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PubMed1996

Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.

Animals↗
PubMed1981

IgM autoantibody to intermediate filaments in infectious mononucleosis.

Sera from 17 patients with infectious mononucleosis reacted with fine cytoplasmic fibrils in monolayer cultures of fibroblasts. In cells pre-treated with vinblastine, the sera reacted with peri- or juxta-nuclear coils of filaments. These staining patterns were similar to those obtained by anti-intermediate filament autoantibody but different from the "actin-cables" stained by anti-actin autoantibody and from the "tubulin paracrystals" stained by anti-microtubule autoantibody. The autoantibodies belonged to the IgM class and ranged in titre from 1 : 16 to 1:512 (mean 1:64). A change in antibody titre was noted in sequential acute and convalescent sera. The results show that IgM autoantibodies to intermediate filaments occur frequently in sera from patients with infectious mononucleosis.

Actins↗
PubMed1993

Keratin intermediate filament structure. Crosslinking studies yield quantitative information on molecular dimensions and mechanism of assembly.

One of the major obstacles to solving the full three-dimensional structure of keratin intermediate filaments (KIF) is the determination of the exact mode(s) of alignment of nearest-neighbor molecules; this in turn requires precise information of the lengths of the non-alpha-helical linker segments within the coiled-coil alpha-helical heterodimer molecule. In this study, we have induced lysine-lysine and cysteine-cysteine crosslinks between keratin intermediate filament molecules in small assembly-competent oligomers, isolated them and then characterized the natures and locations of the crosslinks. Of more than 100 found, 21 quantitatively major crosslinks were used to obtain the relative axial alignments of rod domain segments by least-squares fitting methods. Three dominant modes of alignment were found. In each case the molecules are antiparallel with the first involving molecules in approximate register (stagger = -0.2 nm), the second involving molecules staggered so as to bring the 1B segments into approximate alignment (stagger = -16.1 nm), and the third involving molecules staggered so as to bring the 2B segments into approximate alignment (stagger = 28.2 nm). In addition, the data enable quantitative estimates to be made for the first time of the lengths of the non-coiled-coil segments (L1 = 2.5 nm, L12 = 1.6 nm, L2 = 0.8 nm), and the total length of the rod domain (46.0 nm). Alignment of molecules according to these parameters permits construction of a two-dimensional surface lattice which displays a 1.6 nm (10 or 11 residue) overlap between similarly directed molecules. Together, the data predict six important overlapping sequence regions that recur about 16 times per 46 nm of filament length. Interestingly, synthetic peptides corresponding to these sequences, singly or in combination, significantly interfere with keratin filament structural integrity. These results thus represent the most significant set of structural constraints for KIF yet available and provide insights into how disease-causing mutations disrupt filaments and their organization in cells.

Amino Acid Sequence↗
PubMed1995

Remodeling of the specialized intermediate filament network in mammalian eggs and embryos during development: regulation by protein kinase C and protein kinase M.

The sheets serve as an maternal supply of assembled, cytokeratin, intermediate filaments. They are remodeled at each major developmental transition in mammalian early development, that is fertilization, embryonic compaction, blastocyst formation, and formation of the primitive ectoderm and primitive endoderm during implantation into the uterine wall. Our results indicate that the sheets exist as specialization for placental development as they have a major role in the maintenance of epithelial integrity at the time the embryo is implanting into the uterine wall. They also contribute intermediate filaments to the junctional complexes required for embryonic compaction. Our analyses demonstrate the they are regulated at the time of fertilization by the action of PKC/PKM, a kinase that acts as a cellular chronometer with both temporal and spatial precision that remodels the egg into the zygote.

Animals↗
PubMed1990

Expression of intermediate filaments in neuroendocrine tumors.

Expression of 160-kilodalton (kd) neurofilament, cytokeratin, and vimentin was immunohistochemically investigated in 45 pheochromocytomas, 12 medullary thyroid carcinomas, and 12 pancreatic islet cell tumors. Four mixed neuroendocrine-neural tumors, two multiple endocrine neoplasia type I tumors, and two multiple endocrine neoplasia type II tumors were included in this study. Neurofilament immunoreactivity was demonstrated in 45 (100%) of the pheochromocytomas, 10 (83%) of the medullary thyroid carcinomas, and 11 (92%) of the islet cell tumors. Cytokeratin and vimentin were detectable in 29% and 24% of the pheochromocytomas, 100% and 25% of the medullary thyroid carcinomas, and 67% and 25% of the islet cell tumors, respectively. Neurofilament was the most frequently detected intermediate filament in the neuroendocrine tumors. Cytokeratin was seen in the intra-adrenal but not the extra-adrenal pheochromocytomas. Coexpression of neurofilament and cytokeratin was common in the neuroendocrine tumors, and occasionally vimentin was also expressed simultaneously. There were no characteristic differences in the expression of intermediate filaments between multiple endocrine neoplasia type I or II tumors and sporadic-type tumors or between metastatic tumors and nonmetastatic tumors.

