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PubMed1992

Assembly of a tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo.

Recent reports on the possible contribution of the non-alpha-helical carboxy-terminal domain ("tail") of type III intermediate filament (IF) proteins to IF assembly have been controversial. To examine the importance and role of this domain, we have therefore engineered a Xenopus laevis vimentin cDNA to code for a tail-less polypeptide and have used it in combination with prokaryotic and eukaryotic expression systems. Here we show that tail-less vimentin, isolated from transfected bacteria (Escherichia coli), when used for assembly in vitro, forms normal-looking, loosely packed IFs. By viscometry we demonstrate that this tail-less vimentin assembles at an even higher rate and into longer IFs than wild-type vimentin. In vivo, i.e., by forced expression in transfected type III IF-free cultured epithelial cells, tail-less vimentin was also recovered in short fibrillar structures, in rodlets and in small as well as large spheroidal aggregates ("granules") that did not reveal any IF substructure. Surprisingly, however, spheroidal aggregate structures formed from the tail-deleted vimentin, were seen not only in the cytoplasm but also in the nucleus, indicating a role of the tail in higher order organization and compartmentalization of the vimentin IF system.

Animals↗
PubMed1998

Analysis of eight cDNAs and six genes for intermediate filament (IF) proteins in the cephalochordate Branchiostoma reveals differences in the IF multigene families of lower chordates and the vertebrates.

We report the sequences of seven new cytoplasmic intermediate filament (IF) proteins of the cephalochordate Branchiostoma. The eight sequences currently known describe four subfamilies (A, B, C and D). All eight IF proteins show the short-length version of the coil 1b subdomain found in vertebrates and lack the additional 42 residues present in all nuclear lamins and the protostomic IF proteins. Although the lancelet is considered to be the closest relative of the vertebrates, it is difficult to relate its IF subfamilies unambiguously to a particular type I-IV subfamily of vertebrates. C1 and C2 have tail domains with two 64 residue repeats of coiled coil-forming ability, a structural feature unknown for IF proteins from vertebrates or protostomia. The epidermal protein D1 shows only a slightly better identity score with vertebrate type II keratins than with type III proteins, but the D1 gene organization is that of type III proteins. The same holds for A1, A2, B1, B2 and C2 genes, although the latter has an additional and uniquely positioned intron. Antibodies (Ab) raised against recombinant C2 and D1 proteins reveal these proteins in epidermis, some internal epithelia and parts of the spinal cord. The results on exonic sequences, gene organization and expression suggest that Branchiostoma IF proteins may retain a largely archetypal condition, whereas the vertebrates have established the well-known type I-IV IF system.

Amino Acid Sequence↗
PubMed1990

Alpha-internexin, a novel neuronal intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain.

alpha-Internexin is a 66 kDa protein that copurifies with intermediate filaments (IF) from rat spinal cord and optic nerve. This protein is axonally transported in rat optic nerve along with the neurofilament triplet proteins in slow component a. Polymerization in vitro and distribution in vivo confirm that alpha-internexin is a neuronal IF. We raised 2 highly specific monoclonal antibodies to alpha-internexin which were applied to frozen rat brain sections and Western blots of cytoskeletal extracts. These results indicate that alpha-internexin is primarily an axonal protein found in most, if not all, neurons of the CNS. Immunoreactive proteins of similar molecular weight were found in cytoskeletal extracts of CNS tissue from several additional species, including mouse and cow. While the distribution of alpha-internexin as given by immunocytochemical methods is similar to that of low molecular weight neurofilament protein (NF-L) in the adult, its distribution in the embryo is far more extensive. At embryonic day 16, when the expression of NF-L is still limited to a relatively small number of cells and levels of expression are low, alpha-internexin is already found at much higher levels and in cells not yet expressing NF-L in detectable quantities. Similar results are found at embryonic day 12. These data suggest that neuronal IF in the developing nervous system contain a higher proportion of alpha-internexin than their adult counterparts, and that expression of alpha-internexin precedes that of NF-L in many or most neurons of the developing brain.

Aging↗
PubMed1997

Xefiltin, a Xenopus laevis neuronal intermediate filament protein, is expressed in actively growing optic axons during development and regeneration.

Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as alpha-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, alpha-internexin and gefiltin. alpha-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow.

Animals↗
PubMed1989

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure.

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Animals↗
PubMed1989

Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin.

We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible "neoexpression" during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.

Antibodies, Monoclonal↗
PubMed2000

Intermediate filament protein synemin is transiently expressed in a subset of astrocytes during development.

