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The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.

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A tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes in the cell shape in certain fibroblastic lines. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Two specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. A short (I h) exposure to PMA induced formation of processes in the control cells rather than in the Colcemid treated cells having depolymerized microtubules and the IF that collapsed around the nucleus. A longer (3-4 h) exposure to PMA of the colcemid-treated cells induced a partial reversal of the IF collapse; those parts of peripheral lamellae that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of the IF with the actin system is an essential step in the formation of processes from lamellae.

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This study shows that artificial increase in cell site leads to morphological normalization of transformed fibroblasts. Mouse L cells (clone 171/5) were used. As most transformed cells, they were poorly spread on the substratum, made only dot-like focal contacts with it, rounded quickly at room temperature and did not contain prominent actin cables. Giant cells were obtained by incubation of these cells in the medium supplemented with mitomycin C (0.15-0.20 mcg/ml). DNA synthesis and mitosis were blocked by this treatment, while protein synthesis was changing very slightly. As a consequence, the cell size increased dramatically from 3 to 11 days of the cell incubation in the mitomycin containing medium. The degree of cell spreading per mcg of protein increased significantly in the giant cells. These cells do not round after moderate cooling, and well developed system of actin cables and matured streak-like focal contacts associated with these cables are formed in them. These results, along with our previous data on the restoration of cell spreading and cytoskeleton structure in giant multinucleated cells, provide strong evidences that the increase in cell size per se can induce qualitative changes in cell morphology. It can be suggested that there are some scaling-dependent factors regulating the processes of cytoskeleton assembly and formation of cell-substrate contacts.

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The range of the Djungarian hamster cell lines selected for colchicine resistance in high doses (from 7 to 200 micrograms/ml) was studied. These cell lines are characterized by the different levels of drug-resistance (1000- to 16000-fold). A positive correlation is found between the reversion rate of malignant phenotype and the cellular drug-resistance level. Tumorigenicity and anchorage independence decrease with an increase of the drug-resistance level. The actin-containing bundles of microfilaments in more resistant cells are more organized. The 22 kD protein content increases with the drug-resistance level. The mechanisms of malignancy reversion are discussed.

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Fibroblast spreading was studied using immunofluorescent method that provided visualization of actin structures and adhesion contacts in the same cell. Four stages of actin system formation were observed. 1. Actin concentration in ruffles at the cell periphery. Formation of numerous dot-like contacts along the whole perimeter of the cell. 2. Formation of a circumferential actin bundle. Focal contacts are located at the outer edge of the bundle. 3. Gradual transformation of the circumferential bundle into actin network with triangular meshes. Peripheral (rather than internal) filaments of the network are associated with the focal contacts. 4. Appearance of the system of long straight actin bundles (stress fibers) associated with dash-like focal contacts. The stress fibers are supposed to arise from the triangular actin network which in its turn arises from the circumferential bundle. It is suggested that the formation of actin cytoskeleton is a process driven by the development of tensions in actin structures attached to the focal contacts at the cell periphery.

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Cultured cells attach to the substratum by means of specialized domains of cell surface, called focal contacts. The inner side of the cell membrane is associated in these structures with cytoskeletal elements, while the outer side is connected with extracellular matrix. The present review describes both light and electron microscopic methods of studying the focal contacts and ultrastructure of adhesion plaque, that is the cytoskeletal domain of focal contact. The proteins of adhesion plaque and focal contact membranes are also characterized. The processes of the formation of focal contacts and their association with the bundles of actin microfilaments in normal cultured fibroblasts are described in detail. Association of focal contacts with other cytoskeletal elements microtubules and intermediate filaments is discussed. The neoplastic transformation induced changes of focal contact system and cytoskeletal structures associated with contact sites are described.

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17 cloned cell lines of transformed mouse fibroblasts were used for the evaluation of a correlation between three traits of malignancy: cloning efficiency in semisolid medium (CE); cell dose inducing tumours in 50% of inoculated animals (TD50); degree of cell attachment to a substrate (RT50--rounding time for 50% of the cells under moderate cooling, 18 degrees C). It is shown that an increase in RT50 (better attachment of the cells to the substrate) is accompanied by a decrease in CE and an increase in TD50 (coefficients of rank correlation p = -0.76 and p = 0.58, respectively). A negative correlation between CE and TD50 was also revealed (p = -0.68). A certain increase in the percentage of cells containing actin filament bundles was found in cell clones better attached to the substrate. Mechanisms of disturbances in proliferation and attachment of malignant cells are discussed.

