Roland Hartig

Dexamethasone induces rapid actin assembly in human endometrial cells without affecting its synthesis

Dexamethasone exerts a stimulatory effect of rapid‐onset on the polymerization of actin. This has been documented in human endometrial adenocarcinoma Ishikawa cells, resulting in an acute, dose‐dependent decrease in the G/total‐actin ratio. In the present study we completely characterized this fast and apparently nongenomic effect of dexamethasone on actin assembly. We followed the morphological alterations of actin cytoskeleton and measured the time‐dependent dynamics of actin polymerization both by ruling out any changes of total actin in the cells and by measuring its transcript. Rapid changes in actin polymerization were accurately measured using a highly sensitive and quantitative rhodamine‐phalloidin fluorimetric assay. Ishikawa cells, exposed to 0.1 μM dexamethasone for various time periods up to 24 h, showed a highly significant, rapid, and transient increase in the polymerization of actin starting within 15 min of dexamethasone exposure and lasting 2 h. Treated cells showed a significant (1.79‐fold) enhancement of the fluorescent signal compared to untreated cells at 15 min. This value decreased continuously in a time‐dependent manner, reaching control levels after 120 min and remained so for the next 24 h. Confocal laser scanning microscopy studies confirmed these findings. Intensive coloration of microfilaments over several scanning sections suggested an enhanced degree of actin polymerization in cells preincubated for 15 min with 0.1 μM dexamethasone. Moreover, actin filaments were more resistant to cytochalasin B. Additionally, quantitative immunoblot analysis showed that the content of total cellular actin remained the same during this period, suggesting that the biosynthesis of actin was unaffected. Northern blot analysis showed that the concentration of the actin transcript was also unaffected. Our data suggest that glucocorticoids induce a fast and self‐limited polymerization of actin in human endometrial cells without affecting its synthesis. These findings strengthen the hypothesis that glucocorticoids exert rapid, nongenomic cellular effects and that the actin‐based cytoskeleton is an integral part of this pathway, playing an essential role in receiving and mediating steroid signals for the modulation of cellular responses. J. Cell. Biochem. 65:492–500.

Early alterations of actin cytoskeleton in OK cells by opioids

Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids αS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose‐dependent decrease in the amounts of F‐actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total‐actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and αS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells. J. Cell. Biochem. 70:60–69, 1998.

Colocalization of single ribosomes with intermediate filaments in puromycin‐treated and serum‐starved mouse embryo fibroblasts*

Previous experiments have revealed a relatively weak electrostatic binding capacity of in vitro reconstituted intermediate filaments (IFs) as well as of natural IFs of whole cell mount preparations for purified ribosomal particles of mammalian origin. In order to demonstrate that such associations also occur in vivo, intact cells were subjected to double immunofluorescence microscopy using antibodies directed against vimentin and ribosomal protein S17. Since in proliferating cells the majority of the ribosomal particles are assembled into polyribosomes and these are to a great extent associated with microfilaments, in vitro cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confocal laser scanning microscopy, the ribosomes were detected in colocalization with vimentin IFs. Disassembly of polyribosomes was also achieved by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vimentin IFs being directly associated with the cell nuclei, radiating into the peripheral areas of the cells or showing a stress fiber-like distribution. In both cases, considerable quantities of ribosomal material were seen in close neighborhood to vimentin IFs. Frequently, these ribosome-IF associations were coaligned with microtubules and they also surrounded myosin I-decorated stress fibers. Double labeling with the vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pattern largely superimposable on that of ribosomal protein S17. Treatment of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and the ribosomes stayed in contact with the vimentin IFs. On the basis of these results, it is conceivable that IFs play a role in the storage of ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the often seen coalignment of IFs with microtubules and microfilaments might serve facilitated and directional transport of ribonucleoprotein particles from the nucleus to peripheral areas of the cell.

ORGANIZATION OF FOCAL ADHESION PLAQUES IS DISRUPTED BY ACTION OF THE HIV-1 PROTEASE

Focal adhesion plaques were severely affected in human embryonic fibroblasts permeabilized with digitonin and incubated in buffer containing the human immunodeficiency virus type 1 protease (HIV‐1 PR). A mutant HIV‐1 PR (3271 HIV‐1 PR) had no effect on focal adhesion plaques. Similar effects were seen with cells microinjected with either HIV‐1 PR or 3271 HIV‐1 PR. Immunoblots of the human embryonic fibroblasts demonstrated that a number of focal adhesion plaque proteins were specifically cleaved by HIV‐1 PR. These included fimbrin, focal adhesion plaque kinase (FAK), talin, and, to a lesser extent, filamin, spectrin and fibronectin. Proteins detected by antibodies to β4 integrin and α3 integrin were also cleaved by the HIV‐1 PR. Control experiments demonstrated that the effect and protein cleavages described are due to action of the HIV‐1 PR and not to the action of endogenous host cell proteases.