Search PubMed⌕ Search

Biomedical subjects

M Opas

Publications and source records attributed to M Opas.

At least 73 records · Page 4Linked to original sources
PubMed1986

Effects of iodoacetic acid on cytoskeleton and adhesiveness of the chick RPE cells in vitro.

The retinotoxic, sulfhydryl-binding drug, iodoacetic acid (IAA), affects embryonic chick retinal pigmented epithelial (RPE) cells grown in primary cultures in a reversible, dose-dependent manner. A dose of 5 X 10(-4) M which produces drastic cell shape changes in about 90 min was chosen to study the effects of IAA on the organization of cytoskeleton and adhesiveness in RPE cells. After treatment of cells with IAA microtubules depolymerize and F-actin becomes redistributed from numerous stress fibers to knob-like aggregates. Vinculin is released from focal contacts and adhesions into the cytosol and, at the same time, adhesiveness of the RPE cells to substratum decreases. Since RPE in vitro is susceptible to the action of IAA, it seems possible that also in vivo some of the retinotoxic effects of IAA might be attributed to its damaging influence on the RPE. Thus, the retinotoxic effects of IAA in vivo cannot be solely attributed to the selective degeneration of photoreceptors by this agent.

Animals↗
PubMed1986

Light-microscopical analysis of focal adhesions of retinal pigmented epithelial cells.

Retinal pigmented epithelial (RPE) cells from eyes of chick embryos form colonies in vitro in which cells at the periphery of the colony are unpigmented, undifferentiated, and well spread, while those in center of the colony are cuboidal, polygonal, and pigmented, and resemble RPE cells in vivo. The differentiated RPE cells in the center of the colony display predominantly cell-cell adhesiveness, and their microfilaments are organized in compact, circumferential rings. Undifferentiated RPE cells from the edge of the colony, in contrast, display predominantly cell-substratum adhesiveness and have numerous stress fibers spanning their cytoplasm. The well-spread RPE cells adhere to the substratum with focal contacts and unusually large focal adhesions. The focal adhesions which are typical of the spread chick RPE cells in vitro consist of several closely apposed focal contacts, arranged in a parallel fashion, which are often coalesced with each other along their sides. They occur at the termini of prominent microfilament bundles which contain F-actin and tropomyosin along their entire length. Myosin, which is also present in these bundles, however, is less abundant than actin and tropomyosin in the terminal, focal adhesion-associated parts of these bundles. On the other hand, myosin is more abundant than actin and tropomyosin outside the microfilament bundles in the bulk of the cytoplasm. Both focal adhesions and termini of microfilament bundles coincide with the restricted regions where high concentrations of vinculin, an adhesion-specific protein, are found. In contrast, an actin binding protein, spectrin, is distributed fairly uniformly throughout the entire cortex of RPE cells, and, unlike vinculin, does not seem to participate in the binding of microfilament bundles to the plasma membrane. Although extracellular matrix components laminin, fibronectin, and heparin sulfate proteoglycan are produced and deposited by the more differentiated RPE cells in the center of the colony, heparan sulfate proteoglycan has not been detected along the surface of the flat, undifferentiated RPE cells near the edge of the colony, while both laminin and fibronectin are present in very low amounts, the former along their ventral, and the latter along their dorsal cell surfaces. These data are discussed from a point of view that the formation of highly adhesive membrane-cytoskeleton complexes of the focal type in the spread, undifferentiated RPE cells is brought about by exposure of the cells to the rigid, unyielding substrata, such as glass or plastic.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗
PubMed1985

Spatial distribution of cortical proteins in cells of epithelial sheets.

In the differentiated pigmented epithelial cells of the retina (RPE) of chick embryos cytoskeletal proteins are found in polygonal rings located in the cell cortex. Within the cortical rings of the RPE cells vinculin and spectrin occupy a characteristic position closest to the plasma membrane; actin is found farther away, while tropomyosin and myosin are located farthest from the plasma membrane. The differences in the distribution of these proteins might reflect the functional specialization of different parts of the cortical ring required to develop and transmit tension from individual cells throughout the entire epithelial sheet.

Actins↗
PubMed1985

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro.

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.

Animals↗
PubMed1985

Acute inflammation induced by immune complexes in the microcirculation.

