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B Safiejko-Mroczka

Publications and source records attributed to B Safiejko-Mroczka.

8 recordsLinked to original sources
PubMed2006

Reorganization of actin in K-562 and HL-60 cells treated with taxol.

Influence of taxol, microtubular poison, has been studied on distribution of actin. K-562 and HL-60 cells were treated with taxol in a range of concentrations 0.02-10 microM for 72 hours. The reorganization of F-actin was dependent on its dose. Phalloidin conjugated to TRITC was used to evaluate actin distribution by classical fluorescence and confocal microscopy. Actin was visualized at ultrastructural level by using a postembedding streptavidin-gold method. The treatment of K-562 and HL-60 cells with 2-10 microM of taxol resulted in an increase of F- actin in the cytoplasm, with intense labeling as a ring close to surface of the cell. In HL-60 cells a concentration of F- actin at the site of apoptotic bodies was often observed. Immunogold labeling of actin was localized in the nuclei and cytoplasm in control cells and cells treated with all doses of taxol. At higher doses, compaction of chromatin in the nucleus with strong actin labeling was observed. These observations at the ultrastructural level suggest actin involvement in chromatin reorganization during the process of apoptosis. The present study demonstrated a dose dependent reorganization of actin after treatment with taxol.

Actins↗
PubMed2001

Reorganization of the actin cytoskeleton in the protruding lamellae of human fibroblasts.

To investigate the mechanisms of protrusion in vertebrate cells, the primary event in cell motility, human fibroblasts were treated with neomycin, an inhibitor of the phosphatidylinositol cycle, to induce protrusion. Changes in cell motility and the cytoskeleton were examined by video, fluorescence, scanning electron, and confocal microscopy and by cytofluorometry. Protrusion in neomycin-treated human fibroblasts is correlated with a transient overall decrease in F-actin followed by an increase in F-actin at the leading edge of the protruding lamella. In growing lamellae, F-actin is organized in a marginal band at the leading edge. Although actin is present in the lamella behind the leading edge, very little of it is F-actin. Scanning electron microscopy of detergent-extracted cells reveals a band of dense filaments at the leading edge, corresponding to the marginal band of F-actin seen in fluorescently labeled cells, and a sparse population of short, fragmented filaments, in the rest of the lamella. Gelsolin is colocalized with F-actin in the marginal band and is also present in the lamella where F-actin is largely absent. The data support the hypothesis that the protrusion is initiated by the breakdown of cortical actin filaments, possibly mediated by gelsolin, whereas expansion of the protrusion requires de novo polymerization of actin filaments at the leading edge.

Actins↗
PubMed1998

Distribution of cytoskeletal proteins in neomycin-induced protrusions of human fibroblasts.

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15-20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.

Actins↗
PubMed1996

Bifunctional protein cross-linking reagents improve labeling of cytoskeletal proteins for qualitative and quantitative fluorescence microscopy.

Because permeabilization of the cell membrane is necessary to label intracellular proteins with most fluorescent probes, it is important to optimize the preservation and labeling of the proteins under study. We used qualitative and quantitative fluorescence microscopy to evaluate the effects of six different bifunctional protein cross-linking reagents and several extraction conditions on the labeling of filamentous actin with phalloidin and the immunolabeling of tubulin and gelsolin. The labeling of cytoskeletal and associated proteins can be significantly enhanced by the appropriate combination of bifunctional protein cross-linking reagents and extraction conditions. However, the conditions that give the most intense labeling vary depending on the label used. The greatest intensity of labeling with either phalloidin or antibodies was obtained with the intermediate-length cross-linker DSP. The two-step procedure of cross-linking with DSP and extracting in Triton X-100 in microtubule-stabilizing buffer containing DSP gives maximal labeling with phalloidin. Maximal labeling of gelsolin and tubulin with antibodies is obtained by extracting DSP-cross-linked cells with Triton in Hank's saline containing DSP. Therefore, DSP reproducibly improves preservation of both soluble and filamentous proteins for quantitative and qualitative studies by fluorescence microscopy.

Actins↗
PubMed1995

The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.

Actins↗
PubMed1995

Improved methods for preserving macromolecular structures and visualizing them by fluorescence and scanning electron microscopy.

To determine the optimal procedures to preserve cytoskeletal and other macromolecular structures for microscopic studies we have evaluated the effects of various methods to extract cultured cells. In this report, we compare results using different fixatives, crosslinking reagents, and permeabilization methods on (1) the labeling of cells for fluorescence microscopy with phalloidin or antibody against tubulin; and (2) the morphological preservation of macromolecular structures for scanning electron microscopy. Maximal labeling of F-actin with phalloidin was obtained by fixing cells in 4% paraformaldehyde (PFA) and labeling the unextracted cells with methanolic phalloidin, whereas maximal labeling of tubulin required prefixation with either PFA or the bifunctional protein crosslinking reagent, dithiobis (succinimidylpropionate) (DSP) and extraction with ethanol or Triton in a high salt buffer. However, for both qualitative and quantitative light and electron microscopic studies of intracellular macromolecular structures, prefixation with DSP and extracting with Triton X-100 in a stabilizing buffer is the overall method of choice for both labeling and morphological studies. Although other methods provide maximal labeling or preservation of specific structures, this method provides excellent preservation of morphological structure while allowing proteins to be preserved and labeled by specific probes.

Actins↗
PubMed1993

PDGF and neomycin induce similar changes in the actin cytoskeleton in human fibroblasts.

The addition of platelet-derived growth factor (PDGF) to serum-starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI-cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI-cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent-extracted cells was labelled with TRITC-phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF- and neomycin-induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF-receptors. In conclusion, the present study indicates that an increased turnover of the PI-cycle is not essential for the changes in actin organization induced by PDGF.

3T3 Cells↗