Adenoma, Islet Cell↗
PubMed1984

Antibodies to different intermediate filaments as histogenetic tumormarkers.

Fifty primary carcinomas, nineteen nonmuscular sarcomas, ten rhabdomyosarcomas, one malignant ependymoblastoma and seventeen sympathetic derived tumors have been studied to determine what type of intermediate filaments they express, using affinity purified antibodies to prekeratin, vimentin, desmin, glial fibrillary acidic protein (GFA) and to the different neurofilament proteins. The tissue was ethanol fixed and paraffin embedded before use. In all carcinoma cases the tumor cells are stained by antibodies to prekeratin, whereas tumor cells in nonmuscular sarcomas could only be labeled by antibodies to vimentin. In the cases of rhabdomyosarcomas tumor cells are clearly decorated by antibodies to desmin. The one case of malignant ependymoblastoma was positive when tested with antibodies directed against glial fibrillary acidic protein (GFA). While most of the sympathetic derived tumors react positively with sera directed against the different neurofilament proteins, three neuroblastomas did not react with sera against any of the known intermediate filament types. Wider use of such sera would seem particularly useful in cases such as neuroblastomas, undifferentiated carcinomas, lymphomas and rhabdomyosarcomas.

Antibodies, Neoplasm↗
PubMed2004

Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism.

Cytoskeletons in Sertoli cell play an important role in process of spermatogenesis. The expression and distribution of the intermediate filaments, vimentin, keratin and desmin, were studied in the Sertoli cells of the cryptorchid testis of rhesus monkey. Vimentin was localized in the perinuclear region of Sertoli cells of the normal testis. An intense increase in vimentin immunoreactivity was observed with appearance of disorganized staining in the Sertoli cells of the cryptorchid testes. Cytokeratin 18, a marker of immature Sertoli cells, re-expressed in the cells of the adult cryptorchid testes. Desmin was also observed in the Sertoli cells in addition to the peritubular myoid cells on 30 days after the cryptorchid operation. These data suggest that Sertoli cells in primate can be affected by the heat stress. The altered changes in intermediate filaments could be possible to induce the Sertoli cell functional changes that would partially contribute to the germ cell apoptosis leading to azoospermia or oligozoospermia.

Animals↗
PubMedDate not supplied

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1.

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.

Amino Acid Motifs↗
PubMed1998

Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (vimentin) scaffold.

The temperature-sensitive mutant tsp53val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (39 degrees C), but is rapidly transported into the cell nucleus at the permissive temperature (30 degrees C). tsp53 thus may serve as a model for analysing cellular parameters influencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells differing in their expression of the intermediate filament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate filament deficient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate filament system.

Actins↗
PubMed1989

Developmental expression of intermediate filament proteins in the chick embryo retina: in vivo and in vitro comparison.

An immunocytochemical study was performed on the chick embryo retina to detect the changes in intermediate filament (IF) composition in correlation with development. Results showed that all three major neurofilament subunits (NF L, M and H) were expressed from very immature neurons at the same time. No delayed expression of NF-H was observed. Vimentin (VT) was also present from very early stages in both neuronal and glial cells, but it was not detectable at hatching. Its distribution changed in retinal layers throughout development. It co-existed with NF in neurons, and conversely, it preceded the appearance of the typical glial fibrillar acidic protein (GFA-P), which was detected in radial fibres of Müller cells from day 16 of incubation. These in vivo data were compared with immunocytochemical staining of retinal cultures, grown either on collagen or on polylysine. NF-L and GFA-P were not detectable in vitro. Moreover, the early expression of NF subunits was shown to be influenced by the type of substratum. The contemporary presence of vimentin and neurofilaments was clearly shown both in neuronal cell bodies and processes. The results also show that in the chick retina the pattern of composition of intermediate filaments changes with maturation. Moreover, in vitro conditions influence the expression of IF components. These data are of interest when performing in vitro analysis of the effect of exogenous substances affecting neuronal maturation and differentiation.