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Cultured mouse embryo fibroblasts were extracted with 1% Triton X-100 at pH 6.7 in buffer containing EGTA and stabilizing supplements. Exposed during this extraction cytoskeletons were fixed, dried with the critical point technique, and shadowed with platinum. The platinum replicas of cytoskeleton were used for electron microscopic characterization of the three-dimensional distribution of cytoskeletal fibrils in cytoplasm. Four cytoplasmic regions are revealed with different cytoskeletal structure; these being a "mesh-work zone", a "loose zone", which together formed an active edge of cell lamelloplasm, the "lamella proper" and endoplasm. The two former zones are occupied with dense three-dimensional or loose planar actin network, respectively. The "lamella proper" contains two-dimensional network of different fibrils: microfilaments, microtubules, intermediate filaments and thin connective filaments. The central perinuclear area has a thick microfilament sheath at dorsal cell surface. The most important cytoplasmic elements of fibroblasts are actin microfilament bundles. The fine structure of these bundles, their junctions with each other ("organizing centers") and their terminal parts corresponding to cell-substrate focal contacts are described.

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Several novel variants of mouse transformed L cells are described. A distinctive trait of these variants, isolated by different methods, is a rounding of the majority of cells under the influence of the moderate cooling (at 18 degrees C for 30-60 min). In the serum-free medium, no rounding occurs. The rounding is presumably an active contraction, because it is inhibited by cytochalasin B. The comparison of the phenotype of wild-type and variant cells has shown that the exposure of cultures at 18 degrees leads to a disturbance of cell adhesion to the substratum in both the cell lines. The intensive rounding of variant cells at 18 degrees is due to some defect in their attachment to the substratum, which can be revealed not only at 18 degrees, but also at 37 degrees. By the selection procedure described in this paper, a large group of cell variants defective in adhesion to the substratum may be isolated.

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The cultured mouse kidney cells forming epithelial sheets were studied using an indirect immunofluorescence microscopy with antibodies against tubulin. These cells, as well as fibroblasts, were found to contain a well developed microtubular system sensitive to colcemid. The assembly of microtubules after washing out of colcemid began from one or two perinuclear centers, associated with the cilium-like structure. There were certain differences between the microtubular systems in epithelial cells and fibroblasts: 1) Microtubules in the fibroblasts penetrated the whole cytoplasm including the peripheral lamella whereas in the epithelial cells the lamellar cytoplasm was often free from microtubules. 2) The orientation of microtubules in the epithelial cells, unlike in the fibroblasts, was not correlated with the stable or active state of the cell margin. A possible role of microtubular system in the epithelial cells and fibroblasts is compared and discussed.

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Aggregation of normal mouse fibroblast-like cells plated on the surface of millipore filter was studied quantitatively. It was shown that addition of 10 mug/ml of cytochalasin B (a drug which blocked active cell locomotion) or 0.1 mug/ml of colcemid (a drug which destroyed polarization processes in the cells and therefore interfered with the oriented cell movement) completely inhibited such aggregation. This inhibition was reversible: after the drugs were washed off the cells did aggregate. It was concluded that cell aggregation in this experimental system required active cell movement and cell polarization.

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On the surface of the nirtocellulose membrane filter (pore size 0.3--0.5 mem), normal mouse or hamster embryo fibroblasts formed discrete cell aggregates. Behaviour of transformed fibroblast-like cells of 9 different lines was compared with that of normal cells. Cells of 3 transformed lines grew on this substratum as a uniform monolayer displaying no tendency to aggregation. The following 3 cell lines exposed a slightly "patchy" cell distribution on the 3rd--4th day of cultivation but were unable to form discrete aggregates. The remaining 3 lines did form aggregates but the dynamics of aggregation and the final aggregation pattern for two of them were abnormal. Only one of the 9 investigated transformed lines had the normal aggregation behaviour. Hence, in the course of the neoplastic evolution, cells lose their ability fo form aggregates on the filter surface. Mechanisms of cell aggregation and possible reasons of differencies in the aggregation behaviour between normal and transformed cells, are discussed.

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