The tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes of cell shape in certain fibroblastic lines: motile lamellas are transformed into noncontractile narrow processes; simultaneously, the actin microframent network of lamellas is locally disorganized. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentincontaining intermediate filaments (IF) in the development of PMA-induced reorganizations. PMA readily induced formation of noncontractile processes in Taxol-treated fibroblasts; these cells had a profoundly altered microtubular system but noncollapsed IF. A short (1 hr) exposure to PMA induced formation of processes in control cells but not in the Colcemidtreated cells, which had depolymerized microtubules and IF that collapsed around the nucleus. Longer (3-4 hr) exposure of the Colcemid-treated cells to PMA induced partial reversal of the IF collapse; those parts of the peripheral lamellas that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction ofIF with the actin system is an essential step in the formation of processes from lamellas. The microtubular system controls distribution of IF in the cytoplasm and thus plays an indirect role in the reorganization of the actin cortex.Alterations of the degree of cell segregation into two types of cytoplasmic domains, motile lamellas and nonmotile narrow processes, are characteristic of many morphological differentiations (1). Reversible enhancement of this segregation accompanied by the pronounced formation of nonmotile long processes from motile lamellas is induced in certain fibroblastic and epithelial cultures by phorbol 12-myristate 13-acetate (PMA). This phenomenon is convenient for the experimental analysis of mechanisms controlling segregation (1-5, t). PMA-induced transformation of lamellas into stable processes was found to be accompanied by profound reorganization of the cytoskeleton, especially by the disappearance of the network of actin microfilaments filling lamellas and by the loss ofcontractility ofthe actin cortex (3-5). These alterations of the microfilament system were shown to be microtubule dependent (4, t): their development after shortterm PMA treatment was prevented by preincubation of cells with Colcemid, which caused complete depolymerization of microtubules and was accompanied by perinuclear collapse of vimentin-containing intermediate filaments (IF). These data suggest that the cell has a special microtubule-dependent mechanism controlling the actin microfilament system (4).Experiments presented in this paper show that when the time of exposure of Colcemid-treated cells to PMA is increased to 3-4 hr, microtubules remain depolymerized, but IF collapse is reversed; simultaneously, formation of narrow processes from lamellas takes place. The main cytoskeletal elements pres...

Movements of the receptors of concanavalin A on various parts of the surfaces of substrate-attached cells were compared. Cultured mouse embryo cells of several types were used: epithelial kidney cells and normal and transformed fibroblasts. Initial distribution of receptors was random on the cells of all types. Binding of concanavalin A induced patching of its receptors on all the cell parts. In contrast, directional centripetal movement of receptors was observed only on the surface of certain cell parts, namely, only the surface of peripheral lamellar cytoplasm was cleared of the receptors.Clearing was always initiated in the zone of lamellar cytoplasm located near active cell edges. In epithelial sheets, clearing was not observed on the surface of central cells that had no lamellar cytoplasm. Concanavalin A receptors on the cleared areas of cell surface were gradually restored after the end of incubation. It is suggested that anchoring of the patches of membrane receptors by cortical microfilaments is possible only on the surface of pseudopods and of lamellar cytoplasm but not on the surface of other cell parts. Besides receptor movements, this hypothesis may also explain differences in the adhesive properties of various parts ol the cell surface.The external edge of substrate-spread cultured cells is usually divided into discrete zones of two types: active zones that continuously protrude pseudopods and stable zones that do not form pseudopods. Specialized peripheral structure, lamellar cytoplasm or lamelloplasm, is formed in association with active cell edges (1,2). Distinctive characteristics of lamellar cytoplasm, as opposed to the more central endoplasm, are as follows: (a) lamelloplasm is delimited by an active edge; the entire external edge of lamelloplasm is not active, but usually all the active zones of the edge delimit lamelloplasm; stable zones may delimit either lamelloplasm or endoplasm. (b) Lamelloplasm contains no vesicular organelles visible by phase contrast microscopy. (c) The lower surface of lamelloplasm has numerous sites of attachment to the substrate. (d) The upper surface of the zone near active edges, and, possibly, of the whole lamelloplasm, is adhesive for inert particles (3, 4). Defective formation of lamellar cytoplasm is a characteristic feature of transformed fibroblasts (1, 2).In this paper we compare the movements of concanavalin A (Con A) receptors on the surface of lamelloplasm with those on the other cell parts. Con A and other ligands crosslinking surface receptors induce redistribution of these receptors (5-10). Results obtained with substrate-attached fibroblasts indicate that patches of the receptors may move centripetally from the active cell edges (7-9). Our purpose was to compare receptor movements on the cells in two states: with lamelloplasm and without it. Epithelial cells are especially convenient for such comparison: these cells grow as coherent sheets in which lamelloplasm and active cell edges are formed only by marginal cells but not by the central ce...

