A myoepithelial-like cell line (Rama 401), isolated from rat mammary gland, has been transformed with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV). Rama 401-tsRSV cells adopt a spindle morphology and fail to deposit basement membrane proteins when grown at the permissive temperature (35°C). When switched to the non-permissive temperature (41°C), the cells flatten (with a 5-fold increase in area), and deposit an extracellular matrix containing basement membrane proteins. When the cells are switched from 35°C to 41 °C in the presence of monensin (an ionophore that inhibits protein secretion), basement membrane proteins are no longer deposited extracellularly although the cells flatten, their area increasing by ninefold. Cells switched from 35 °C to 41 °C in the presence of cycloheximide still flatten and deposit basement membrane proteins, whereas the morphological change on switching from 41 °C to 35 °C is inhibited by cycloheximide. These experiments indicate that the ability of Rama 401-tsRSV cells to spread on a plastic substratum is not dependent on the de novo synthesis and deposition of basement membrane proteins.

The ducts of the mature, virgin rat mammary gland consist of a layer of luminal epithelial cells that are separated from the basement membrane by an apparently continuous layer of myoepithelial cells (Radnor, 1972a,b; Warburton et al. 19826). In vivo and in vitro evidence both indicate that myoepithelial cells are largely responsible for the synthesis of basement components (Liotta et al. 1979 ; Warburton et al. 1981, 1982a). The origin of myoepithelial cells is not entirely clear. Initial studies (Radnor, 1972a) indicated that myoepithelial cells develop from undifferentiated pale cells that descend towards the basement membrane. More recent studies (Williams & Daniel, 1983; Ormerod & Rudland, 1984) have suggested that undifferentiated ‘cap cells’, located in the basal cell layer at the tip of the terminal-end buds, differentiate into myoepithelial cells during ductal elongation. These two mechanisms are not mutually exclusive; both may occur at different stages and at different locations within the developing ductal system. Myoepithelial differentiation includes the development of the myofilament system and cellular spreading, and is associated with changes in both the structure, composition and enhanced deposition of the basement membrane (Warburton et al. 19826; Williams & Daniel, 1983). It is therefore likely that myoepithelial cell-basement membrane interactions are important in, and may even determine, myoepithelial characteristics. In this paper, we examine the correlation between cell spreading and basement membrane deposition using a virally transformed rat mammary myoepithelial cell line.

Cell culture

Rama 401 is a single-cell-cloned, myoepithelial-like cell line established from a primary culture of a neonatal rat mammary gland (Warburton et al. 1981). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 5 % foetal calf serum (Gibco, Paisley) and 50μgml−1 ascorbic acid. The procedure for the transformation of Rama 401 cells with Rous sarcoma virus (RSV) will be described in detail elsewhere (Warburton et al. unpublished). Briefly, cultures were infected with ≈107 focus forming units ml−1 of a temperature-sensitive mutant of RSV, tsLA339 (Wyke, 1973) in medium containing 1μgml−1 dexamethasone and lμgml−1 polybrene. After 2h of absorption at 35°C the medium was replaced, and again after 24 h. Cells were transferred after 4, 7 and 10 days, by which time all the cells appeared to be spindle-shaped. Cells were then transferred every 4 days at a ratio of 1:10. In some experiments 1 μM-monensin (Calbiochem) or cycloheximide (1 μgml−1) was included in the culture medium. In these experiments, the foetal calf serum was depleted of fibronectin by two cycles of chromatography on gelatin-Sepharose.

Cell areas were compared by tracing the outline of cells onto a digitizing tablet (Hypad, Sintron, Reading, UK) input into an Apple lie microcomputer.

Cell attachment and spreading were determined in bacteriological dishes coated with various substrata. The dishes were coated by adding a solution of the component (10μgml−1) and allowing them to air-dry overnight. After several washes with phosphate-buffered saline (PBS), ≈ 10s Rama 401 cells were added, and the dishes incubated at 37°C for 3 h in serum-free medium. The medium was then removed and PBS was pipetted vigorously over the surface of the dishes to remove loosely adherent cells. The number of attached cells was determined, after trypsinization, with a Coulter counter. In all other experiments cells were grown on either polystyrene coverslips or Nunc Petri dishes to which they readily attach without addition of exogenous basement membrane proteins.