The aims of the studies presented in this publication were to elucidate the morphology and quantitate the kinetics of an inflammatory reaction elicited by immune complexes and to ascertain the role of complement in the reaction. The hallmark of both the direct active (DAA) and reversed passive (RPA) Arthus reactions was the accumulation of immune precipitates and polymorphonuclear leukocytes (PMNs) in and around vessels. Using fluoresceinated antigen as a tracer, immune complexes localized in the lumina and walls of venules and small veins in the DAA and in the wall of vessels and perivascularly in RPA. PMNs accumulated at these same sites, phagocytosed the fluoresceinated complexes and became degranulated. The precise localization of immune complexes was achieved by examining the same tissue sections first by fluorescence microscopy, followed by conventional staining and examination by light microscopy. Marked stasis of the microcirculation was observed, particularly in DAA, in which a few immune complex-containing PMNs were entrapped in a mass of densely packed red blood cells. Some edema was observed in early lesions and definitive separation of collagen fibers was noted in lesions older than 2 hr. Hemorrhage became the dominant characteristic of both types of reactions from 2 hr onward. By administering radiolabeled cells, proteins, and microspheres as a "pulse," given at various times before sacrifice, the quantitation and kinetics of the inflammatory lesions elicited by immune complexes could be elucidated. In RPA all parameters quantitated reached a peak soon after elicitation of the reaction (2-4 hr), which is in keeping with other forms of acute inflammation. In DAA there was some difficulty in assessing the quantitation because of interanimal variations and because of progression of the inflammatory lesions, as the antigen diffused peripherally from the site of its injection. Peak activities occurred in 4- to 8-hr-old lesions. These observations and a comparison of the center and periphery of the lesions, strengthen the contention that the RPA and DAA have common features and features which differ. In common are immunological mechanisms (antigen-antibody interaction and complement activation) and cellular events (polymorphonuclear leukocyte chemotaxis, phagocytosis, and release of lysosomal contents). Different features are the site of immune complex formation and its sequelae. In RPA they form primarily in the wall of venules and small veins and hence have a marked effect on increase in vessel permeability. In the DAA most of the complexes form and the leukocytes accumulate in the lumen of the same vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The focal adhesions of chick retinal pigmented epithelial cells.

Retinal pigmented epithelial (RPE) cells obtained from the eyes of chick embryos from colonies in vitro in which cells at the periphery of the colony express an undifferentiated, well-spread morphology and develop extremely large areas of cell-substratum adhesion. These adhesions can be classified as focal on the basis of the following: (a) their black surface reflection interference image, the contrast of which is not affected by changes in either the wavelength of the incident light or the refractive index of the immersion medium; (b) their association with the termini of actin-containing microfilament bundles; and (c) their ability to be labelled with antiserum against vinculin, a protein specific for adhesions of the focal type. The focal adhesions of RPE cells comprise laterally associated individual focal contacts, the mechanism by which this association is achieved and maintained is yet unknown. Because of the unusually large size and excellent microscopical definition of their focal adhesions, I used RPE cells to investigate the role of other actin-associated proteins in adhesion complexes. One of these, nonerythroid spectrin (fodrin), a protein suggested to play a role in anchoring actin filaments to the plasma membrane, was neither concentrated in nor excluded from the focal adhesions of RPE cells. Thus, at least in this cell type, spectrin seems unlikely to serve as a link between the major actin-containing microfilament bundles and the plasma membrane in the regions of cell-to-substratum contacts.

Actin Cytoskeleton↗
PubMed1985

Multiple labeling of cellular constituents by combining surface reflection interference and fluorescence microscopy.

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along the ventral aspect of cells adhering to a glass substratum, we feel that this simple and rapid technique has great potential in studies of cell-substratum adhesiveness and of adhesion-related cytoskeletal organization.

Animals↗
PubMed1985

Distribution of spectrin and lectin-binding materials in surface lamina of RPE cells.

Explants of embryonic chick retinal pigmented epithelium (RPE) form colonies in primary cultures in which the cells occupying the center of the colony are cuboidal, pigmented, and resemble closely the differentiated RPE cells found in vivo, while those towards the periphery gradually become less differentiated, flatter, nonpigmented, and motile. In the present article we describe the distribution of alpha-spectrin in cells in different parts of the RPE colony and compare it with that of the surface lamina components that bind the lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA). Spectrin is present predominantly in the cell cortex in both differentiated and undifferentiated cells and seems to be accumulated especially heavily in the differentiated RPE cells in the colony center that have acquired the in vivo morphology. The distribution of lectin-binding materials in the surface lamina of the RPE cells is similar but not identical to that of alpha-spectrin. This is particularly evident in the flatter RPE cells along the edge of the colony in which surface lamina components visualized by fluorescein isothiocyanate (FITC)-WGA are distributed in the punctate pattern that is distinctly different from the reticular pattern in which alpha-spectrin is distributed. Also, in cells of the intermediate and central zones, strong staining along the edge of the cells was seen with antibodies to alpha-spectrin but not with FITC-WGA. Thus, a codistribution similar to that reported for the spectrin-like proteins and WGA-binding material in mesenchymally derived cells is not found in RPE cells.

