Localization of actin in Dictyostelium amebas by immunofluorescence.

Antibody prepared against avian smooth muscle actin has been used to localize actin in the slime mold, Dictyostelium discoideum. The distribution of actin in migrating cells is different from that in feeding cells. Migrating amebas display fluorescence primarily in advancing regions whereas feeding amebas show uniform fluorescence throughout. The reaction is specific for actin since the fluorescence observed is blocked when the antibody is absorbed by actin purified from avian skeletal muscle, human platelets, and Dictyostelium. These results, in addition to describing the distribution of actin in D. discoideum, demonstrate that actins from these diverse sources share at least one common antigenic determinant.

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum.

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.

Assembly of keratin onto PtK1 cytoskeletons: evidence for an intermediate filament organizing center.

Purified keratin, solubilized in 8 M of urea, was added to Triton X-100-extracted PtK1 cells in 5 mM PIPES buffer. The buffer conditions induced assembly of keratin filaments which appear to associate with nuclei of extracted cells. These keratin fibers extend beyond the original margin of the cells and frequently form bridges between adjacent cells. Electron microscopy shows that keratin filaments associate closely with the surface of the nucleus. We suggest that the site of association between keratin and the nucleus may represent an intermediate filament organizing center.

Association of creatine phosphokinase with the cytoskeleton of cultured mammalian cells.

Using an antibody specific for creatine phosphokinase (CPK), we have discovered an association between that enzyme and the cytoskeleton. Immunofluorescence observations show that CPK is associated with intermediate filaments in PTK cells and BALB/3T3 cells. The CPK distribution also follows intermediate filaments when cells are treated with colchicine.

Altering the state of phosphorylation of rat liver keratin intermediate filaments by ethanol treatment in vivo changes their structure.

Dephosphorylation of keratin intermediate filaments (IF) in livers from ethanol-fed rats relative to controls occurs concurrently with a reorganization of the distribution of IF in the cells. One possible molecular mechanism for this reorganization is a phosphorylation-induced conformational change in the keratin that propagates as a change in the polymerization of the keratin subunits. To test this hypothesis, the structure of liver keratin IF, from both control and alcohol-fed rats, was explored by circular dichroism (CD), tryptophan fluorescence quenching, and 13C nuclear magnetic resonance (NMR). Keratin IF were isolated from livers of control rats and from livers of rats that had ethanol included in their feed for 6-40 weeks. A significant decrease in the intensity of the CD spectrum of keratin IF from livers of ethanol-treated animals, relative to controls, was observed. These data suggested either that a change in conformation or an increase in conformational motility in the keratin IF from ethanol-treated animals occurred as a result of the ethanol-induced dephosphorylation. 13C NMR data were obtained to distinguish between these two possibilities. An increase in resonance intensity of some 13C NMR resonances was observed in the keratin IF from livers of ethanol-treated animals, relative to controls. The CD and NMR data were therefore consistent with an increase in conformational motility of the rod domain in these keratin IF. No significant change was observed in the quenching of tryptophan fluorescence by KI. The change in protein dynamics detected in these experiments could be the molecular basis for the alteration of keratin IF organization in alcoholic hepatitis.

Alteration of intermediate filament distribution in PtK1 cells by acrylamide.

PtK1 cells were treated with low concentrations of acrylamide resulting in disruption of intermediate filament networks. An optimum treatment, 5 mM acrylamide in culture medium for 4 h, resulted in formation of a juxtanuclear aggregate containing both keratin and vimentin intermediate filaments. Actin-containing stress fibers and microtubules appeared normal after this treatment. Cells recovered when acrylamide was washed out of the cultures, and normal keratin and vimentin networks reappeared. These cells were capable of proliferation and grew to confluence. Acrylamide-treated cells appeared to locomote normally, showing membrane ruffling and changes in shape, but cytoplasmic organelles did not appear to move normally throughout the cell but remained at the cell center. These observations indicate that acrylamide is a useful intermediate filament inhibitor that does not affect other cytoskeletal elements.

Microtubules in mammalian erythroblasts. Are marginal bands present?

The purpose of this study was to determine if marginal bands, such as those present in mature nucleated red blood cells of other non-mammalian vertebrates and in primitive mammalian erythrocytes are present in definitive mammalian erythroblasts. In a small number of erythroblasts examined from mouse spleen, a bundle of 5-8 microtubules could be seen. These microtubules appeared similar to those previously identified by others as marginal band microtubules in liver and marrow erythroblasts. However, it was difficult to distinguish these bundles from remnants of mitotic spindle microtubules, or bundles of microtubules which extend to the midbody, a structure which is seen quite frequently in sections of erythroid cells. Triton extraction, a process which renders cytoskeletal elements such as microtubules more visible, also failed to confirm the presence of conventional marginal bands in these cells. It is suggested that use of the term "marginal band" be restricted to those cases in which it can be unequivocally demonstrated that a bundle of microtubules encircles the perimeter of the cell.