Immunofluorescence staining

Cells on polystyrene coverslips (Lux Scientific Corp.) were fixed and made permeable to the antibodies by incubation in methanol at 4°C for 2h. The coverslips were washed with PBS and incubated with the first antibody (diluted 1:100 with 0·5% bovine serum albumin in PBS) for 40min at room temperature and then washed seven times with PBS. Incubation was continued with fluorescein (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) (diluted 1:40, Nordic Immunological Laboratories) for 40 min at room temperature. After further washing with PBS, the coverslips were mounted in Hydromount (National Diagnostics Inc.) and fluorescence was observed with a Reichert Polyvar fluorescence microscope. Photographs were taken on Ilford XP1 film. The production and characterization of polyclonal antibodies to type IV collagen, laminin and fibronectin have been described previously (Warburton et al. 1981).

Analysis of secreted proteins

Cultures of Rama 401-tsRSV cells, growing at 35°C and then switched to 41°C were labelled with [3H]proline (10μCiml−1) for 24h. After addition of protease inhibitors, the culture medium was dialysed against water, lyophilized, and analysed by electrophoresis through a 7 % poly-acrylamide-sodium dodecyl sulphate gel. To compare the amount of fibronectin attached to the culture dish, radioactively labelled cells were detached by incubation with 0·1 % Triton X-100 in PBS for 5 min. The dishes were washed twice with PBS, and material attached to the surface of the culture dish was dissolved in 4 M-guanidine-hydrochloride, 50mM-Tris-HCl, pH 7·4. After dialysis against PBS, fibronectin was immunoprecipitated as described (Warburton et al. 1982a).

Electron microscopy

Cells grown on Thermanox coverslips (Lux Scientific Corp.) were washed with PBS, fixed in 2% glutaraldehyde for 1 h, and postfixed in 1 % osmium tetroxide for 2h. Both fixatives were phosphate-buffered (pH7·2–7·4), and the osmotic pressure was adjusted to 350mosM by addition of sucrose. Samples were dehydrated in ethanol and embedded in Epon/Araldite via propylene oxide. Semithin sections (1μm) for light microscopy were stained with Toluidene Blue, and regions were selected for electron microscopy. Ultrathin sections were cut on a Reichert 0MU4 ultratome, stained with uranyl acetate and lead citrate, and examined in a Philips EM400 electron microscope.

Effect of basement membrane proteins on cell attachment to bacteriological dishes

Rama 401 cells did not attach to uncoated bacteriological dishes or dishes coated with bovine serum albumin (BSA). However, when the dishes were coated with either fibronectin or laminin the cells readily attached and spread (Table 1). Control experiments indicated that maximum cell spreading occurred within 3 h and that most (≈90%) of the attached cells had spread and attained their polygonal morphology (data not shown). Type I collagen was ineffective at promoting Rama 401 cell attachment, whereas type III and IV collagens had a small effect.

Table 1.

Spreading of Rama 401 cells on various substrates

Spreading of Rama 401 cells on various substrates
Spreading of Rama 401 cells on various substrates

Effect of temperature on cell spreading and basement membrane protein deposition

Rama 401 cells normally grow as flat, irregularly shaped polygonal cells surrounded by a peripheral extracellular matrix that contains basement membrane proteins (Warburton et al. 1981). This morphology and ability to deposit basement membrane proteins extracellularly was maintained at both 35 °C and 41 °C (data not shown). After transformation with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV), the cells became spindle-shaped when grown at the permissive temperature for viral transformation (35 °C) but reverted to the flat, polygonal morphology when switched to the non-permissive temperature (41 °C) for 24h. When grown at 35 °C, Rama 401-tsRSV cells were no longer able to deposit an extracellular matrix containing laminin, fibronectin (Fig. 1A,C) and type IV collagen (not shown) but were able to do so when switched to 41 °C (Fig. 1B,D). Cells grown at 35 °C appear to have stronger diffuse, cytoplasmic staining than cells grown at 41 °C.

Fig. 1.