Animals↗
PubMed1984

Astrocyte cell lineage. III. The morphology of differentiating mouse astrocytes in colony culture.

Disaggregated cells of newborn DBA/1J mouse neopallium were grown in colony cultures, and colonies of cells at various stages of differentiation along the astrocyte cell lineage were examined after 3 days, 1, 2 and 4 weeks by electron microscopy and by NBD-phallacidin which demonstrates the distribution of microfilaments. The earliest astrocyte precursor cells or glioblasts are closely apposed epithelial cells that rarely have junctions. Their scanty cytoplasm contains many free ribosomes but few microfilaments. The cells in the next stages of astrocyte lineage or proastroblasts are flat and are separated from each other to a variable degree. They have intercellular junctions associated with microfilaments and contain singly dispersed intermediate filaments. The proastroblasts gradually differentiate into astroblasts which have a similar morphology except that in addition to the singly distributed intermediate filaments they also contain intermediate filaments arranged into bundles of various sizes. The mature fibrous astrocytes have well-defined processes and distinct perikarya. They form from astroblasts in culture and also contain numerous bundles of intermediate filaments. The dibutyryl-cyclic AMP (dBcAMP)-induced astrocytes in culture in contrast are large stellate cells similar to reactive astrocytes found around sites of injury in the brain. On the basis of these and previous immunocytochemical studies of the formation and distribution of intermediate filaments in the cytoplasm of differentiating astrocytes, criteria are proposed for identification of different cells along the astrocyte lineage.

Animals↗
PubMed1984

Astrocyte cell lineage. IV. Changes in the organization of microfilaments and adhesion patterns during astrocyte differentiation in culture.

The organization of microfilaments using NBD-phallacidin and cell adhesion to substratum by surface reflection interference microscopy was examined during differentiation of astrocytes in colony cultures and correlated with motile behaviour of cells. Disaggregated cells from neopallium of 12-day-old or newborn DBA/1J mouse embryos were used to establish colonies and astrocyte precursor cells at various stages of differentiation along the astrocyte lineage were examined after 3 days, 1, 2 and 4 weeks in culture. The earliest astrocyte precursor cells, the glioblasts, are stationary and form epithelial-type colonies which adhere to the substratum primarily around the edge where large microfilament bundles are found. Bundles of microfilaments are also present around the apical ends of closely packed cells. As the epithelial cells start to separate and transform into flat proastroblasts, adherens-type junctions which have a zig-zag appearance and are associated with microfilaments form between adjacent cells. In the highly motile astroblasts these junctional regions break down into multiple smaller regions where the separated cells remain in contact through fine processes. The astroblasts also have stress fibres, focal contacts with substratum, foci from which microfilament bundles radiate and a complex pattern of fine, circumferentially oriented bundles of microfilaments. This elaborate organization of microfilaments disappears as the motile astroblasts differentiate into stationary fibrous astrocytes that have little polymerized actin and lack focal contacts. These results show that stationary astrocyte precursor cells in vitro go through a highly motile stage having a characteristic distribution of microfilaments and focal contacts before becoming stationary again. We consider that the motile stage could correspond to the stage in vivo when astrocyte precursor cells migrate from the ventricular and subventricular regions to take up position in different parts of the developing brain.

Actins↗
PubMed1984

Surface reflection interference microscopy: a new method for visualizing cytoskeletal components by light microscopy.

Surface reflection interference microscopy of detergent resistant residues of cultured cells stained with protein dyes can be used to obtain high resolution images of the cytoskeleton. We have compared the images obtained using different dyes and have examined the effect of four of these dyes on the visualization of different parts of the cytoskeleton in detail. The dependence of contrast of the images obtained on the illuminating numerical aperture and the wavelength of incident light was determined. Staining with Acid Yellow 36, Guinea Green B and Naphtol Blue Black produces images from the entire cytoskeleton and contrast in these images is relatively insensitive to changes in the incident wavelength. Coomassie Brilliant Blue R250 images, on the other hand, result primarily from reflection from the lower surface of the cytoskeleton and the contrast of these images is sensitive to changes in incident wavelength dropping abruptly in the region of the transmission peak of the stain. From the different spectral sensitivities of the reflection images obtained and from differential interference effects at low and high illuminating numerical apertures, we conclude that the reflection images obtained using the first three stains result from modulation of the reflection by interference effects. In contrast, in the case of Coomassie Brilliant Blue R250 the resulting image originates mainly from selective reflection of wavelength near the absorption range of the dye.

