity is a specific characteristic of astrocytes in the vertebrate CNS, not all astrocytes contain detectable levels of glial filaments, particularly during early development. We have isolated a monoclonal antibody termed 7B11 which binds to the surface of astrocytes and glial precursors, but not to other major types of neural cells. The 7B11 antigen is expressed by astrocytes in the adult CNS in vivo and in cultures of developing CNS tissue, but not on cells of the peripheral nervous system. During early development, 7B11 immunoreactivity appears prior to the expression of GFAP and is retained as punctate staining on the surface of most classes of astrocytes. During CNS maturation, however, 7B11 immunoreactivity is lost from the surface of Bergmann glia in the cerebellum, suggesting that differentiative events lead to functionally and antigenically distinct classes of CNS glial cells. In the adult spinal cord, biochemical analysis suggests that the epitope recognized by 7B11 is associated with a group of polypeptides of apparent molecular weights 200-160, 140, and 92 kD. The cellular distribution of 7B11 expression suggests that astrocytes and their precursors share a distinct cell surface antigenic property and that the expression of 7B11 immunoreactivity may be a useful operational marker for astrocytes in the absence of detectable GFAP expression.

Expression of Intermediate Filaments in Choromas: An Immunocytochemical Study of Five Cases

An immunocytochemical investigation has been carried out on five cases of chordoma (2 of the sacrum, 2 in the spheno-occipital bone and 1 in the parapharyngeal area) to study the expression of the 5 classes of intermediate filaments (IF): cytokeratin (CK), desmin (DES), GFAP, neurofilaments (NF) and vimentin (VIM). Our results show that constant coexpression of CK, NF and VIM does occur in chordomas, whereas DES and GFAP are not demonstrable in tumor cells. The three detected IF are invariably present in all cell types but not in intracellular vacuoles or in the extracellular mucoid substance. The pattern of immunoreactivity of chordomas appears very unique as very few other neoplasms demonstrate the simultaneous occurrence of 3 distinct IF. Only choroid plexus tumors have been shown to manifest CK-NF-VIM immunoreactivity. The complex immunophenotype of chordomas may be related to their supposed origin from the notochord which normally undergoes conspicuous changes in location and morphology during embryonal development. Such changes might require the contemporary presence of multiple IF; IF expression, in fact, is known to be related to cell function and morphology. Notochordal cells and their neoplastic counterpart may consequently express an IF pattern which reflects unique architectural and morphological variations occurring during embryonal and tumor growth. Together with the speculative value of the detection of CK, NF and VIM in chordomas, the unusual immunocytochemical pattern of these tumors might provide useful diagnostic tool in differential diagnosis.

Cytoskeletal Filament Typing of Human Corneal Endothelial Cells

Flat mounts of human corneal endothelial cells (HCECs) were examined immunohistochemically by using a wide assortment of monoclonal antibodies against the five classes of intermediate filaments (IFs) and actin and myosin. HCECs showed uniform immunostaining with monoclonal antibodies against the 40-kD (CK 19) and 45-kD (CK 18) cytokeratin (CK). Only part of the endothelial cells reacted with monoclonal antibodies against the 52-kD (CK 8) and 54-kD (CK 7) cytokeratin polypeptides and with monoclonal antibodies against vimentin. Monoclonal antibodies against the low- and middle-molecular-mass neurofilament proteins produced positive staining of all HCECs. No positivity was obtained with antibodies against desmin or glial fibrillary acidic protein. In addition, positive immunostaining with monoclonal antibodies against actin and slow myosin demonstrate that these proteins form part of the cytoskeleton of HCECs. The results of this study show that immunostaining of flat cell preparations is very useful for studies on HCECs. HCECs display an unusual combination of cytokeratin IFs and neurofilaments, together with vimentin, and are heterogeneous with respect to their IF makeup. These findings are discussed in relation to the presumed origin of HCECs.