Effect of temperature on the immunofluorescence localization of basement membrane proteins. Rama 401-tsRSV cells grown at either 35°C (A,C) or 41 °C (B,D) were incubated with antibodies to laminin (A,B) or fibronectin (C,D). Note the increase in cell spreading and extracellular deposition of basement membrane proteins when the cells are grown at 41 °C. Bar, 30 μm.

Fig. 1.

Effect of temperature on the immunofluorescence localization of basement membrane proteins. Rama 401-tsRSV cells grown at either 35°C (A,C) or 41 °C (B,D) were incubated with antibodies to laminin (A,B) or fibronectin (C,D). Note the increase in cell spreading and extracellular deposition of basement membrane proteins when the cells are grown at 41 °C. Bar, 30 μm.

Effect of monensin on cell spreading and deposition of basement membrane protein

When cells are grown at 35 °C in the presence of monensin for 24 h, they become more rounded, and immunofluorescence staining reveals several bright, cytoplasmic vacuoles containing basement membrane proteins. When switched to 41 °C for 24 h in the presence of monensin, the cells become larger, and the number of cytoplasmic vacuoles increases. However, immunofluorescence staining failed to reveal any extracellular deposits containing basement membrane proteins (Fig. 2). These experiments were carried out in the presence of fibronectin-depleted serum. Quantitative morphometric analysis indicated that cell area increased fivefold in control Rama 401-tsRSV cells, and by ninefold in monensin-treated cells after switching from 35 °C to 41 °C (Table 2).

Table 2.

Quantitative morphometric analysis of cell areas

Quantitative morphometric analysis of cell areas
Quantitative morphometric analysis of cell areas
Fig. 2.

Effect of monensin on the immunofluorescence localization of basement membrane proteins. Rama 401-tsRSV cells grown at either 35°C (A,C) or 41 °C (B,D) in the presence of 1 μM-monensin were incubated with antibodies to laminin (A,B) or fibronectin (C,D). Note the increase in cell spreading but lack of extracellular deposition of basement membrane proteins when the cells are grown at 41 °C. Bar, 30μm.

Fig. 2.

Effect of monensin on the immunofluorescence localization of basement membrane proteins. Rama 401-tsRSV cells grown at either 35°C (A,C) or 41 °C (B,D) in the presence of 1 μM-monensin were incubated with antibodies to laminin (A,B) or fibronectin (C,D). Note the increase in cell spreading but lack of extracellular deposition of basement membrane proteins when the cells are grown at 41 °C. Bar, 30μm.

Analysis of proteins secreted by control Rama 401-tsRSV and monensin-treated cells 24 h after switching from 35 °C to 41 °C by polyacrylamide gel electrophoresis demonstrated that there was a dramatic decrease in the secretion of all proteins (Fig. 3). Quantitative measurement of the incorporation of [3H]proline into trichloroacetic acid-insoluble protein indicated a 95 % inhibition of protein secretion by 1 /ZM-monensin during the 24 h labelling period. Analysis of the material attached to the culture dish after the removal of cells with detergent revealed a marked decrease in the amount of fibronectin in monensin-treated cells (Fig. 4). Scanning densitometry of the autoradiograph indicated an eightfold decrease in the amount of substrate-attached fibronectin. The amounts of laminin and type IV collagen in the substrate-attached material were too small for reproducible quantification in this way, although decreased deposition was observed in monensin-treated cells (not shown).

Fig. 3.

Effect of monensin on protein secretion. Proteins secreted by Rama 401-tsRSV cells grown in the absence (A) or presence (B) of monensin were analysed by polyacrylamide gel electrophoresis. The migration positions of molecular weight markers (×10−3) are shown at the left.

Fig. 3.

Effect of monensin on protein secretion. Proteins secreted by Rama 401-tsRSV cells grown in the absence (A) or presence (B) of monensin were analysed by polyacrylamide gel electrophoresis. The migration positions of molecular weight markers (×10−3) are shown at the left.

Fig. 4.

Effect of monensin on substrate-attached fibronectin (fn). Material attached to the culture dish was solubilized after removal of the cells with Triton X-100. The extract, from cells grown in the absence (A) or presence (B) of monensin, was immunoprecipitated with antibodies to fibronectin, and analysed by polyacrylamide gel electrophoresis. The arrow marks the migration position of rat plasma fibronectin.

