When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on released type I collagen gels show greatly enhanced mRNA levels and secretion rates of ,B-casein and of some other milk proteins. We show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows >90% of cells to produce high levels of 13-casein. By 1-3). Until a decade ago, most investigators cultured cells on plastic surfaces, which led to drastic alterations of morphology and function from the parent tissue (4). Following the example of Emerman and Pitelka (5), we and others have shown that several aspects of functional epithelium can be maintained when primary mouse mammary epithelial cells (PMME) are cultured on "released" (RG or "floating") collagen type I gels instead of plastic. These include polarization of organelles, appearance of apical microvilli, formation of a basal lamina (5, 6), changes in glucose metabolite pattern (7), lumina formation (8), altered synthesis and compartmentalization of extracellular matrix (ECM) components (9, 10), enhancement ofmost milk protein synthesis and secretion (11), and increases in p-casein (12) and transferrin (13,14) mRNA levels. These results signify the importance of cell-substratum interactions in regulating tissue-specific functions and point to a possible regulatory role for ECM in vivo.Cells on released gels (and not on plastic or flat gels) were shown to synthesize an intact basement membrane (5) containing high levels of heparan sulfate and other sulfated glycosaminoglycans (9), type IV collagen, and laminin (10). We reasoned that the released gel may act through the influence of newly synthesized basement membrane components. To test the mechanism of cell-ECM interaction more directly, we have analyzed the consequences of culturing PMME cells on a reconstituted basal lamina derived from Engelbreth-Holm-Swarm (EHS) tumor (15) and on some of its individual components.MATERIALS AND METHODS PMME from 14-to 17-day pregnant BALB/c mice and collagen from rat tail tendon were prepared as described (5, 10, 11). Tumors (EHS) from normal or lathyritic mice were extracted with high salt and 2 M urea as described by Kleinman et al. (15). Dialyzed EHS extract (100-200 1.d) was spread either directly on 35-mm dishes or on top of rat-tail collagen gels. Collagen gels were released 3-4 days after seeding. Matrigel, an EHS preparation from Collaborative Research (Waltham, MA), was used for some experiments. Laminin and type IV collagen (Bethesda Research Laboratories) were spread at 2.5-10 pug/cm2. Heparan sulfate proteoglycan (HSPG; low density form) was prepared from EHS tumors as described by Hassell et al. (16) and was spread at 1-5 ,ug/cm2 or was added to the medium at 5 ,ug/ml every other day. The latter was less toxic but also less effective. EHS (10 ,ug/ml) and individual substrata other than HSPG we...

The initial events during phagocytosis of latex beads by mouse peritoneal macro-=; phages were visualized by high-resolution electron microscopy of platinum replicas of freezedried cells and by conventional thin-section electron microscopy of macrophages postfixed with I% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These results suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

Induction of the neutral proteinase, collagenase, is a marker for a specific switch in gene expression observed in rabbit synovial fibroblasts . A variety of agents, including 12-0-tetradecanoylphorbol-13-acetate, cytochalasins B and D, trypsin, chymotrypsin, poly(2-hydroxyethylmethacrylate), and trifluoperazine induced this change in gene expression . Induction of collagenase by these agents was always correlated with a marked alteration in cell morphology, although the cells remained adherent to the culture dishes . The amount of collagenase induced was positively correlated with the degree of shape change produced by a given concentration and, to some extent, with the duration of treatment . Altered cell morphology was required only during the first few hours of treatment with inducing agents ; after this time collagenase synthesis continued for up to 6 d even when agents were removed and normal flattened cell morphology was regained. All agents that altered cell morphology also produced a characteristic switch in protein secretion phenotype, characterized by the induction of procollagenase (M r 53,000 and 57,000) and a neutral metalloproteinase (M r 51,000), which accounted for approximately 25% and 15% of the protein secreted, respectively . Secretion of another neutral proteinase, plasminogen activator, did not correlate with increased collagenase secretion . In contrast, synthesis and secretion of a number of other polypeptides, including the extracellular matrix proteins, collagen and fibronectin, were concomitantly decreased . That changes in cell shape correlated with a program of gene expression manifested by both degradation and synthesis of extracellular macromolecules may have broad implications in development, repair, and pathologic conditions. The neutral proteinase, collagenase, which specifically degrades interstitial collagens, is induced by a variety of agents, including the phorbol diester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)' (2, 10), cytochalasin B (CB) (23), and proteolytic enzymes (52), as well as by phagocytosis (54). We have demonstrated that TPA triggers an alteration in gene expression in rabbit synovial fibroblasts that is marked by induction of several secreted proteins, including collagenase and a wide-spectrum neutral metalloproteinase, and by reduction of the synthesis of other secreted proteins, including 'Abbrevations used in this paper.