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles.

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.

Acrylamide treatment of PtK1 cells causes dephosphorylation of keratin polypeptides.

Treatment of PtKl cells with 5 mM acrylamide for 4 hr results in alterations in the distribution of keratin filaments within the cells. This effect is reversible within 18 hr. Labeling of PtKl cells with 32P demonstrates that there are four phosphorylated keratins, having Mr of 56 k, 53 k, 45 k, and 40 k. Phosphate associated with these polypeptides appears to turn over with a t1/2 of 12 hr. Incubation of labeled cells in 5 mM acrylamide results in approximately 50% dephosphorylation of keratins within 2 hr, which is 3 times faster than normal turnover. Recovery of cells from acrylamide is accompanied by rephosphorylation of keratins within 18 hr. Analysis by 31P NMR spectroscopy shows that acrylamide treatments are accompanied by a transient decrease in soluble inorganic phosphate. This is followed by a rapid increase in Pi which gradually returns to normal levels. These studies show a strong correlation between phosphorylation of PtKl cell keratins and morphological response of keratin filaments to acrylamide. These observations suggest that normal distribution of keratin filaments may be, in part, mediated by protein phosphorylation.

Role of phosphorylation in ethanol-induced aggregation of keratin intermediate filaments.

BACKGROUND: Keratins are members of a diverse group of tissue-specific cytoskeletal components known as intermediate filaments. Regulation of the structure and intracellular distribution of intermediate filaments is known to be related to the phosphorylation state of their structural subunits. It also is known that disruption of the keratin filaments of hepatocytes in response to chronic ethanol ingestion is characteristic of alcoholic liver disease. METHODS: To characterize the mechanism of ethanol-induced keratin filament reorganization and dephosphorylation, cells were grown in culture with and without ethanol, and then were treated at the end of the incubation period for 1 hr with either 8-bromo-adenosine 3':5'-cyclic monophosphate (8Br), water-soluble forskolin (ws-forskolin), H-89 diHCL, or okadaic acid. Morphology of the cells was examined by immunofluorescence microscopy, and keratin phosphorylation levels were determined by analysis of 32p labeling. RESULTS: We found that treatment of hepatoma cells with 300 mM ethanol results in disruption and aggregation of the keratin network in the vicinity of the nucleus as well as a hypophosphorylation of keratin subunits from ethanol-treated cells compared with non-ethanol-treated controls. 8Br and ws-forskolin treatment of ethanol groups restored keratin phosphorylation to control levels and reversed the ethanol-induced aggregation of keratin filaments. When H-89, an inhibitor of A-kinase, was added to control cells, keratin filament disorganization and dephosphorylation was observed. H-89 produced only a slight additional decrease in keratin phosphorylation in ethanol-treated cells, with no change in keratin distribution. Okadaic acid treatment of control cells produced hyperphosphorylation and filament network disruption, whereas in ethanol groups a reversal of the ethanol-mediated hypophosphorylation was observed but without reversal of the keratin filament aggregation. CONCLUSIONS: These results suggest that site-specific phosphorylation of keratin filaments is important in maintaining their integrity and that activation of the A-kinase system can antagonize the effects of ethanol, whereas its inhibition results in filament dephosphorylation and reorganization, mimicking effects of ethanol treatment.

The effect of cytochalasin B on the enucleation of erythroid cells in vitro.

The process of nuclear extrusion continues when erythroid cells of mouse spleen are placed in vitro, although the process may be considerably retarded. This allows visualization of unusually large numbers of enucleating cells after 30 min in vitro. With this system, the mechanism of enucleation can be analyzed both qualitatively and quantitatively. We have used this system to study the effects of cytochalasin B and have found it to be a potent inhibitor of enucleation. These results suggest that microfilaments are involved in the process of enucleation.

A reevaluation of the process of enucleation in mammalian erythroid cells.

In this paper, we have analysed cells which are in the process of enucleation in mouse spleen. We have provided additional support for the hypothesis that erythroid cells can autonomously undergo enucleation. The presence of a constriction in the nucleus and in the plasma membrane during this process has bee confirmed. Microfilaments have been described for the first time in the cytoplasm of maturing erythroid cells. Their position during enucleation is suggestive of an active role for these elements in this process.

Microvascular response to angiography.