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Substrate-attached normal mouse fibroblasts, transformed mouse fibroblasts (L strain), and epithelial cells (MPTR strain) were incubated with two ligands crosslinking different groups of the surface receptors: concanavalin A and cationic ferritin. Surface-attached ligands were revealed by an indirect immunofluorescence method. Incubation of control cells with these ligands induced the patching of corresponding surface receptors and the clearing of these receptors from the surface zones located on thelamelar cytoplasm near cell edges actively protruding pseudo dia. Effects of three antitubu ins (microtubule-destroying drugs:Colcemid, colchicine, and vinblastine) on the ligand-induced redistribution of receptors were investigated and compared with the previously described effects of these drugs on the distribution of active cell edges. Incubation of normal and transformed fibroblasts with these antitubulins led to the disappearance of nonactive cell edges; the whole cell perimeter became active. Correspondingly, the clearing pattern of the surface receptors of fibroblasts was altered by antitubulins: the cleared area in antitubulin-treated cells formed a circular band along the whole peripheral cell edge. In epithelial cultures, in contrast to fibroblastic ones, antitubulins changed neither the distribution of the active sites of the surface nor the distribution of the areas cleared of crosslinked receptors. Thus, the specific ability of the surface areas located near the active cell edges to become cleared of crosslinked receptors is characteristic not only for the cells with intact microtubules, but also for the cells with microtubules destroyed with antitubulins.Crosslinking of surface receptors by polyvalent ligands, such as lectins or antibodies, may lead to redistribution of these receptors: patches of receptors are formed, and certain zones of the surface are cleared of these patches; this second process is often designated as capping (1-3). Experiments with substrate-attached fibroblasts and epithelial cells have shown that only specific zones of the cell surface are cleared of the receptors crosslinked by concanavalin A (Con A) (4-7). These are the zones of substrate-attached lamellar cytoplasm located near active cell edges, i.e., near the edges where pseudopodia and ruffles are continuously formed. This paper describes the results of experiments testing the effects of three different microtubule-destroying drugs (antitubulins including colchicine, Colcemid, and vinblastine) on the redistribution of crosslinked surface receptors of fibroblasts and epithelial cells. As previously described (8), incubation of fibroblasts with these antitubilins leads to disappearance of the nonactive parts of the cell edges; all of the external edge of these cells becomes active. In contrast, distribution of stable and active edges in the sheets of cultured epithelial cells is not altered by antitubulins. Correspondingly, localization of the surface zones cleared of crosslinked receptors is altered by antitubulins ...

Summary-3H-thymidine labelling and autoradiography were used to compare density dependent inhibition of growth in the cultures of two transformed lines of hamster fibroblasts and in primary cultures of their parent normal cells. Similar manifestations of density dependent inhibition were found in the isolated cultures of normal and neoplastic cells: at saturation densities these cultures had low labelling indices; these indices considerably increased when the cells migrated into the wound from the dense sheet, prelabelled cells seeded on the dense sheets of unlabelled homologous cells did not proliferate. However, proliferation of neoplastic cells was not inhibited when they were seeded on the dense sheet of normal fibroblasts. Thus, neoplastic hamster fibroblasts of both lines retained sensitivity to the inhibiting effect of homologous neoplastic cells but completely lost sensitivity to the inhibiting effect of normal fibroblasts. The possible significance of this selective loss of the sensitivity to normal cells is discussed briefly.INSENSITIVITY to the local inhibiting effect of homologous cells (loss ofthe homologous density dependent inhibition of growth) is usually regarded as one of the characteristic traits of transformed fibroblasts. At the same time, certain lines of transformed cells retain sensitivity to heterologous inhibition: their growth is inhibited by normal fibroblasts (Stoker, 1964;Ponten and MacIntyre, 1968;Weiss and Njeuma, 1971 The cells were grown in small (10 ml)