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Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.
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PMID:An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes. 138 Dec 90

Although it has been shown previously that an acidic (type I) "soft" keratin can interact with many basic (type II) "soft" keratins to form 10-nm intermediate filaments, it has been unclear whether "soft" keratins are compatible with the "hard" keratins typically found in hair and nail. To address this issue and to generate more structural information about hard keratins, we have isolated and sequenced a cDNA clone that encodes a mouse hair basic keratin (b4). Our sequence data revealed new information regarding the structural conservation of hard keratins as a group, being significantly different from soft keratins. Using expression vectors containing appropriate cDNA inserts, we studied the expression of this basic (b4) as well as an acidic (a1) mouse hair keratin in HeLa cells. The expression of these alien hair keratins in the transfected cells was surveyed using a panel of monoclonal and polyclonal antibodies. Our results indicated that the basic and acidic hair keratin readily incorporated into the existing endogenous soft keratin network of HeLa cells. Overproduction of hair keratin, however, occasionally led to the formation of cytoplasmic aggregates containing both hard and soft keratins. These data suggest that although small amounts of newly synthesized hair keratins can incorporate into the "scaffolding" of the preformed soft keratin filament network, possibly through dynamic subunit exchange, overproduction of hard keratins can lead to the partial collapse of the soft keratin network. These observations, along with the deduced amino acid sequence data, support and extend the concept that hard and soft keratins, although closely related, are divergent enough to justify their being divided into two separate subgroups.
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PMID:Transient expression of mouse hair keratins in transfected HeLa cells: interactions between "hard" and "soft" keratins. 171 23

The murine keratins Endo B and Endo A, which are homologs of the human keratins K18 and K8, constitute the intermediate filaments (IFs) that are found in all simple epithelia of the adult and in the first epithelial derivatives of the early embryo. The cellular role of simple epithelial keratins in development and differentiation was investigated by inducing filament collapse in HR9 endoderm and F9 embryonal carcinoma cells in which mutant Endo B protein was constitutively expressed. By immunolocalization techniques a perturbation of the keratin network was revealed as well as concomitant disruption of vimentin IFs and displacement of surface desmosomal proteins, demonstrating an intimate structural association of Endo B/A filaments with these cellular components. In aggregates of differentiating F9 cells displaying altered Endo A/B IFs, the formation of a compact, polarized visceral endoderm layer was significantly compromised. These results indicate that an intact keratin network influences the three-dimensional formation of cell-cell or cell-substratum contacts in embryonic visceral endoderm.
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PMID:Disruption of keratin filaments in embryonic epithelial cell types. 171 95

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.
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PMID:Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments. 241 18

Expression of intermediate filament (IF) isotypes was studied in six human and two murine melanoma cell lines. With one exception, these lines expressed IFs only of the vimentin type; neurofilament peptides, desmin and GFAP were not detected. However, the M5 human melanoma line also expressed extensive cytokeratin tonofilament arrays, as visualized by immunofluorescence with a panel of eleven monoclonal antibodies and hetero-antisera to cytokeratins; only the keratin 19-specific antibody BA16 did not react. By 2 D gel electrophoresis, five major keratin peptides were detected (keratins 7, 8, 13, 17 and 18), and an additional 57 kD peptide was detected on immunoblots with several antikeratin antibodies. Also observed in M5 cells was focal collapse of tonofilament arrays in mitotic cells. All the melanoma lines tested were positive for S100; M5 and two other cell lines were also positive for the 220-240 kD neuroectoderm-associated cell-surface differentiation antigen defined by monoclonal antibody UJ 127:11. In all the melanoma cell lines, secretion of extracellular matrix proteins (fibronectin, laminin and collagen type IV) was sparse or absent, and all were negative for the epithelial cell markers HMG-1 and HMG-2. Co-expression of keratin and vimentin by a melanoma cell line is discussed in the light of recent controversy concerning expression of cytokeratins by other neoplasms of putative neuroectodermal origins.
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PMID:Phenotypic analysis of cultured melanoma cells. Expression of cytokeratin-type intermediate filaments by the M5 human melanoma cell line. 242 48