A controlled laboratory study using matched groups fo pure-bred Sprague-Dawley rats evaluated the histologic microvascular response to contrast angiography. The femoral vessels were studied by both light and transmission electron microscopy (TEM) following injection of the infrarenal aorta with Renografin-60 or saline at intervals from 2 hours to 56 days. None of the specimens studied showed significant cellular, interstitial, or intravascular injury. TEM demonstrated electron-dense particles which migrated from the vessel lumen into the endothelial cytoplasm and basal lamina and subsequently diffused back into the cytoplasm. This study demonstrates the absence of microvessel damage from a radiopaque contrast medium administered by techniques designed to parallel the clinical model.

Combined immunofluorescence and high-voltage electron microscopy of cultured mammalian cells, using an antibody that binds to glutaraldehyde-treated tubulin.

An antibody against tubulin that binds specifically to microtubules in glutaraldehyde-fixed cells has been prepared. Sodium dodecyl sulfate-denatured tubulin treated with glutaraldehyde was used to immunize rabbits. In glutaraldehyde-fixed cells the fluorescent image of this antibody reveals a fine lattice of microtubules around the nucleus of PtK1 (Potorous tridactylis) cells and many uniformly fluorescent microtubules in the peripheral cytoplasm. In cells fixed with formaldehyde the microtubules appear to have a similar distribution, but the fluorescent image is much less uniform. Combined high-voltage electron microscopy and immunofluorescent studies reveal that microtubules are found in the cytoplasm in the same region as the fluorescent antibody stain.

Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP-dependent phosphorylation.

Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.

Vimentin filaments in spreading, randomly locomoting, and f-met-leu-phe-treated neutrophils.

Human neutrophils contain intermediate filaments of the vimentin type. A cytoskeletal preparation, produced by high-salt and Triton X-100 extraction of human neutrophils, reveals a major band at 57000 Mr that comigrates with 3T3 cell vimentin on one-dimensional gels. Two-dimensional gel electrophoresis of whole neutrophils illustrates the presence of vimentin but not desmin- or keratin-filament subunits. The presence of vimentin in neutrophils is also shown by its specific staining with avian vimentin antiserum by two-dimensional gel immunoautoradiography. Indirect immunofluorescence studies show that vimentin antiserum labels an area on one side of the nucleus in spreading neutrophils. This bright area appears as a loose knot of vimentin filaments; a few filaments may radiate from the knot. In contrast to spreading neutrophils, those undergoing random locomotion contain a fine network of filaments that are located in the cytoplasm between the nucleus and the trailing end of the cell. Similarly, in chemoattractant-treated neutrophils, vimentin filaments are bundled in the uropod. Transmission electron microscopy of human neutrophil monolayers confirms the intracellular distribution of intermediate filaments as shown by immunofluorescence in spreading and randomly locomoting cells.

Site-specificity of ethanol-induced dephosphorylation of rat hepatocyte keratins 8 and 18: A 31P NMR study.

Chronic feeding of ethanol to rats results in disorganization of the keratin intermediate filament network within hepatocytes. Previous studies from this laboratory have shown that intermediate filament organization in cultured cells is related to the phosphorylation state of the proteins. Therefore, we have examined the phosphorylation state of hepatocyte keratins from control and ethanol-fed rats. Feeding ethanol to rats results in dephosphorylation of one site on keratin 8 and one site on keratin 18 at all time points beginning with 6 weeks of ethanol treatment. Dephosphorylation was detected by phosphate analysis and by two-dimensional electrophoresis in which a change in isoelectric point of keratins from ethanol-fed rats was observed. These observations indicate that dephosphorylation of keratins in ethanol-fed animals may be an early step in alcoholic hepatitis which has occurred by 6 weeks of ethanol treatment. To further characterize keratin dephosphorylation in ethanol-fed rats, we used 31P NMR spectroscopy to classify the dephosphorylation site(s). Hepatocyte keratins were purified and solubilized in 9.5 M urea, 10 mM Tris-Cl, pH 8.1. 31P NMR spectra were obtained at 109 MHz, in 10 mm tubes at 30 degrees C. Samples of hepatocyte keratins were phosphorylated with A-kinase, protein kinase C, casein kinase II or Ca/CAM kinase and these samples were analyzed by 31P NMR spectroscopy. The resulting spectra were used as standards to compare the 31P chemical shifts of the resonances produced by these kinases with the phosphorus resonances of control and experimental samples. The 31P NMR spectrum of control hepatocyte keratins shows three resonances at 0.7, 4 and 5 ppm. In vitro phosphorylation by A-kinase produces a resonance at 4 ppm which is distinctly different from the resonance produced by each of the other kinases. In hepatocyte keratins from ethanol-fed animals, the resonance at 4 ppm was missing from the spectrum. These observations indicate that the keratin site that is dephosphorylated in ethanol-fed rats is characterized by the same 31P chemical shift as the keratin site that is phosphorylated by A-kinase.

Phosphorylation modulates keratin structure.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.