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On the presence of high glycogen concentration and intermediate filaments in the flat cells of the electroreceptive epidermis of mormyrid fish.

The cytoplasm of the flat cells of the electroreceptive epidermis of Mormyrids was examined in the light and the electron microscope, in order to reveal the presence of glycogen and to study its distribution. In the electroreceptive epidermis, which consists of three layers, the periodic acid Schiff reaction used to stain polysaccharides is strongly positive in the superficial polyhedral cells and in the flat cells of the intermediate layer. Polysaccharides are absent in the basal polyhedral cells. Pre-incubation with alpha-amylase shows that glycogen is present only in the intermediate cell layer. In the electron microscope, after reaction with periodic acid, thiocarbohydrazide and silver proteinate, glycogen is seen in the form of rosettes of monoparticles. These rosettes occupy both the central region of the cytoplasm of these cells, and the more peripheral parts, where alignments of desmosomes are found. In the cytoplasm of certain flat cells, the rosettes are grouped to form accumulations of glycogen which cover several mu2. Observation in the electron microscope reveals that in addition to glycogen, these cells contain tonofilaments or intermediate filaments, common to epithelial cells, which may group themselves in bundles. Glycogen and the intermediate filaments are thus the principal constituents of the cytoplasm of the flat cells of the electroreceptive epidermis of Mormyrids. The possible role of the filaments, and especially of the glycogen which is a polysaccharide high in energy, in the flat cells which apparently have a low metabolic rate, is discussed.

Animals↗

Spindle cell carcinoma of the renal pelvis. Immunohistochemical and ultrastructural study of a case demonstrating coexpression of keratin and vimentin intermediate filaments.

A carcinoma of the renal pelvis characterized histologically by a spindle cell sarcomatoid morphological growth pattern was studied by electron microscopy and immunohistochemical techniques. Ultrastructural examination revealed abundant perinuclear cytoplasmic tonofilament bundles in association with prominent rough endoplasmic reticulum. Immunohistochemical study demonstrated coexpression of keratin and vimentin, two intermediate filaments thought to be specific for epithelial and nonepithelial cells, respectively. It is proposed that the spindle transformation of the epithelial cells in such cases may be explained on the basis of the development by the tumor cells of nonepithelial characteristics, such as the expression of vimentin intermediate filaments, that may be responsible for the adoption of the morphological growth pattern characteristic of neoplasms following mesenchyme-derived lines of differentiation.

Aged↗

An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments.

Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.

Actin Cytoskeleton↗

Molecular biology of neuronal intermediate filaments.

In the past few years, several neuronal intermediate filament proteins have been characterized. While ongoing investigations have continued to shed light on their developmental expression, the importance of different domains of the proteins for assembly, the elements in their genes necessary for tissue-specific expression, and the role of phosphorylation of neurofilaments, the function(s) of these structures remain a matter of speculation.

Animals↗

In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.

Intermediate filaments (IF) from baby hamster kidney (BHK-21) cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21 degrees C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 3000 nm. Thus, turbidity can be used as a quantitative assay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly/reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.

Animals↗

Heterogeneity of keratin intermediate filaments expression in human glioma cell lines.

Keratin intermediate filaments (Ifs) are specific for epithelial cell differentiation. This study demonstrates the presence of keratin in two recently established human glioblastoma cell lines 8-MG-BA and 42-MG-BA. Immunofluorescence staining was performed on cells within passage 230 to 235 using monoclonal pan-cytokeratin antibodies. The cells were analyzed during several DIV at different cell density. Keratin-positive stained cells reached 5 to 7% in 8-MG-BA and less than 0.1% in 42-MG-BA cell line. The presence of keratin-positive cells was independent on cell density and days in vitro. Keratin-positive cells appeared unevenly distributed in both cell lines. They were observed as single or areas of keratin-positive cells. The morphological features of keratin-positive and keratin-negative cells were similar. The results are discussed with respect to previous studies on glial fibrillary acidic protein (GFAP) and vimentin to show the heterogeneity of IFs expression in glioma cell lines.

Brain Neoplasms↗

Expression of vimentin intermediate filament cytoskeleton in acute nonlymphoblastic leukemias.

Since vimentin intermediate filament (IF) expression in hemopoietic cells varies with the cell lineage as well as the state of differentiation of the cells, we studied the vimentin cytoskeleton by direct immunofluorescence and electron microscopy in 50 cases of acute nonlymphocytic leukemias. We found that malignant cells tend to reproduce the vimentin organization characteristic of their normal cellular counterpart. Thus, in M2 and M3 leukemias (French-American-British classification), vimentin was often reduced to a juxtanuclear bundle of filaments contrasting with the rich filamentous network expressed by M4 or M5 leukemias. In erythroblastic leukemias (M6) and megakaryoblastic leukemias, both identified by the expression of lineage-specific antigens, the absence of vimentin IFs could be correlated with the level of differentiation reached by the blasts. M1 leukemias displayed an abnormal pattern of vimentin organization with aggregated filaments giving a ring-like structure. However, no abnormality of the vimentin polypeptide could be detected by two-dimensional electrophoresis. These results show that the expression of the vimentin IF cytoskeleton may be a useful marker of differentiation in the study of leukemic cells.