Animals↗
PubMed1984

Microfilament distribution and adhesion patterns in cultured cells after glutaraldehyde-formaldehyde fixation.

To study the relationship between microfilament distribution and adhesion patterns in the same cultured cell, we have employed a simple glutaraldehyde-formaldehyde fixation technique followed by permeabilization of the cells in buffered Triton X-100. This method gives an excellent preservation of cellular morphology in general and of adhesion patterns in particular for examination with surface reflection interference microscopy. It also permits the concomitant use of the actin-specific fluorescent probe NBD-phallacidin to visualize the distribution of microfilaments.

Cell Adhesion↗
PubMed1983

Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro.

The distribution of microtubules (MT), microfilaments (MF), and patterns of cell-to-substratum adhesion were studied by tubulin antibody labeling, NBD-phallacidin staining and by reflection interference contrast (RIC) microscopy respectively in colonies of differentiating RPE cells obtained from explants after 10 days in culture. In each colony three zones could be identified: a central zone of packed well-differentiated cuboidal cells (zone 1), an intermediate zone of more flattened, pleomorphic cells (zone 2) and a peripheral zone of very spread cells at the edge of the colony (zone 3). As visualized with antibodies to tubulin, the MT distribution in cells of each zone was distinctly different and correlated well with differences in cell shape. Changes in the distribution of MF were more striking. In the cuboidal well-differentiated cells of zone 1, prominent cortical bands but no stress fibers were observed after staining with NBD-phallacidin and RIC microscopy showed that the cells lacked strong adhesion to the substratum. Stress fibers, in addition to cortical bands of MF, were seen in the more spread, less differentiated cells of zone 2 and focal contacts were observed when these cells were examined by RIC microscopy. The flattened least differentiated cells in zone 3 lacked cortical bands but had prominent stress fibers. These cells displayed a variety of adhesion forms ranging from a mosaic of far and close contacts to numerous focal contacts and broad focal adhesions. Our results show that as the RPE cells display less differentiated morphologies, i.e. are more flattened and less densely packed towards the edge of the colony, there is a gradual decrease in the cortical bands of MF and an increase in the number and prominence of stress fibers. This increase in numbers of stress fibers is correlated with an increase in the cell adhesiveness to the substratum, as estimated by RIC microscopy. These results strongly support the general observation that normal epithelial cells in colonies tend to adhere to the substratum more strongly by marginal cells than by the more differentiated centrally located cuboidal cells which have well developed intercellular contacts.

Animals↗
PubMed1983

Immunocytochemical studies of intermediate filament aggregates and their relationship to microtubules in cultured skin fibroblasts from patients with giant axonal neuropathy.

Giant axonal neuropathy (GAN) is a severe autosomal recessive disease affecting both the peripheral and central nervous systems. It is characterized by segmental axonal ballooning due to large neurofilamentous masses and abnormal aggregation of filaments in other cell types including glial cells. Coomassie blue staining of the detergent-resistant cytoskeleton of cultured skin fibroblasts from three patients with GAN revealed the presence of large cytoplasmic filamentous aggregates in the great majority of cells. The aggregates were birefringent when viewed under polarization microscopy and electron microscopy showed that they were composed of aggregates of 8 to 10 nm intermediate filaments. The aggregates stained with antisera specific for vimentin but did not stain with antibodies to actin, tubulin, or the high molecular weight (HMW) microtubule associated protein. Examination of the fibroblasts containing the vimentin aggregates with antibodies to tubulin and the HMW protein showed that they had a normal distribution of microtubules and that the microtubules present were normally associated with the HMW protein. The results suggest that giant axonal neuropathy is a generalized inborn error of organization of intermediate filaments and that a defect in microtubules or their association with HMW protein is not responsible for the observed aggregation of intermediate filaments in this disease. Further study of GAN may be useful in understanding the function of intermediate filaments.

Child↗
PubMed1978

Holographic microscopy of glycerination of Amoeba proteus.

The process of glycerination of Amoeba proteus was observed under the holographic microscope with coherent noise elimination. It was found that during glycerination redistribution of cell material occurs, and is accompanied by reversible deformation of the cell cortex. The rôle of the cortex in shape maintenance in glycerinated models was demonstrated.

Amoeba↗
PubMed1978

Interference reflection microscopy of adhesion of Amoeba proteus.

A simple method of observing the adhesive behaviour of large cells by means of interference reflection microscope is described, and some observations of monopodial Amoeba proteus are presented. Amoebae may contact with the substratum at any point along the long axis of the cells. Points of contact are usually few, small and temporary. Frequently the cell surface within the contact points oscillates, changing the separation distance from the substratum.

Amoeba↗