The nucleoside analog 3'-deoxyadenosine (cordycepin) rapidly collapses the intermediate filaments into juxtanuclear caps in interphase fibroblasts and keratinocytes. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA) to inhibit its degradation is required to see these effects. This is the same concentration required for cordycepin to arrest cells at the onset of mitosis and depolymerize the microtubules to small asters. Cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP with a concomitant drop in ATP levels. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on either the intermediate filaments or microtubules. In addition, similar effects are not produced by a variety of other adenosine analogs with alterations in the 2'- and 3'-ribose positions. Although other pharmacological reagents result in alterations of the fibroblastic intermediate filaments, cordycepin is unusual because of the rapidity with which the fibroblastic intermediate filaments collapse into the juxtanuclear caps. The juxtanuclear caps have a morphology different from that of the perinuclear bundles of intermediate filaments that arise after long-term depolymerization of the microtubules. The keratin fibers in the epidermal cells retract to a perinuclear ring when treated with cordycepin.
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PMID:Cordycepin rapidly collapses the intermediate filament networks into juxtanuclear caps in fibroblasts and epidermal cells. 245 49

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.
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PMID:Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthesis during the heat shock response in mammary epithelial cells. 246 40

The distribution of cytoskeletal structures has been studied by electron and immunofluorescence microscopy in human embryonic epithelial cells (EUE cells) exposed to a hypertonic medium containing 0.274 M NaCl. A first noticeable effect involved an increase of cell size. Microtubules, microfilaments and intermediate filaments were also considerably changed under these experimental conditions. The most marked effect was on intermediate filaments of the keratin type which formed very thick bundles around the nucleus and gave rise to an intracellular cagework which is likely i) to increase mechanical resistance and ii) to avoid cell collapse in conditions of hyperosmolarity. A remarkable increase in complexity of the microfilamentous network was also found: stress fibers became thicker and more densely arranged and vinculin-containing streaks at focal cell-substratum contacts increased in number and size; this indicated improved cellular adhesion. The phenotypic adaptation of EUE cells to conditions of hyperosmolarity is slowly reversible under defined experimental conditions.
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PMID:Effects of hypertonic conditions on cytoskeletal and adhesion structures of EUE cells. 307 3

We have compared the appearance and preservation of molecular and supramolecular structures in preparations that were dried in vacuo at room temperature or freeze-dried. Fibrinogen and brain spectrin molecules appear similar in both types of preparation provided that drying at room temperature is performed in the presence of glycerol, which results in an even and reproducible distribution of such molecules (Shotton et al., 1979, J. Mol. Biol. 131, 303-329; Fowler and Erickson, 1979, J. Mol. Biol. 134, 241-249). In the case of crystalline actin sheets, actin filaments, and keratin filaments, freeze-drying preserves structural details that are often completely lost during drying at room temperature, whether or not glycerol is used. On the other hand, keratin filaments prepared by drying in the presence of glycerol display a beaded axial repeat that is probably due to "glycerol decoration." We conclude that although freeze-drying has no clear advantage over glycerol spraying/vacuum-drying in the case of single extended molecules, it may provide insight into the multiple effects of glycerol in specimen preparation. In the case of supramolecular assemblies such as filaments or crystalline sheets, freeze-drying preserves significantly more substructure and surface detail. The loss of such detail during drying at room temperature, probably through collapse phenomena such as distortion and flattening, cannot be prevented by glycerol.
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PMID:Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing. 619 49

Cytoskeletal intermediate filaments (IFs) constitute a diverse family of proteins whose members are expressed in tissue-specific patterns. Although vimentin IFs are normally restricted to mesenchyme, a variety of cell types express vimentin alone or together with cell-specific IFs during growth, differentiation, and neoplasia. In this study, we have investigated the influence of increased vimentin expression on the simple epithelial cell phenotype. An expression vector encoding a human vimentin cDNA was transfected into murine HR9 endoderm and F9 embryonal carcinoma cell lines, which serve as models for early extraembryonic epithelial differentiation. Stable clones that expressed varying levels of human vimentin were characterized by human vimentin were characterized by immunofluorescence and biochemical analysis. A relatively high level of vimentin expression in HR9 and differentiated F9 epithelial cells resulted in aberrant vimentin structures with co-collapse of keratin K8/K18 filaments and lowered amounts of keratin protein. In F9 epithelial cells, the desmosomal proteins DP I/II did not appear to localize to cell surface desmosome s but rather but rather co-aggregated with the perturbed IFs. Although overall cell morphology was not dramatically altered, individual nuclei were distorted by excess intracellular vimentin. Furthermore, cell proliferation as well as the cell spreading response time were slowed. Ther appears to be a threshold effect regarding overall vimentin levels as cells that expressed lower amounts of the human vimentin exhibited no obvious structural nor biological effects. Our results demonstrate that wild-type vimentin can act as a "mutant" protein when present at high intracellular levels, inducing a variety of phenotypic changes.
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PMID:Structural and biological consequences of increased vimentin expression in simple epithelial cell types. 867 30


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