Acute Disease↗

PKC activation by melatonin modulates vimentin intermediate filament organization in N1E-115 cells.

Melatonin enters cells and causes cytoskeletal rearrangements in unicellular organisms, plants and vertebrates. This pineal secretory product causes microtubule enlargement and neurite outgrowth by a calmodulin antagonism in N1E-115 cells. Recently, direct in vitro activation of protein kinase C by melatonin was described. Vimentin intermediate filaments are attached to microtubules and their organization depends on both microtubule distribution and phosphorylation of specific proteins. Protein kinase C is a serine threonine kinase which phosphorylates vimentin and through this mechanism causes intermediate filament disassembly. In this work the effects of melatonin on protein kinase C activation, content, and subcellular distribution were studied in N1E-115 cells. Also, melatonin effects on vimentin phosphorylation and subcellular distribution were explored. The results show that melatonin both activates and increases protein kinase C content in the membrane cytoskeletal fraction. Melatonin protein kinase C activation was followed by an increase in both vimentin phosphorylation and by vimentin subcellular redistribution. Moreover, staurosporine, a serine threonine kinase inhibitor, prevented increased vimentin phosphorylation elicited by melatonin. Similar effects to those caused by melatonin were obtained with the protein kinase C activator phorbol 12-myristate 13-acetate. Data support the idea that melatonin modulates vimentin organization through protein kinase C activation.

Animals↗

Intermediate filaments from Chinese hamster ovary cells contain a single protein. Comparison with more complex systems from baby hamster kidney and mouse epidermal cells.

Intermediate filaments (IF) were prepared under identical conditions from Chinese hamster ovary (CHO), baby hamster kidney (BHK), and primary mouse epidermal cells. CHO and BHK cells each have a major IF protein (Mr = 55,000); these proteins comigrate on two-dimensional gels. BHK cells have an additional protein constituent (Mr = 54,000) which is absent from CHO. The IF preparation from the epidermal cells was considerably more complex, containing at least five proteins with molecular weights between 50,000 and 70,000, none of which has a mobility similar to the CHO protein. If assembled in vitro from crude CHO IF preparations were morphologically indistinguishable from filaments assembled from other cell types. Analysis of these in vitro polymerized filaments from CHO cells using circular dichroism and wide angle x-ray diffraction revealed that the CHO filaments were structurally similar to filaments prepared from BHK or epidermal cells. The Mr = 55,000 proteins from CHO and BHK cells were found to have identical one- and two-dimensional peptide maps, demonstrating that their amino acid sequences must be very similar. These studies indicate that CHO cells possess the simplest system thus far described for studying the assembly of intermediate filaments.

Animals↗

Microfilaments, microtubules, and intermediate filaments in cultured corneal fibroblasts.

Corneal stromal fibroblasts play an important role in wound healing. Proteins from all three cytoskeletal classes (microfilament, microtubule, and intermediate filament) are involved in the control of various cellular events, such as motility, cell adhesion, shape changes, intracellular transport, and mitosis. By epifluorescent light microscopy, we studied the intracellular distributions of actin (microfilament), tubulin (microtubule), and vimentin (intermediate filament), as well as vinculin (a junctional protein connecting microfilaments to the cell membrane), in cultured corneal fibroblasts. Mutual positional relationships between actin and the other cytoskeletal proteins were investigated by double-labeling. Particular attention was paid to the leading edge of spreading or migrating fibroblasts and to their cell-to-cell contacts.

Actins↗

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.

Actinin↗

Expression of intermediate filament proteins and neuronal markers in the human fetal gut.

The human enteric nervous system (ENS) derives from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. During development of the NCC, a rearrangement of various cytoskeletal intermediate filaments such as nestin, peripherin, or alpha-internexin takes place. Although all are related to developing neurons, nestin is also used to identify neural stem cells. Until now, information about the prenatal development of the human ENS has been very restricted, especially concerning potential stem cells. In this study the expression of nestin, peripherin, and alpha-internexin, but also of neuronal markers such as protein gene product (PGP) 9.5 and tyrosine hydroxylase, were investigated in human fetal and postnatal gut. The tissue samples were rapidly removed and subsequently processed for immunohistochemistry or immunoblotting. Nestin could be detected in all samples investigated with the exception of the 9th and the 12th week of gestation (WOG). Although the neuronal marker PGP9.5 was coexpressed with nestin at the 14th WOG, this could no longer be observed at later time points. Alpha-internexin and peripherin expression also did not appear before the 14th WOG, where they were coexpressed with PGP9.5. This study reveals that the intermediate filament markers investigated are not suitable to detect early neural crest stem cells.