Synemin, a developmentally regulated protein first described in muscle cells, has recently been recognized as an intermediate filament (IF) protein. Because IF proteins are invaluable markers of cell origin within the nervous system, we were interested in determining the expression pattern of synemin in the brain. Our results show that, during development of the rat cortex, synemin is expressed only in a subpopulation of astrocytic cells expressing GFAP as well as vimentin and nestin. Unlike GFAP, however, synemin is not expressed in mature astrocytes and, unlike vimentin and nestin, synemin is not present in astrocytic precursors before GFAP expression. Taken together with morphological evidence, the time course of synemin expression, as determined by Western blotting, suggests that synemin is expressed in radial glial cells undergoing morphological transformation into astrocytes. Studies of synemin expression in vitro demonstrate that, early in primary culture, the majority of polygonal astrocytes are derived from synemin(+) radial glial cells. With time in culture, however, polygonal astrocytes either stop expressing synemin or are overgrown by cells not expressing synemin. The unique pattern of synemin expression, both in vivo and in vitro, suggests that the use of synemin as a marker will add a new dimension to studies of astrocytic differentiation.

Animals↗
PubMed1994

Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks.

Desmoplakins (DPs) are the most abundant proteins in the innermost portion of the desmosomal plaque and have been proposed to play a role in the attachment of intermediate filaments (IF) to cell-cell contact sites. Our previous results suggest that the globular end domains of DP perform dual functions: first, to target DP to the desmosome via the NH2 terminus and second, to attach IF to the desmosomal plaque via the COOH terminus. When ectopically expressed in most cultured cells, the COOH terminus plus the rod domain (DP. delta N.SerC23) exhibits striking coalignment with keratin IF networks. However, in certain cell types (e.g. PtK2) or in cells treated with forskolin to activate protein kinase A, DP. delta N.SerC23 exhibits a diffuse cytoplasmic distribution. A variant molecule (DP. delta N.GlyC23) in which a serine located 23 amino acids from the COOH terminus is altered to a glycine, thereby disrupting a protein kinase A consensus phosphorylation site, co-localizes with keratin IF networks regardless of cell type or forskolin treatment. Analysis of the phosphopeptide maps of these DP variants and endogenous DP is consistent with the phosphorylation of the serine 23 residues from the COOH terminus. These results suggest that phosphorylation of a specific residue in the DP COOH terminus may negatively regulate its interaction with keratin IF networks.

Amino Acid Sequence↗
PubMed1998

Association of S100B with intermediate filaments and microtubules in glial cells.

Previous in vitro studies have shown that the Ca2+-regulated S100B protein modulates the assembly-disassembly of microtubules (MTs) and type III intermediate filaments (IFs). In the present report, by double immunofluorescence cytochemistry S 100B was localized to both GFAP/vimentin IFs and MTs as well as to centrosomes in U251 glial cells. In cells treated with the MT-depolymerizing agent, colchicine, S100B remained associated with the rearranged GFAP IFs throughout the cell and, at the cell periphery, vimentin IFs. In cells treated with the MT stabilizing agent, taxol, S100B followed partly the rearrangement of MTs and partly the rearrangement of IFs. Under the latter condition, bundles of MTs with their associated S100B appeared surrounded and/or flanked by rearranged IFs with their associated S100B. Colocalization of S100B with closely arranged IFs and MTs was best evident in cells manipulated with taxol and in triton-cytoskeletons. In these cases, MTs and their associated S100B appeared surrounded and/or flanked by and/or intermingled with IFs and their associated S100B. Also, a preferential association of S100B with GFAP vs. vimentin IFs could be observed near the nucleus where colocalization of S100B with MTs was also maximal. Condensation of IFs and alteration of the MT network caused by treatment of cells with the phosphatase inhibitor, okadaic acid, resulted in a concomitant condensation/alteration of the S100B immunoreactivity. The present results lend support to the possibility that S100B may be an important factor implicated in the regulation of the dynamics of MTs and IFs.

Calcium-Binding Proteins↗
PubMed1992

The role of intermediate filaments in early Xenopus development studied by antisense depletion of maternal mRNA.

The effects of depleting a maternal cytokeratin mRNA on the developing embryo are described. Cytokeratins are members of the intermediate filament family of cytoskeletal proteins, and are expressed in a cortical network of the superficial cytoplasm of the oocyte. After fertilisation, a new cortical network is built up, which comes to occupy only the most superficial cells of the blastula. The maternal cytokeratin mRNA is abundantly translated, both during oogenesis, and during oocyte maturation and after fertilisation. Depletion of the mRNA results in depletion of the cortical filaments at the blastula stage and leads to gastrulation abnormalities. We discuss the various possible control experiments required for antisense oligo depletion studies and the implications of these results for cytokeratin function.

Animals↗
PubMed1994

Effects of 6-dimethylaminopurine on the length of the cell cycle and on the state of phosphorylation of putative intermediate filament proteins in sea urchin embryos.

The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 microM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase. To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 microM 6-DMAP and with 200 microCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly.

Adenine↗
PubMed1989

Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.

We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.

Animals↗
PubMed1995

A role for intermediate filaments in the establishment of the primitive epithelia during mammalian embryogenesis.