Animals↗
PubMed1986

Intermediate filament proteins in classic and variant types of small cell lung carcinoma cell lines: a biochemical and immunochemical analysis using a panel of monoclonal and polyclonal antibodies.

The intermediate filament protein (IFP) characteristics of a panel of lung cancer cell lines including adenocarcinoma (two cell lines) and small cell lung cancer (SCLC, three classic and three variant cell lines) were examined using one- and two-dimensional gel electrophoretic techniques, immunocytochemical techniques and immunoblotting assays. A panel of 28 monoclonal and polyclonal antibodies to the five different types of IFP were used. The results of our studies indicate that these human lung adenocarcinoma, classic SCLC and variant SCLC cell lines can be differentiated on the basis of their pattern of IFP. The main conclusions from this study can be summarized as follows. The two adenocarcinoma cell lines contain cytokeratins 7, 8, 18, and sometimes 19, next to vimentin intermediate filament (IF). The three classic-type SCLC cell lines contain only cytokeratin IFs but not vimentin IF or neurofilaments (NFs). Cytokeratin polypeptides 7, 8, 18 and 19 could be detected. All three variant-type SCLC cell lines do not contain detectable amounts of cytokeratins. In contrast, two out of three variant SCLC cell lines contain neurofilament proteins. All three variant-type SCLC cell lines contain vimentin IF. Using immunoblotting assays with monoclonal and polyclonal antibodies to defined NF proteins the presence of the 68 X 10(3) Mr and the 160 X 10(3) Mr NF polypeptide could be demonstrated in two variant SCLC cell lines. As patients with SCLC-variant phenotype have a poorer prognosis after cytotoxic therapy than patients with 'pure' SCLC, the use of antibodies to IFP in staining fresh lung tumours, especially anaplastic ones, may differentiate the two subtypes of SCLC. Such a distinction would have a major impact on therapy selections and may be of prognostic importance.

Antibodies, Monoclonal↗
PubMed1985

Immunofluorescence microscopic evaluation of the intermediate filament expression of the adrenal cortex and medulla and their tumors.

Normal adrenal glands (10 specimens) and adrenal gland tumors (58 cases) were immunohistochemically evaluated for different types of intermediate filament (IF) proteins. Some of the normal cortical cells showed cytokeratin positivity, and no positivity was seen for epidermal keratin or other types of IF. In the adrenal medulla, neurofilament positivity was seen in nerve axons, some ganglion cells, and chromaffin cells; and cytokeratin-positive cells could not be detected. Only the vascular and connective tissue elements showed vimentin positivity in both cortical and medullary areas. In half of the cortical carcinomas (13/25), cytokeratin-positive tumor cells were found. Furthermore, vimentin-positive tumor cells were present in 10 of 25 cases, in some of them together with cytokeratin-positive cells. Thus, the results show heterogeneity among the adrenal cortical carcinomas. Interestingly, many benign adrenal cortical tissues and some carcinomas lacked immunoreactivity for all types of IF, suggesting a poorly developed IF system in these tissues. In contrast to adrenal cortical tumors, pheochromocytomas contained neurofilamentlike immunoreactivity. These results reflect the different cellular nature of adrenal cortical and medullary tumors, which apparently can be distinguished from each other with antibodies to intermediate filament proteins.

Adenoma↗
PubMedDate not supplied

Cytoskeletal identification of intermediate filaments in the inner ear of the jerker mouse mutant.

The expression of the five main groups of intermediate filaments (IF) and their subgroups, especially cytokeratins, was analysed in cryosections of the labyrinth of the jerker mouse mutant and compared with normal CBA/CBA controls. Fourteen different well characterized monoclonal antibodies were used. In principle the same pattern of IF was found in the two mouse strains. IF were not found in hair cells of the mouse inner ear, which may reflect a disparity as compared with human fetal hair cells. Chain-specific (epitope-specific) differences were visualized in cytokeratin no. 8 with regard to cells in epithelia involved in the homeostasis of inner ear fluids. Differences in immuno-staining occurred between adjacent cells in the same epithelium, for instance in the semicircular canals.

Animals↗
PubMedDate not supplied

Mucoepidermoid carcinoma of the salivary glands: immunohistochemical distribution of intermediate filament proteins, involucrin and secretory proteins.