Adolescent↗

Immunohistochemical demonstration of simple epithelia-type keratin intermediate filament in a case of Merkel cell carcinoma.

A case of Merkel cell carcinoma that developed on the right cheek of a 77-year-old woman is reported. The diagnosis was ultrastructurally made by demonstrating dense-core granules in the cytoplasm of the tumor cells. Immunohistochemically, the tumor cells were shown to possess simple epithelia-type keratin intermediate filaments, but no neurofilaments. This finding was in accordance with that obtained on normal Merkel cells. No bioactive peptides examined could be detected in the tumor cells. Typing of intermediate filaments in tumor cells may be one of the important markers, along with the ultrastructural findings, in diagnosing Merkel cell carcinoma.

Adenocarcinoma↗

Keratin incorporation into intermediate filament networks is a rapid process.

The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.

Animals↗

Expression of histocompatibility antigens, transferrin receptors, intermediate filaments, and alkaline phosphatase by in vitro cultured rat placental cells and rat placental cells in situ.

Rat placental cells (RPCs) derived from the chorioallantoic placenta of day-12 Holtzman rats were tested for the expression of class I and class II RT I histocompatibility antigens, transferrin receptors, intermediate filaments, and alkaline phosphatase. The binding of mouse monoclonal antibodies to those antigens by RPCs was compared with the binding of the same reagents to rat placental cells in situ. RPCs expressed low levels of class I antigens and failed to express detectable levels of class II antigens. RPCs resisted up-regulation of expression of class I antigens by interferon-gamma, and did not express class II antigens following exposure to medium containing interferon. Transferrin receptors; cytokeratin intermediate filaments, and alkaline phosphatase were universally expressed by RPCs. Taken together with the patterns of expression of the same antigens by rat placental cells in situ, the results suggest that RPCs comprise labyrinthine trophoblast cells. Those cells may provide a valuable new approach for studying the structures and functions of trophoblast cells in vitro.

Alkaline Phosphatase↗

A cationic detergent, cetyltrimethylammonium bromide (CTAB), selectively dissociates the intermediate filament of the fibroblast.

We investigated conditions for selective solubilization of the intermediate filament (IF) of BHK-21 cells, and found that a cationic detergent, cetyltrimethylammonium bromide (CTAB), was effective for rapid dissociation of IF into the monomeric form. More selective dissociation was performed with a combination of CTAB and Tween 40. The CTAB-dissociated vimentins were unstable, but the breakdown of them was successfully blocked by leupeptin. Thus, with our extraction buffer, composed of 1% CTAB, 1% Tween 40, 10 mM Tris-HCl (pH 7.4) and 25 micrograms/ml leupeptin, almost all of the vimentin as well as the desmin were solubilized, while two thirds or more of actins were retained in the CTAB/Tween-insoluble fraction.

Animals↗

Expression of intermediate filaments and actin in the embryonic human inner ear.

The immunoreactivity for intermediate filaments (IF) and F-actin was documented in serially cryosectioned human inner ears aged 14-19 gestational weeks. An individual immunoreactivity for IF was documented for different cell types in both the cochlear and vestibular parts of the labyrinth. All secretory epithelia showed a similar expression of the cytoskeleton. In outer hair cells, immunoreactivity for vimentin was documented, but not for other IF types. The cytoskeletal composition is similar in the tectorial membrane and in supporting cells of the great epithelial ridge. Strong immunoreactivity for F-actin occurs close to the surface of all vestibular organs and Kölliker's organ, but not to the same extent in other epithelia lining the endolymphatic space.

Actins↗

[Distribution of the intermediate filaments in rat Sertoli cells: an electron microscopic study].

The distribution of intermediate filaments (IFs) in the Sertoli cells of rat testis was studied under a transmission electron microscope. The results showed that IFs in Sertoli cells were centrally arranged around the nucleus in a criss-cross manner. From the perinuclear region the IFs had widespread extensions and were connected with the following structures: 1) with the mitochondria, Golgi apparatus, endoplasmic reticulum and other subcellular structures; 2) with the desmosome-like and hemi-desmosome-like junctions with adjacent Sertoli and spermatogenic cells; 3) with the microfilaments (MFs) in the ectoplasmic specialization (ES) surrounding the head of elongated stage I-V; spermatids; 4) with the MFs in ES between adjacent Sertoli cells. In conclusion, IFs form a delicate intracellular network in Sertoli cell with its center in the perinuclear region and stretching and connecting with many subcellular structures in the cytoplasm and cell membrane.

Animals↗