Fig. 4.

Effect of monensin on substrate-attached fibronectin (fn). Material attached to the culture dish was solubilized after removal of the cells with Triton X-100. The extract, from cells grown in the absence (A) or presence (B) of monensin, was immunoprecipitated with antibodies to fibronectin, and analysed by polyacrylamide gel electrophoresis. The arrow marks the migration position of rat plasma fibronectin.

Effect of cycloheximide on cell spreading and deposition of basement membrane protein

Cells switched from 35 °C to 41 °C in the presence of cycloheximide were still able to flatten and assemble extracellular deposits of basement membrane proteins (Fig. 5). However, both the morphological changes and loss of the extracellular matrix observed when the cells are switched from 41 °C to 35 °C were inhibited by cycloheximide. Cycloheximide, at a concentration of 1/zgml-1, inhibits protein synthesis by 90–95 % in Rama 401-tsRSV cells.

Fig. 5.

Effect of cycloheximide on the immunofluorescent localization of basement membrane proteins. Rama 401-tsRSV cells were switched from 35°C to 41°C (A,B) or from 41 °C to 35°C (C,D) in the presence (A,C) or absence (B,D) of cycloheximide and incubated with an antibody to laminin. Cycloheximide does not inhibit cell spreading and laminin deposition during the switch from 35°C to 41 °C, but it does inhibit the morphological change and loss of extracellular laminin when the cells are switched from 41 °C to 35°C. Bar, 30μm.

Fig. 5.

Effect of cycloheximide on the immunofluorescent localization of basement membrane proteins. Rama 401-tsRSV cells were switched from 35°C to 41°C (A,B) or from 41 °C to 35°C (C,D) in the presence (A,C) or absence (B,D) of cycloheximide and incubated with an antibody to laminin. Cycloheximide does not inhibit cell spreading and laminin deposition during the switch from 35°C to 41 °C, but it does inhibit the morphological change and loss of extracellular laminin when the cells are switched from 41 °C to 35°C. Bar, 30μm.

Electron microscopy

Electron microscopy of cells grown at 35 °C in the presence of monensin revealed large cytoplasmic vesicles filled with amorphous material apparently composed of two components differing in electron density (Fig. 6). The membranes of these vesicles were lined with ribosomes indicating that they originated from rough endoplasmic reticulum. Occasional normal Golgi were seen. These vesicles were not observed in control cells incubated without monensin. However, control cells contained smaller, smooth-membraned vesicles that were devoid of electron-dense material. Other studies have indicated that in monensin-treated fibroblasts, extracellular matrix components largely accumulate in electron-lucent, smooth-membraned vesicles (Ledger et al. 1980), provided that incubation with monensin is carried out for a relatively short time (<5 h). The accumulation of secretory deposits in the rough endoplasmic reticulum observed in Rama 401-tsRSV cells is probably a consequence of prolonged (24 h) incubation with monensin.

Fig. 6.

Ultrastructure of monensin-treated cells. Monensin-treated cells (A) are characterized by large cytoplasmic vacuoles composed of two components differing in electron density. The membranes of these vesicles are lined with ribosomes. Such vacuoles are not present in control cells (B). However, these cells contain distended rough endoplasmic reticulum and smooth-membraned, clear vesicles. Bars: A, 0·5 μm; B, 0·8μm.

Fig. 6.

Ultrastructure of monensin-treated cells. Monensin-treated cells (A) are characterized by large cytoplasmic vacuoles composed of two components differing in electron density. The membranes of these vesicles are lined with ribosomes. Such vacuoles are not present in control cells (B). However, these cells contain distended rough endoplasmic reticulum and smooth-membraned, clear vesicles. Bars: A, 0·5 μm; B, 0·8μm.