Abstract. Agents that alter the morphology of rabbit synovial fibroblasts induce synthesis of the metalloendopeptidases, collagenase and stromelysin. We studied the relationship of cytoskeletal changes to the commitment to expression of these metalloendopeptidases. Cells treated with cytochalasin B (CB) or 12-0-tetradecanoylphorbol-13-acetate rounded, and only cells that had lost their stress fibers expressed collagenase and stromelysin, as determined by immunofluorescence. We concentrated on the effects of CB because of its rapid reversibility. When CB was added for 1-24 h, then removed, the cells respread within 30-60 min. The minimum period of CB treatment that committed cells to the subsequent synthesis of collagenase and stromelysin was 3 h. After initial treatment with 2 #g/ml CB for 3-24 h, or with various concentrations of CB (0-2 vg/ml) for 24 h, both enzyme activity and biosynthesis of the proenzymes showed a graded increase when measured at 24 h. Even after treatment with 2 ug/ml CB for only 3 h, >85% of all cells were positive for both collagenase and stromelysin when cells were monitored by immuno fluorescence. In contrast, when the dependence of collagenase and stromelysin expression on the inducing concentration of CB was examined, there was a dose-dependent increase in the number of cells positive for collagenase and stromelysin, as determined by immunofluorescence. Thus, at low concentrations of CB (<0.5 ug/ml), a heterogeneous population response was observed. These results suggest that the commitment of fibroblasts to induction of the metalloproteinases is a stochastic process in which a second signal that correlates with the disruption of the actin cytoskeleton may be rate-limiting for coUagenase and stromelysin gene expression. emerging body of knowledge from a variety of experimental systems indicates that there is a correlation between cytoskeletal architecture and the expression of certain genes (2,4,6,7,11,12,15,(24)(25)(26)(27)(28)31). Many of these cases have involved the terminal differentiation of developing systems with a permanent reorganization of the actin cytoskeleton. For example, in induction of chondrogenesis (6, 25, 27, 31) and adipogenesis (24) and in mammary epithelial differentiation (11, 15), the acquisition of a rounded cell shape and the reorganization of the actin cytoskeleton are prerequisites for initiation of expression of genes that encode differentiated cell products.In previous work using rabbit synovial flbroblasts (l, 2, 9, 16) we studied a reversible alteration in collagenase gene expression that correlated with changes in cell shape. Recently, we described a second metalloendopeptidase, stromelysin (9, 13), that is secreted by fibroblasts along with collagenase. Our data suggested a model in which an agent must be present initially for several hours for induction of the proteinases to take place and must cause alterations in cell morphology during this period. Synthesis and secretion of collagenase and stromelysin, as their zymogen precursors procollagenase ...

The structure, distribution and composition of the extracellular matrix present around the human oocyte and in the cumulus was examined following fixation in the presence of ruthenium red. An extracellular matrix comprising granules and filaments is present in the cumulus layer, in the corona radiata, in the outer pores of the zona pellucida and in the perivitelline space surrounding unfertilized oocytes. In replicate samples, the extracellular matrix comprised filaments which were mostly very long, occasionally cross-connected by shorter filaments, and usually decorated with numerous small granules. Enzymatic digestion with affinity-purified trypsin or Streptomyces hyaluronidase removes the granules and filaments, respectively, at all levels of the oocyte-cumulus complex. These results are interpreted to mean that protein and hyaluronic acid are present in all extracellular compartments of the human oocyte-cumulus complex. The significance of this distribution of hyaluronic acid with respect to the role of sperm hyaluronidase in fertilization is discussed.