Investigations of the cytoskeleton in mammalian eggs and embryos have revealed the existence of an unusual array of crosslinked intermediate filaments composed of cytokeratins 5, 6, 16, and 'Z' that are referred to as cytoskeletal sheets. We have been investigating the function of these cytoskeletal sheets during embryogenesis. In this investigation we report the rapid appearance of extensive arrays of tonofilaments extending across blastomeres and in association with intercellular desmosomal junctions appearing at the time the embryo hatches from its zona pellucida, through the time of implantation of the embryo into the uterine wall. Just prior to the time of gastrulation these tonofilaments disappear. Electron microscopy and immunoconfocal microscopy demonstrate that the tonofilaments are composed of cytokeratins characteristic of the type found earlier in development, that is types 5 and 6; whereas, cytokeratin type 8 which has been shown to be synthesized in blastocysts is localized primarily at perinuclear regions. Cytokeratins 8 and 18 are synthesized to about the same extent as actin at the time the tonofilaments appear whereas the synthesis of cytokeratins 5 and 6 is greatly reduced. Our results suggest that cytokeratins 5 and 6 in the tonofilaments may arise from the stored form of cytokeratins in the cytoskeletal sheets. Consequently, our results suggest that the sheets may serve as a maternal reserve of cytokeratin employed by the embryo at the time of implantation to form extensive arrays of tonofilaments in the embryo that likely provide structural integrity to the embryo as it is subjected to mechanical stress during invasion and implantation into the uterine wall.

Animals↗
PubMed2005

The three-dimensional structure of trichocyte (hard alpha-) keratin intermediate filaments: features of the molecular packing deduced from the sites of induced crosslinks.

The spatial distribution of the crosslinks that can be induced between lysine residues in trichocyte (alpha-) keratin intermediate filaments (IF) using disulfosuccinimidyl tartrate has been analyzed in detail and the results used to provide information about the three-dimensional (3-D) structure. The pattern of inter-molecular interactions derived from earlier studies is essentially two-dimensional in that it involves projection on to a cylinder followed by unwrapping to give a sheet. Crosslinks are observed between molecular strands four apart and it is shown that this can only occur if the paths of the molecular strands through the IF are systematically distorted. These crosslinks are clustered axially at intervals of around 15 nm, a value closely related to the pitch length of the constituent coiled-coil molecules in the rod domains. The number of crosslinks between adjacent molecular strands shows a striking difference depending on lateral direction and provides support for the concept of a head-to-tail stacking of tetramers defined by the A(CN) mode of packing to form protofilament substructures in the fully formed IF. Each protofilament would consist of a pair of oppositely directed molecular strands stabilized by A(11) and A(22) interactions identified in earlier work. A detailed model for the IF in the reduced state comprising a ring of eight protofilaments is suggested. When combined with earlier studies of crosslink formation in the oxidized state, the present findings lead to the conclusion that there is a major reorganization of the molecular packing within the protofilaments during keratinization in vivo. Taken in conjunction with existing X-ray data on the fully keratinized structures, the new evidence for a protofilament substructure also enables a detailed 3-D model for the mature IF to be suggested.

Animals↗
PubMed2005

Mutation analysis of patients with neuronal intermediate filament inclusion disease (NIFID).

Abnormal neuronal aggregates of alpha-internexin and the three neurofilament (NF) subunits, NFL, NFM, and NFH have recently been identified as the signature lesions of neuronal intermediate filament (IF) inclusion disease (NIFID), a novel neurological disease of early onset with a variable clinical phenotype including frontotemporal dementia, pyramidal and extrapyramidal signs. In other neurodegenerative diseases in which protein aggregates contribute to disease pathogenesis, mutations in the encoding protein cause the hereditary variant of the disease. To determine the molecular genetic contribution to this disease we performed a mutation analysis of all type IV neuronal IF, SOD1 and NUDEL genes in cases of NIFID and unaffected control cases. We found no pathogenic variants.

Adult↗
PubMed1993

Intermediate filament protein expression in early developmental stages of the mouse. A confocal scanning laser microscopy study of in vitro fertilized and in vitro cultured pre-implantation mouse embryos.

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.

Animals↗
PubMed1981

Hypertrophy-induced increase of intermediate filaments in vascular smooth muscle.

The distribution of filaments was studied in hypertrophied rabbit vascular smooth muscle. Hypertrophy was induced by partial ligation of the portal-anterior mesenteric vein. 14 d after ligation, there was an approximately threefold increase in the number of intermediate filaments per cross-sectional area, as compared to control values. The actin:intermediate:myosin filament ratio was 15:1.1:1 in control and 15:3.5:0.5 in hypertrophied portal-anterior mesentric vein vascular smooth muscle. Comparison of the filament ratios with the increase in volume density of the hypertrophied cells suggests that the number of myosin filaments per cell profile remained approximately the same as in controls, whereas the number of actin filaments increased in proportion to the increase in cell volume.

Actins↗