The ability of the myoepithelial cell line, Rama 401, to attach and spread on a plastic surface is mediated by both laminin and fibronectin. Other extracellular matrix components investigated (collagens types I, III and IV) had little or no effect. Although it was originally suggested that laminin promoted the attachment and spreading of epithelial cells, whereas the effects of fibronectin were restricted to spreading of epithelial cells, whereas the effects of fibronectin were restricted to mesenchymal cells, it now seems clear that these proteins can be involved in the attachment of both epithelial and mesenchymal cells (Grinnell & Field, 1979; Terranova et al. 1980; Couchman et al. 1983). In the case of Rama 401 cells, this type of assay probably reflects promotion of attachment as ≈ 90 % of the cells that attached within 3 h had also spread on the substratum. To obtain a better indication of the role of basement membrane proteins in myoepithelial cell spreading, we have examined the deposition of these proteins during the spreading of Rama 401 cells infected with a temperature-sensitive mutant of Rous sarcoma virus as the cells are switched from the permissive to the non-permissive temperature for transformation.

The results obtained with the virally transformed cells clearly dissociate the ability of Rama 401 cells to spread on a plastic substratum from the de novo synthesis and deposition of basement membrane proteins. The ability of Rama 401-tsRSV cells to spread on the plastic substratum when switched from 35 °C to 41 °C is not impaired by the presence of monensin at a concentration that effectively inhibits secretion and extracelluar deposition of basement membrane proteins. Several studies have shown that monensin inhibits the passage of extracellular matrix molecules and secretory proteins within the Golgi complex, probably by perturbing intracellular monovalent cation levels (Ledger et al. 1980; Uchida et al. 1980; Tartakoff, 1983; Kay et al. 1984). The cell spreading experiments were carried out in the presence of fibronectin-depleted serum and it therefore seems unlikely that serum-derived fibronectin adsorbed onto the plastic surface can be involved in the spreading process. Serum contains other cell attachment and spreading factors, e.g. vitronectin (Hayman et al. 19856). However, the presence of such factors alone cannot account for cell spreading as Rama 401-tsRSV cells maintain their spindle morphology when grown at 35 °C in the presence of whole serum.

The ability of Rama 401-tsRSV to deposit basement membrane proteins when switched to the non-permissive temperature in the presence of cycloheximide suggests that there is an intracellular pool of basement membrane protein in cells grown at 35 °C that can be secreted and organized extracellularly in the absence of de novo protein synthesis. This observation suggests that the inability of Rama 401-tsRSV cells to deposit basement membrane proteins at 35 °C results from a defect in the extracellular assembly of these proteins rather than inhibition of their synthesis. Inhibition of the morphology changes and loss of extracellular basement membrane deposits by cycloheximide during the switch to the permissive temperature may result from the inability to synthesize pp60v-src under these conditions. Similarly, whatever surface components are involved in the spreading of these cells, they must also be present (at 35 °C) in an intracellular pool at sufficient levels to enable the cells to spread when switched to 41 °C in the absence of de novo protein synthesis.

Attachment and spreading of cells have been extensively investigated, especially in fibroblastic systems (Grinnell, 1978). Cells use multiple spreading mechanisms. Attachment and spreading can be determined by the extracellular matrix, serum proteins, and cell surface components (Damsky et al.,1982; Stenn, 1981; Stenn et al. 1983; Hayman et al. 1985b). Cell spreading on culture dishes coated with extracellular matrix or serum proteins rarely approaches 100%, indicating that even in relatively homogeneous populations of cells several mechanisms must be utilized to obtain maximum spreading. Fibroblasts, for example, can attach and spread by fibronectin-dependent (Grinnell & Field, 1979) or independent mechanisms (Harper & Juliano, 1981). Experiments with monensin-treated fibroblasts have also demonstrated that this type of cell can spread independently of fibronectin deposition (Virtanen et al. 1982; Lehto & Virtanen, 1985). The physiological significance of fibroblast spreading is not entirely clear. However, the spreading of myoepithelial cells has been demonstrated to coincide with the acquisition of the differentiated phenotype and with changes in the synthesis and structure of the basement membrane (Williams & Daniel, 1983; Ormerod & Rudland, 1984). Attached Rama 401-tsRSV cells spread equally in the presence or absence of de novo basement membrane protein synthesis and deposition, suggesting that the nature of the substratum is not the predominant factor that determines cell spreading in this cell type.

We thank Dr J. Wyke for his generous gift of viruses. M. J. Warburton is supported by a grant from the Cancer Research Campaign.

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