Mucoepidermoid carcinomas of the salivary gland comprising low (n = 6), intermediate (n = 11) and high grade malignant (n = 11) tumors were evaluated for immunohistochemical reactivity of cytokeratin (monoclonal antibody KL1, PKK1, K8.12), vimentin, involucrin and secretory proteins (lysozyme, LY; lactoferrin, LA; alpha 1-antitrypsin, alpha 1-AT and alpha 1-antichymotrypsin, alpha 1-Ach). Keratin expression was usually confined to intense staining in epidermoid tumor cells, but was negative in almost all mucous cells. Vimentin was coexpressed with keratin only in epidermoid tumor cells. Great heterogeneity of intermediate filament proteins was found in intermediate and epidermoid tumor cells. Involucrin in epidermoid tumor cells was particularly abundant in well keratinized cells but was lacking in intermediate and mucous forming cells. The frequency of occurrence of positive staining for LY, LA, alpha 1-AT and alpha 1-Ach was relatively low in mucoepidermoid carcinoma with positive immunohistochemical staining for these secretory proteins in mucous forming tumor cells, while varying expression was observed in epidermoid tumor cells.

Antibodies, Monoclonal↗
PubMed1989

[Circulating anticellular antibodies in male patients: coincidence of antibodies to intermediate filaments and HLA-B27 in Reiter syndrome].

Evaluating 57 male patients with anticellular antibodies (detected on HEp-2 cells) we could find an association of Reiter's syndrome (17/57), IgG antibodies to intermediate filaments (vimentin type) of the cytoskeleton (17/17) and HLA-B27 (13/17). We conclude, that both, genetic and hormonal factors may influence the humoral immune response in males with Reiter's syndrome. IgG antibodies to intermediate filaments might be precious additional markers for diagnosis of Reiter's syndrome in males.

Arthritis, Reactive↗
PubMed1994

IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of intermediate filaments to these junctions.

The distribution of IFAP 300, a protein previously characterized as cross-linking vimentin intermediate filaments (IF), has been investigated in epithelial cells. In frozen sections of bovine tongue epithelium the staining obtained with IFAP 300 antibodies is concentrated in the peripheral cytoplasm of keratinocytes, including the entire peripheral region of basal cells. Further immunofluorescence studies reveal that in primary cultures of mouse keratinocytes the distribution of IFAP 300 is similar to that of the desmosomal protein desmoplakin. In rat bladder carcinoma 804G cells the staining pattern of IFAP 300 antibodies coincides with that obtained with antibodies against the hemidesmosomal protein BP 230. By immunogold electron microscopy IFAP 300 is mainly located at sites where IF appear to attach to desmosomes and hemidesmosomes. Morphometric analyses of the distribution of the gold particles show that IFAP 300 overlaps with desmoplakin and BP 230, but also that it extends deeper into the cytoplasm than these latter two proteins. The staining reaction seen in epithelial cells by immunofluorescence and immunogold is specific for IFAP 300 as shown by immunoblotting. Immunoblotting also reveals that IFAP 300 is present in both cell-free preparations of desmosomes and hemidesmosomes. These morphological and biochemical results are intriguing since, in recent years, the proteins appearing in these two types of junctions have been found to be different. One possible exception is plectin, a protein that has been suggested to be very similar to IFAP 300. However, we show here that IFAP 300 differs from plectin in several respects, including differences at the primary sequence level. We also show that purified IFAP 300 pellets with in vitro polymerized IF prepared from desmosome-associated keratins under conditions in which IFAP 300 alone is not sedimentable. This indicates that IFAP 300 can associate with keratin IF. These data, taken together with the immunogold results, suggest that IFAP 300 functions in epithelial cells as a linker protein connecting IF to desmosomes as well as to hemidesmosomes, possibly through structurally related proteins such as desmoplakin and BP 230, respectively.

Amino Acid Sequence↗
PubMed2007

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.

Animals↗
PubMed2000

Improved ultrastructure of the desmosome-intermediate filament complex in MCF-7 breast cancer cells.

We report on the application of variations to the traditional TEM processing methods, which provide improved clarity of the desmosome-cytoskeleton complex of MCF-7 human breast cancer cells. A more comprehensive understanding of the ultrastructure is presented, which in the past has been demonstrated in diagrammatic form based on numerous electron micrographs. Ultrastructural analysis shows that intermediate filament bundles do not terminate at the desmosome structure, but instead are continuous into the cytoplasm. Furthermore only a minor proportion of individual filaments are in actual contact with the desmosome plaque. Intermediate filaments were also observed throughout the cytoplasm and to the surface of the nuclear membrane. Extraction protocols allowed clear identification of other cellular features such as nuclear pores, which are approximately 80-85 nm in diameter, and were best viewed in sections cut tangentially to the nuclear surface.

Breast Neoplasms↗